Tese de Doutorado

UNIVERSIDADE FEDERAL DE GOIÁS

PROGRAMA DE PÓS-GRADUAÇÃO EM MEDICINA TROPICAL

E SAÚDE PÚBLICA

ADELIANE CASTRO DA COSTA

AVALIAÇÃO DA MODULAÇÃO DA RESPOSTA IMUNE INDUZIDA POR

VACINA CONTRA TUBERCULOSE: rBCG-CMX

Goiânia,

2016

i

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1. Identificação do material bibliográfico: [ ] Dissertação [ x ] Tese

2. Identificação da Tese ou Dissertação

Autor (a): Adeliane Castro da Costa

E-mail: adeliane.castrodacosta@gmail.com

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Vínculo empregatício do autor Aluno

Agência de fomento: Sigla: CNPq

País: Brasil UF: GO CNPJ:

Título: AVALIAÇÃO DA MODULAÇÃO DA RESPOSTA IMUNE INDUZIDA POR VACINA

CONTRA TUBERCULOSE: rBCG-CMX

Palavras-chave: Tuberculose, Vacinas, BCG e rBCG-CMX

Título em outra língua: EVALUATION OF THE IMMUNE RESPONSE MODULATION

INDUCED BY VACCINE AGAINST TUBERCULOSIS: rBCG-CMX

Palavras-chave em outra língua: Tuberculosis, Vaccine, BCG and rBCG-CMX

Área de concentração: Imunologia

Data defesa: (dd/mm/aaaa) 01/03/2016

Programa de Pós-Graduação: Programa de Pós Graduação em Medicina Tropical e Saúde Pública

Orientador (a): Prof. Dr. Ana Paula Junqueira Kipnis

E-mail: apkipnis@gmail.com

Co-orientador (a):* Prof. Dr. André Kipnis

E-mail: akipnis@gmail.com

*Necessita do CPF quando não constar no SisPG

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ii

ADELIANE CASTRO DA COSTA

AVALIAÇÃO DA MODULAÇÃO DA RESPOSTA IMUNE INDUZIDA POR

VACINA CONTRA TUBERCULOSE: rBCG-CMX

Tese de Doutorado apresentada ao

Programa de Pós-Graduação em

Medicina Tropical e Saúde Pública da

Universidade Federal de Goiás para

obtenção do Título de Doutor em

Medicina Tropical e Saúde Pública.

Orientador: Profa. Dr. Ana Paula

Junqueira Kipnis

Co-orientador: Prof. Dr. André

Kipnis

Goiânia,

2016

iii

iv

Programa de Pós-Graduação em Medicina Tropical e Saúde Pública

da Universidade Federal de Goiás

BANCA EXAMINADORA DA DEFESA DE DOUTORADO

Aluno (a): Ms. Adeliane Castro da Costa

Orientador (a): Profa. Dr. Ana Paula Junqueira Kipnis

Co-orientador (a): Prof. Dr. André Kipnis

Membros:

1. Membro 1 (Presidente da Banca): Profa. Dr. Ana Paula Junqueira Kipnis

2. Membro 2: Profa. Dr. Jaime Martins de Santana

3. Membro 3: Profa. Dr. Luciana Cezar Cerqueira Leite

4. Membro 4: Profa. Dr. Mariane Martins de Araújo Stefani

5. Membro 5: Profa. Dr. Mara Rúbia Nunes Celes

Data: 01/03/16

v

DEDICATÓRIA...

Este trabalho é dedicado

Á Deus, meu pai, companheiro e amigo fiel.

“A Ele Toda Honra e Toda Glória”

Aos meus pais

Antônio Dias da Costa e

Aidê Castro da Costa

Por serem o alicerce da minha vida.

Aos meus irmãos

Vagner Castro da Costa e

Graciélia Castro da Costa,

Por me apoiarem em minhas escolhas.

"Há mais mistérios entre o céu e a terra do que pode sonhar a nossa vã filosofia".

(Shakespeare)

vi

AGRADECIMENTOS

À professora Drª Ana Paula Junqueira-Kipnis, por ser mais que orientadora. Ao me dar

crédido para me orientar no doutorado, investiu em mim suas expectativas, sendo

exigente em suas cobranças. Com ela aprendi fazer minhas escolhas, com objetividade

e foco. Em todo o tempo que estive trabalhando com ela, tive muitas conquitas, mas

também tivemos perdas, porém, todas as perdas foram compensadas com mais

conquistas. Quando falo que conquistamos, parece até que foi fácil, porém foram as

mais árduas tarefas para obtermos as vitórias. Porém, por mais difíceis que tenha sido

a caminhada, a cada vitória vibramos juntas, com muita alegria. Mas não acabou, pois

não foram apenas orientações, há muita amizade envolvida, ao ponto de apenas ela

perceber que eu estava com problemas. Apenas ela conseguiu me mostrar alegrias onde

apenas havia tristezas. Até em minha saúde ela me orientou, onde parecia não haver

saída. Hoje posso dizer que renascemos, que a caminhada foi árdua mas estamos

vencendo. Muito obrigada por ser tudo isso em minha vida. Meus sinceros

agradecimentos.

Ao professor Drº André Kipnis não existem palavras para agradecer o quanto foi

importante... não somente no desenvolvimento deste trabalho, mas sobretudo no meu

crescimento como ser humano. Com seu empenho, dedicação e paciência conduziu a

orientação deste trabalho de maneira sábia. Sabemos que passamos por muitas

dificuldades, muitas no desempenhar do trabalho em si, mas muitas nas relações entre

os colegas... porém com sua sabedoria conseguimos vencer ao ponto de chegarmos na

defesa deste trabalho o qual é muito importante para mim. Além disso, a colaboração

de seu laboratório foi essencial no desenrolar de todo o processo, uma vez que foi

cedido seu tempo, orientações e materiais ao laboratório. Não há dúvidas do quanto o

senhor é importante neste trabalho. No mais, tenho muito a agradecê-lo por fazer parte

deste trabalho.

À colega, companheira e amiga Sarah Veloso Nogueira por fazer parte deste momento,

tanto a elaboração dos trabalhos quanto em sua amizade que foi preciosa. Fui muito

vii

feliz em ter sua presença no laboratório e em nossas atividades. Passamos por

momento difíceis, porém sua amizade me deu forças para continuar. Muito obrigada.

Ao colega e amigo Ms. Danilo Pires de Resende por estar presente durante a

realização dos experimentos. Com sua paciência, bom ânimo e bom coração me

proporcionou forças para continuar e até a conclusão deste trabalho.

Ao colega e amigo Bruno de Paula Oliveira, muitas vezes fui motivada por seu ânimo e

determinação. Deus te deu o dom de iluminar o ambiente em que você está, além de

trabalhar muito bem. Foi muito bom trabalhar com você e conviver com você desde o

primerio momento em que esteve no laboratório. Você foi peça fundamental na

construção deste trabalho. Muito Obrigada.

Ao colega e amigo Fábio Muniz de Oliveira por participar diretamente das atividades

deste trabalho. Com seu entususiasmo, boa vontade e bom ânimo fez com que este

trabalho se tornasse prazeroso em ser realizado. Obrigada por sua amizade sincera e

palavras sábias nos momentos mais difíceis. Muito obrigada.

À colega, companheira e amiga Monalisa Martins Trentini. Podemos dizer que

começamos juntas. É umas das pessas mais inesquecíveis que há em minha vida. Por

ser uma pessoa muito esforçada e animada, trouxe ao laboratório um novo momento,

momento este que se prolonga até hoje. Passamos por muitos momentos juntas, e

compartilhamos muitas alegrias, tristezas e sobretudo conquistas. Levarei suas

lembranças por toda a vida. Muito obrigada por me ensinar, por me animar e por estar

por perto sempre.

Aos colegas Dr. Lorena Cristina dos Santos, Dr. Alxexander Algusto da Silveira e Ms.

Abadio de Oliveira Costa Júnior por terem realizado a construção e produção em larga

escala da vacina rBCG-CMX.

Ao colega e amigo Lázaro Moreira Marques Neto, muito obrigada por proporcionar

bons momentos de profundo conhecimento, visto que é uma pessoa genial e muito

agradável convivência. Por muitos momento, foi responsável por acalentar situaçãoes,

viii

pois é uma pessoa de muita paz de espírito. Muito obrigada por fazer parte da minha

vida.

André França Correa. Muito obrigada por me ensinar a trabalhar com o citômetro de

fluxo FACS Verse da UNB. Agradeço toda paciência e boa vontade. Muito Obrigada.

Ao colega Eduardo Martins de Sousa, muito obrigada por se lembrar de mim, mesmo

depois de passarmos momentos difíceis juntos. Que Deus possa te abençoar

grandemente e possa te fortalecer para que continue conquistando seus objetivos.

A colega e Amiga Adrielle Zagmignan por ter me ajudado na formatação final da tese.

Muito obrigada.

A colega e amiga Viviane Lopes que esteve por perto todo esse tempo, auxiliando o

laboratório, e com seu auxílio foi possível ter o suporte técnico para a realização dos

trabalhos contidos nesta tese. Não somente participação técnica, a Viviane já foi anjo

de Deus em minha vida, em momentos difíceis. Muito obrigada por fazer parte desta

conquista.

Aos colegas e amigos Rogério Coutinho das Neves, Lucilandia Maria Bezerra, Adrielly

Zagmignan, Tatiane Marlene Galvez Sanches, Stella Francy Vicente de Assunção,

Victor Oliveira Procopio, Clayson Moura Gome, Lucila Àvila, Michelle Cristina

Guerreiro dos Reis, e todos os que já passaram pelo laboratório, que de alguma

maneira tenham contribuído diretamente ou indiretamente para este conquista: Aléx,

André, Bruna, Danilo, Duanne, Fernando, Letícia, Marcos, Mayara, Patrícia, Juliana,

Camilla, Matheus, Vanessa, Aline, Lucas, Joyce, Beatriz, Rayanny, Camila, Larissa,

Thaiz. Agradeço ao companheirismo, a compreensão e a todos os momentos que

passamos juntos, todos contribuíram diretamente para meu crescimento pessoal e

profissional. Muito Obrigada.

A todos os professores do IPTSP especialmente do Setor de Imunologia. Muito

obrigada.

ix

Aos funcionários da secretaria geral e da secretaria do curso de pós-graduação:

Senhor Fernando, Valéria, Divina, José Clementino, Kariny. Agradeço toda

assistência.

Aos Amigos: Louvable Nunes Folha, Estevão Marcos Ferreira, Leandro, Zélia de

Oliveira Meira, Teodora Ataíde, Edmilson Barbosa, Donizete+, Adriana Cândido,

Leonardo, Poliana Candido, Maria A. Borges, Glêisson J. de Jesus, Jailma Bastos,

Patrícia S. de Souza Hélio S. de Souza, Edivânia, Edivân, Jean Carlos pela amizade e

bom ânimo. Muito obrigada.

Às agências de fomento CNPq, Sectec e FAPEG pelo financiamento do projeto.

Certamente seria inviável realizar este trabalho sem este apoio. Muito obrigada.

Ao CNPQ pela bolsa de Doutorado. Muito obrigada.

A todos que contribuíram direta ou indiretamente para realização deste trabalho.

Muito obrigada.

x

SUMÁRIO

1 INTRODUÇÃO.............................................................................................................. 1

1.1 TUBERCULOSE........................................................................................................ 1

1.2 Resposta Imune ao Mycobacterium Tuberculosis........................................................ 2

1.3 Modulação da Resposta Imune Inata por Antígenos de Mycobacterium tuberculosis

e produtos micobacterianos................................................................................................

1.3.1. Proteínas de M. tuberculosis reconhecidas por TLR-2.....................................

1. 1.3.2. Proteínas de M. tuberculosis reconhecidas por TLR-4.............................................

1.3.3. Proteínas de M. tuberculosis reconhecidas que interagem com TLR-2, TLR-4 e

e outros receptores..................................................................................................................

6

7

8

9

2 JUSTIFICATIVA.......................................................................................................... 11

3 OBJETIVOS.................................................................................................................. 12

3.1 OBJETIVO GERAL.................................................................................................. 12

3.2 OBJETIVOS ESPECÍFICOS................................................................................... 12

4 ARTIGOS....................................................................................................................... 13

Artigo 1. Recombinant BCG: Innovations on an old vaccine. Scope in BCG strains and

strategies to improve long lasting memory.........................................................................

14

Artigo 2. A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector

Cells with Higher Protective Efficacy against Tuberculosis.............................................

42

Manuscrito. Modulation of the immune response induced by the recombinant fusion

protein CMX involves IL-6 and TGF-β production and TLR-4

stimulation..........................................................................................................................

83

5 DISCUSSÃO.................................................................................................................. 115

6 CONCLUSÕES.............................................................................................................. 120

7 REFERÊNCIAS............................................................................................................. 121

8 ANEXOS....................................................................................................................... 132

xi

TABELAS, FIGURAS E ANEXOS

Table 1

Artigo 1

BCG sub strains genetic background used for recombinant BCG

vaccines development and ability to induce memory and protection

against tuberculosis.

20

Table 2 Description of strains and antigens used in the papers visited for this

review. References published and indexed in PubMed from 2008

April 2013

28

Figure 1

Artigo 2 Plasmid construction and CMX expression for three different

rBCG-CMX vacines

53

Figure 2 Stability of rBCG-CMX in vivo 55

Figure 3 Levels of phagocytosis by peritoneal macrophages of BCG and

rBCG-CMX after infection (MOI = 10)

57

Figure 4 Immunogenicity of rBCG-CMX in BALB/c mice 59

Figure 5 Levels of CD4+IFN-y+ T cells induced by ex vivo stimulation

with recombinant Ag85, MPT51, and HspX

61

Figure 6 Levels of CD4+IL-17+ T cells induced by ex vivo stimulation with

recombinant Ag85, MPT51, and HspX

62

Figure 7 Levels of polyfunctional CD4+ T cells induced by BCG and rBCG-

CMX vacines

64

Figure 8 Bacterial load in the lungs of BALB/c mice 45 days after

Mycobacterium tuberculosis challenge

66

Figura 9 Representative lung pathology of BALB/c mice after challenge 68

Figure 1

Manuscrito

The ex vivo and in vitro induction of cytokines involved in Th17

differentiation

93

Figure 2 In vivo induction of macrophage profile 94

Figure 3 The rBCG-CMX vaccine induces more macrophage apoptosis and

better vaccine processing than does BCG-Moreau

96

Figura 4 Production of cytokines by rAg85c, rMPT51, rHspX and rCMX

proteins in RAW cels and BMMs

98

Figura 5 Production of cytokines by rAg85c, rMPT51, rHspX and rCMX

proteins in BMMs

99

Figure 6 Production of cytokine IL-6 by rAg85c, rMPT51, rHspX and rCMX

proteins in alveolar and peritoneal macrophages

100

Figure 7 TLR receptors related to the recognition of rAg85c, rMPT51, rHspX

and rCMX in BMM from TLR-2 KO and TLR-4 KO mice

101

Suplementar

1

rBCG-CMX vaccine expresses the CMX protein 24 h after infection

in macrophages

102

xii

SÍMBOLOS, SIGLAS E ABREVIATURAS

AERAS-402 Vacina de Adenovirus 35 (rAd35) expressando Ag85A, Ag85B e TB10.4

Ag Antígeno

AIDS Síndrome da Imunodeficiência Adquirida

AP-1 Activator Protein 1

APC Aloficocianina (do inglês Allophycocyanin)

APCs Células Apresentadoras de Antígeno

BAAR Bacilo Álcool Ácido Resistente

BCG Bacillus Calmette–Guérin

BM Medula óssea (do inglês Bone Marrow)

C3 Proteína do Complemento

CCR7 C-C chemokine receptor type 7

CD1 Claster de Diferenciação (do inglês Cluster of Differentiation)

CEUA Comitê de Ética para Uso Animal

CFP-10 Culture Filtrate Protein 10

CFU Unidade Formadora de Colônia (do inglês Colony-Forming Unit)

CO2 Dióxido de Carbono

ConA Concavalina A

COX-2 Cicloxigenase 2

CpG DNA DNA contendo dinucleotídeos CpG não-metilados

cPLA2 Calcium-dependent Phospholipase A2

CR Receptor do Complemento

cRPMI Meio RPMI contendo antibiótico, SBF, Piruvato e Glutamina

CTLs Células T Citolíticas

DAB 3,3'-diaminobenzidina

DATIN Dormancy Associated Translation Inhibitor

DCs Células Dendríticas

DMEM Meio Eagle Modificado por Dulbecco (do ingles Dulbecco's Modified

Eagle's Medium)

DNA Ácido Desoxirribonucleico

E. coli Escherichia coli

ELISA Ensaio Imunoenzimático

ELISA Ensaio Imunoezimático

ESAT-6 Early Secreted Antigen Targed 6

ESX-1 type VII secretion system

F4/80 Marcador de macrófago de camundongo

FACS Fluorescence-activated Cell Sorting

fbp Proteína ligadora de fibronectina (do ingles Fibronectin-binding

protein)

Fc Fragmento Cristalizável (Região do anticorpo)

FITC Isotiocianato de Fluorceína (do inglês Fluorescein Isothiocyanate)

GM-CSF Fator Estimulador de Colônia de Monócitos e Granulócitos

H2O2 Peróxido de Hidrogênio

H37Rv Cepa do Mycobacterium tuberculosis

HE Hematoxilina e Eosina

HIV Vírus da Imunodeficiência Humana (do inglês Human Immunodeficiency

Virus)

xiii

HspX Proteína de Shoque Térmico X (do inglês Heat shock protein X)

IFN-γ Interferon –γ

IgG Imunoglobulina

IL Interleucina

iNOS Óxido Nítrico Sintase Induzível

IPTG Isopropil β-D-1-tiogalactopiranosídio

kan Canamicina

kb Kilobases

LB meio de cultura Lúria Bertanii

LPS Lipopolissacarídeo

LXA4 Lipoxina A4

M. bovis Mycobacterium bovis

mc2-CMX Mycobacterium smegmatis expressando a proteína de fusão CMX

MCP-1 Proteína 1 Quimioatraente de Monócitos (do ingles Monocyte

Chemoattractant Protein-1)

MHC Complexo de Histocompatibilidade Principal (do ingles Major

Histocompability Complex)

miR microRNA

MOI Multiplicidade de Infecção

Mpt-64 uma proteína de 24-kDa do Mtb

MR Receptor de Manose

Mtb Mycobacterium tuberculosis

MTBVAC Vacina de Mtb atenuado

Myd88 Myeloid Differentiation Primary Response 88

NF-κB Nuclear Factor κB

NO Óxido Nítrico

nRD18 non-Region of Differentiation 18

OADC Ácido oléico, dextrose e catalase

ORF Frase de leitura aberta ( do inglês Reading Frames)

PAMPs Padrões Moleculares Associados a Patógeno

PBMC Células Mononucleares do Sangue Periférico ( do ingles Peripheral

Blood Mononuclear Cell)

PBS Tampão Fosfato Salina (do ingles Phosphate Buffered Saline)

PC Positive Control

PCR Reação em Cadeia da Polimerase

PE Ficoeritrina (do ingles Phycoerythrin)

PE35 Membro da família PE M. tuberculosis

PE-PGRS Polymorphic GC-rich Sequences

PERCP Peridina Clorofila ( do inglês Peridinin Chlorophyll)

PGE2 Prostaglandina E2

PI Iodeto de Propídeo

PPD Derivado Proteico Purificado (do ingles Purified Protein Derivative

Test)

PPE Ácido Prolina-prolina-glutâmico

PPE68 Membro da família PPE M. tuberculosis

rBCG AFRO-1 BCG expressing Ag85A, Ag85B and TB10.4

rBCG BCG recombinante

rBCG:30 r30-Ag85B

rBCG-AE rBCG expressing the fusion protein Ag85A-ESAT-6

rBCG-AMM BCG expressing Ag85B-MPT64190-198-Mtb8.4

xiv

rBCG-CMX BCG recombinante expressando a proteína de fusão CMX

rBCGΔureC::hly BCG recombinante delta uréase C expressando lysteriolisina

rCMX Antígeno constituído total ou parcialmente pelo Ag85C, MPT51 e HspX

RD Regions of Difference

RNA Ácido Ribonucleico

RPMI Meio Roswell Park Memorial Institute

SBCAL/COBEA Sociedade Brasileira de Ciência em Animais de Laboratório

SBF Soro Bovino Fetal

SDS–PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SEAP do ingles Secreted Embryonic Alkaline Phosphatase

SigI Fator de Sigma Alternativa de RNA Polimerase ( do ingles Alternative

RNA Polymerase Sigma Factor)

SOC do ingles Super Optimal broth with Catabolite repression

SSC do ingles Side Scatter

TB Tuberculose

TCM Célula T de Memória Central ( do ingles T Central Memory Cell)

TEM Célula T de Memória Efetora ( do ingles T Effector Memory Cell)

TGF-β Fator de crescimento tumoral do tipo β (do ingles Tumor Growth Factor-

β)

Th1 T helper 1

Th17 T helper 17

TLR Receptor do tipo Toll ( do ingles Toll Like Receptors)

TNF-α Fator de Necrose Tumoral α

TRAF Fator associado ao receptor de TNF (do ingles TNF receptor-associated

factor)

TRIF TIR-domain-containing adapter-inducing interferon-β

TST Teste de Sensibilidade a Tuberculina ( do inglês Tuberculin Skin Test)

UFC Unidade Formadora de Colônia

ureC Urease C

WHO Organização Mundial de Saúde - OMS (do ingles World Health

Organization)

xv

RESUMO

A Tuberculose (Tb) é uma doença infecto contagiosa, causada pelo Mycobacterium

tuberculosis (Mtb). Apesar de ser uma doença antiga, a Tb continua sendo um dos principais

problemas de saúde pública. A Organização Mundial de Saúde acredita que cerca de um

terço da população mundial está infectado com Mtb, gerando milhões de mortes por ano.

Uma das medidas que podem melhorar a prevenção e bloquear a transmissão do Mtb é o

desenvolvimento de novas vacinas que previnam o estabelecimento e a progressão da TB em

humanos. Embora exista a vacina BCG que é eficiente contra formas graves de TB na

infância, existe a necessidade do desenvolvimento de novas vacinas para controlar a

disseminação da TB, que sejam mais eficientes e seguras que a BCG. Com este intuito, o

objetivo deste trabalho é avaliar a proteção e a modulação da resposta imune induzida por

BCG recombinante expressando espítopos imunodominantes Ag85C, MPT-51 e HspX do

Mycobacterium tuberculosis induzida em modelo murino. Nossos resultados demonstram

que a inserção da proteína CMX na vacina BCG recombinante (rBCG-CMX) foi um fator

determinante para indução de resposta Th1 e Th17, além de células polifuncionais que

possivelmente foram responsáveis pela redução das lesões inflamatórias no pulmão de

camundongos BALB/c, reduzindo significantemente a carga bacilar em comparação com

imunização com BCG Moreau. Além disso mostramos neste trabalho que a proteína rCMX

é capaz de modular a vacina BCG e ativar a imunidade inata para a indução de uma melhor

resposta protetora. Nossos resultados demonstram que a vacina rBCG-CMX induz ativação

de macrófagos pulmonares por meio da expressão de moléculas de ativação CD86 e CD206.

O aumento da expressão dessas moléculas é acompanhada por produção de TGF-β e IL-1α,

sendo prováveis responsáveis pela menor indução de necrose e maior indução de apoptose

pela vacina rBCG-CMX. Este fenômeno pode estar proporcionando a esta vacina maior

capacidade de sobrevivência celular, colaborando para um melhor processamento e

apresentação por MHC-II. Devido a proteína rCMX ser capaz de induzir produção de IL-1α,

IL-6 e TGF-β por uma via que parece haver a participação de TLR-4. In vivo demonstramos

que a vacina rBCG-CMX depende de TLR-2 e TLR-4 para induzir respostas Th1 e Th17,

após imunização de camundongos com esta vacina. Neste trabalho hipotetizamos que a

proteína CMX pode modular a resposta imune inata e adaptativa, por uma via em que há a

participação do TLR-4. Esta pode ser a via pela qual a CMX, quando expressa por BCG

favorece uma boa resposta protetora em animais desafiados com Mtb.

xvi

ABSTRACT

In the first chapter of this thesis we demonstrate, in a review article, some of the

successful strategies employed in the construction of Bacillus Calmette-Guérin (BCG)

vaccines, among others being: overexpression of promising Mycobacterium tuberculosis

(Mtb) immunodominant antigens already expressed by BCG introduction of Mtb

immunodominant antigens not expressed by BCG, such as antigens in the regions of

difference (RD) 1 thru 16; combination of overexpression and introduction of novel antigens

to BCG; BCG modification to skew immune response toward TCD8+, as for example

recombinant BCG (rBCG) expressing cytokines. In the second chapter, we demonstrate that

the recombinant fusion protein CMX is capable of aggregating important immunogenic

properties to vaccine vectors, by inducing an effective response for the control of Mtb

infection in the mouse tuberculosis infection model. It is hypothesized that the introduction

of the rCMX protein in the BCG vaccine could add immunological properties that are absent

in BCG, thus leading to the induction of important cell populations for the control of Mtb

infection. Our results demonstrate that the introduction of the rCMX in the BCG vaccine,

resulting the recombinant BCG vaccine (rBCG-CMX) was an important factor for the

observed Th1 and Th17 responses, as well as polyfunctional cells, that could be responsible

for the reduced inflammatory lesions seen in the lungs of Mtb infected BALB/c mice,

significantly reducing the bacillary load in comparison to in comparison to mice immunized

with BCG Moreau vaccine. Lastly, in the third chapter of this thesis we propose that rCMX

protein could be responsible for modulating the BCG vaccine to activate a more adequate

and protective innate immunity. Our results show that the rBCG-CMX vaccine induces the

activation of alveolar macrophages by means of expression of activation-associated

molecules CD86 and CD206. The increase in the expression of those molecules are

accompanied by the production of TGF-β e IL-1α which in turn could be responsible for the

decreased necrosis and higher apoptosis induction promoted by rBCG-CMX vaccination.

This phenomenon could be providing a higher cellular survival rate of the recombinant

vaccine, leading to a better processing and presentation by MHC-II. As rCMX was shown to

induce the production of IL-1α, IL-6 e TGF-β by a pathway that seems to involve the

participation of TLR-4, we hypothesize that this recombinant protein could be modulating

the BCG vaccine to induce a more appropriate and protectiveresponse for Mtb infection.

xvii

PRÓLOGO

Meu nome é Adeliane Castro da Costa, sou graduada em Biomedicina pela Pontifícia

Universidade Católica de Goiás (PUC-2009/2). Durante a graduação, em meados de

2008, realizei estágio no Laboratório de Imunopatologia das Doenças Infecciosas, sob a

supervisão da professora Dr. Ana Paula Junqueira Kipnis, com a qual fui iniciada nas

atividades de pesquisa científica. No ensejo, tive a oportunidade de realizar um curso de

Imunologia de verão na USP, sendo de grande importância, por me fazer entender como

funciona um ambiente em que se aspira ciência. Ao concluir a graduação (2009/2)

retornei ao laboratório e tive a oportunidade de ocupar o cargo de Técnico de Nível

Superior do CNPQ. Durante este tempo, aprendi a trabalhar com algumas técnicas de

laboratório e fui responsável pela realização de compras e auxílio em prestação de

contas de projeto de pesquisa. Esse processo foi muito importante, uma vez que me

promoveu a base de como administrar um projeto de pesquisa e seus recursos. Ao final

deste período tive a oportunidade de realizar o processo seletivo para o Mestrado

(2010).

Durante o mestrado realizei a padronização de ELISA para diagnóstico de

Tuberculose, utilizando proteínas de Mtb (rGroES e rCMX). Foi uma importante etapa,

uma vez que participei da finalização da caracterização de proteínas de Mtb para o

diagnóstico da TB. Como resultado deste trabalho com a proteína rGroES, após um ano

de mestrado, publicamos meu primeiro artigo (Revista de Patologia Tropical e Saúde

Pública, 2011). Ao mesmo tempo, participei do início de outra pesquisa no laboratório,

que veio a ser a utilização da proteína de fusão rCMX no diagnóstico e em modelos

vacinais. Deste trabalho com a proteína rCMX publicamos meu segundo artigo (PLOS

one, 2012). Este momento representou o início de minhas atividades em pesquisa, sendo

muito importante em minha carreira, uma vez que tive a oportunidade de participar da

geração de patente da proteína CMX. Além disso, durante o mestrado tive a

oportunidade de participar de outros projetos de pesquisa. Dentre esses posso citar uma

participação em projeto com TB humana, do qual obtive minha terceira publicação

(Immunology Letters, 2014). Em outra linha de pesquisa, trabalhamos com DPOC

(Doença Pulmonar Obstrutiva Crônica), numa colaboração com o Pneumologista

professor Dr. Marcelo Fouad Rabahi, com o qual estive vinculada a um projeto de

xviii

mestrado e um projeto de doutorado. Por meio do trabalho de mestrado, obtivemos

minha quarta publicação (International Journal of COPD, 2014).

Após finalizarmos o meu mestrado, tive a oportunidade de trabalhar durante um ano

na Clínica do Aparelho Respiratório e Medicina do Sono (CLARE), cujo responsável é

o professor Dr. Marcelo Fouad Rabahi. Nesta clínica atuei no diagnóstico de distúrbios

respiratórios e relacionados ao sono, realizando exames de Espirometria e

Polissonografia. Na oportunidade, fui inserida em outro projeto, o qual havia

investimentos de Indústrias Farmaceuticas, nos proporcionando mais uma publicação,

sendo meu quinto artigo (International Journal of COPD, 2015).

Ao final de um ano (2013), retornei ao laboratório da professora Dr. Ana Paula

Junqueira Kipnis, pela qual fui convidada a fazer o Doutorado. Na oportunidade, o

laboratório estava finalizando a construção de uma vacina BCG recombinante

expressando a proteína CMX (rBCG-CMX). A rCMX é uma proteína de fusão

construída por nosso grupo, composta por epítopos imunodominantes dos antígenos

Ag85c, MPT51 e HspX inteiro de Mtb. Esta vacina ativou a resposta imune em

camundongos e de ser antigênica em indivíduos com TB ativa (de Sousa et al., 2012).

Com o intuito de utilizar a rCMX em um modelo vivo de crescimento rápido, a proteína

foi expressa por vetor Mycobacterium smegmatis (mc2-CMX), por meio da qual mostrou

ser boa indutora de resposta imune do tipo Th1 e Th17 em pulmão de camundongos

imunizados, sendo também boa indutora de anticorpos IgG1 e IgG2a (Junqueira-Kipnis

et al, 2013). Diante do contexto de que a rCMX favorece uma resposta eficaz contra a

TB, a proteína foi, então, expressa no vetor vivo BCG (rBCG-CMX). Desta maneira,

realizei no meu doutorado a avaliação da resposta imune e proteção induzida por esta

vacina. Durante o primerio ano, realizamos uma revisão de literatura, do qual gerou meu

sexto artigo (Frontiers in Immunology, 2014) e também capítulo de livro da revista

Frontiers in Immunology. No ensejo, testamos a eficácia da vacina rBCG-CMX em

modelo murino, gerando minha sétima publicação (PLOS one, 2014). Seguindo este

período, avaliamos a capacidade da proteína CMX em modular a resposta imune inata,

por meio do qual submetemos um outro artigo, o qual será abordado nesta tese.

Após 6 anos de trabalho com pesquisa (2010-2016) tive a oportunidade estar em 7

artigos publicados. Além da participação em uma patente e capítulo de livro, podemos

contar como produto mais de 35 resumos apresentados em congressos nacionais e

internacionais. Concomitantemente, realizei minha primeira orientação de Trabalho de

xix

Conclusão de Curso, cujo aluno defendeu recentemente (2016). Atualmente (2015-

2017), ocupo o cargo de Professor Substituto do setor de Imunologia do Instituto de

Patologia Tropical e Saúde Pública, no qual ministro aulas para os cursos de

Biomedicina, Farmácia e Medicina.

1

1. INTRODUÇÃO

1.1. TUBERCULOSE

O agente causador da Tuberculose (TB) é o bacilo de Koch Mycobacterium

tuberculosis (Mtb), o qual foi descoberto em 1882 por Robert Koch (Prémio Nobel em

1905), é responsável por mais mortes do que qualquer outro patógeno (OTTENHOFF, 2009;

KAUFMANN, HUSSEY e LAMBERT, 2010). O Mtb pertencente à ordem

Actinomycetales, subordem Corynebacteriaceae, família das Mycobacteriaceae e gênero

Mycobacterium, e a um complexo denominado complexo Mycobacterium tuberculosis, no

qual faze parte: M. africanus, M. bovis, M. canettii, M. microti, M. pinnipedii e M. caprae.

Entretanto, somente M. africanus, M. bovis e M. tuberculosis (Mtb) causam TB humana

(ROBERTS et al., 1991). A TB pode afetar vários órgãos, mas acomete principalmente os

pulmões (TB Pulmonar), por meio do qual os pacientes acometidos apresentam tosse

produtiva com mais de 15 dias, febre vespertina baixa, suor noturno, dor no tórax e perda de

peso (WHO, 2015).

A TB é uma doença infecto contagiosa que causa, no mundo, em torno de 9 milhões

de novos casos e 1,5 milhões de morte no ano. O Brasil faz parte do grupo que responde por

quase 50% dos casos de TB no mundo. Dentro deste grupo encontra-se Brasil, Federação

Rússia, India, China e África do Sul (BRICS). O Brasil apresenta uma população em torno

de 200,4 milhões de habitantes, sendo notificados 83,310 mil novos casos de TB (WHO,

2015). Dentre esses, 41,885 mil novos casos foram de TB pulmonar bacteriologicamente

diagnosticada, 18,303 mil casos de TB pulmonar diagnosticados clinicamente e 10,148 mil

casos de TB extrapulmonar, dentre outros (WHO, 2015). No Brasil, Goiás é o segundo

estado com menor número de casos, no entanto, esses não mostram redução, sendo

notificados em torno de 867 casos, sendo 546 de TB pulmonar (TB primária). Dados do

Sinan revelam que Goiânia apresenta em torno de 16 casos para cada 100.000 habitantes

(BRASIL, 2014). Uma das principais preocupações da Organização Mundial de Saúde

(OMS) em relação a TB são os casos em que a doença está associada ao Vírus da

Imunodeficiência Humana (HIV). Segundo a WHO, em 2014 a TB é responsável por 1,5

milhões de mortes, sendo que desses 0.4 milhões eram HIV positivas (WHO, 2015).

O tratamento para TB é baseado na administração de Rifampicina, Pirazinamida,

Isoniazida e Etambutol por 2 meses, seguido pela administração de Rifampicina e

Isoniazida por 4 meses. O paciente com TB que apresenta resistência a, no mínimo,

rifampicina e isoniazida é definido como Multi-Droga Resistência (MDR) enquanto que

2

aquele resistente a, no mínimo, fluoroquinolona e a uma segunda linha injetável é definido

como Extensivamente Droga Resistente (XDR). Em geral 3,5% dos novos casos de TB

pulmonar notificados no mundo e 20,5 % dos casos previamente tratados são pacientes TB

Multi Droga Resistentes (MDR). Dos casos de MDR e XDR representam em torno de 9%

dos casos de TB notificados no mundo. Esses dados refletem em torno de 480 mil novos

casos de pacientes com TB MDR, dos quais desencadearam 210 mil mortes em 2013

(WHO, 2015).

Atualmente, a vacina utilizada para prevenção da TB é a BCG (Bacilo Calmette-

Guérin), uma cepa atenuada derivada do Mycobacterium bovis, a qual foi atenuada após

mais de 13 anos de cultura in vitro, sendo utilizada desde 1921 (CALMETTE et al., 1929).

É uma das vacinas mais largamente administradas mundialmente e a única vacina disponível

que previne infecções contra M. tuberculosis (RAPPUOLI e ADEREM, 2011), sendo

produzida em vários laboratórios no mundo. Apesar de ser a única vacina aprovada para uso

humano, e conferir proteção em crianças contra meningite tuberculosa e TB miliar, seu

efeito protetor continua questionável, uma vez que não protege adultos contra TB pulmonar

(WHO, 2015).

Neste ultimo ano (2014), o Brasil investiu 79 milhões de dólares no controle da TB,

sendo que 87% deste investimento foi a partir de financeamento interno e 2% de

financiamento internacional. No entanto, há um fraco financiamento no desenvolvimento de

novas vacinas, a qual tem sido desenvolvida com base em recursos próprios do país (WHO,

2015). Diante deste cenário, o desenvolvimento de novas vacinas para a prevenção da TB é

de extrema urgência, uma vez que a vacina utilizada atualmente apresenta uma variação na

proteção de indivíduos na fase adulta (WHO, 2015).

1.2. Resposta Imune ao Mycobacterium tuberculosis

Após a entrada de Mtb nos pulmões, os macrófagos alveolares são as primeiras

células a interagirem com o bacilo. Tanto em humanos como em camundongos os

macrófagos alveolares reconhecem os PAMPs (Padrões Moleculares associados a

Patógenos) do Mtb por meio de receptores de reconhecimento do padrão (PRR’s) presentes

nessas células, permitindo a ativação e fagocitose do bacilo. Entre os PRRs podem-se citar

os receptores para Fc de imunoglobulinas, complemento, manose, proteína surfactante,

CD14, e CD43 (STURGILL-KOSZYCKI, SCHLESINGER, CHAKRABORTY et al.,

1994; PETERSON, GEKKER, HU et al., 1995; ZIMMERLI, EDWARDS e ERNST, 1996;

3

RANDHAWA, ZILTENER, MERZABAN et al., 2005). Fragmentos da proteína C3 do

complemento são capazes de opsonizar antígenos do Mtb, permitindo uma interação com os

receptores CR1, CR3 e CR4 presentes nos macrófagos (MAGLIONE e CHAN, 2009). É

importante observar que, independente do receptor utilizado na interação com o macrófago,

o colesterol, presente na membrana celular, favorece a ancoragem e assim a internalização

da bactéria (GATFIELD e PIETERS, 2000). Ao interagir com os bacilos de Mtb, por meio

dos receptores Fc de imunoglobulinas, os macrófagos aumentam a produção de

intermediários reativos de oxigênio, permitindo que ocorra a fusão dos fagossomos com os

lisossomos (ARMSTRONG e HART, 1975). Em contrapartida, quando as bactérias

interagem com o CR3 há impedimento da explosão respiratória, em um processo que

impede a fusão dos fagossomos com os lisossomos (STURGILL-KOSZYCKI,

SCHLESINGER, CHAKRABORTY et al., 1994).

Uma outra maneira que Mtb utiliza para interagir com os macrófagos é por meio da

Trealose de mycolato (TDM) presente em sua parede celular. TDM ancora em MARCO

(Scanveger receptor) SR e CD14, presente nos macrófagos promovendo a sinalização nos

receptores TLR2. Por meio destes receptores, TDM modula a resposta dos macrófagos

induzindo a ativação de NF-B e promovendo a produção de IL-6, IL-1 e TGF-. A

afinidade de TDM com MARCO é muito grande, o que induz alto recrutamento de MARCO

na membrana do macrófago, favorecendo a ativação de TLRs e a indução de fagocitose

(BOWDISH, SAKAMOTO, KIM et al., 2009). A capacidade de inibir a fusão do fagossoma

com o lisossoma, a modulação do padrão de morte de células infectadas, além da

propriedade de se reproduzir dentro do compartimento endossomal, promovendo o retardo

da acidificação fagossomal, dá ao bacilo a capacidade de sobrevivência dentro dessas células

(FLYNN e CHAN, 2001).

Um importante mecanismo que Mtb utiliza para sobreviver dentro dos macrófagos é

sua capacidade de sobreviver dentro de vacúolos (RUSSELL, MWANDUMBA e

RHOADES, 2002). Dentro dos macrófagos esta bactéria promove um processo de

dislipidemia tanto dos macrófagos, quanto das células presentes naquele microambiente da

infecção. Após a análise do transcriptoma do granuloma humano, foi possível observar mais

de 30 genes ativos relacionados com o metabolismo lipídico (KIM, WAINWRIGHT,

LOCKETZ et al., 2010). Alguns desses genes promovem o sequestro de lipídio do

hospedeiro para dentro do macrófago. O acúmulo de lipídio dentro do macrófago gera uma

alteração celular conhecida como foamy macrophage. Os lipídeos presentes nos macrófagos

4

servem de fonte de alimento para Mtb, permitindo sua sobrevivência nessas células

(PEYRON, VAUBOURGEIX, POQUET et al., 2008). Em macrófagos provenientes de

granuloma humano foi demonstrado que a presença de colesterol que está envolvendo Mtb,

bem como a presença de proteínas não dobradas (UPR) induz stress do retículo

endoplasmático (RE) e a ativação de CHOP (fator de transcrição ativado em resposta a

proteínas não dobradas). Consequentemente, a ativação de CHOP promove a indução de

apoptose celular (SEIMON, KIM, BLUMENTHAL et al., 2010).

Após a entrada no bacilo no macrófago, este continua interagindo com a célula.

Dentro do fagossoma, Mtb libera vesículas, conhecidas como vesículas de membrana

bacteriana (BMV). Estas vesículas são ricas em lipoproteínas, como lipomananas,

lipoarabnomananas, DNAK dentre outras, capazes de induzir resposta pro-inflamatória ao

interagir com TLR-2 (PRADOS-ROSALES, BAENA, MARTINEZ et al., 2011). 2011).

Estas vesículas são liberadas para o meio extracelular e desempenham atividades pró-

inflamatórias ao interagir com TLR-2 e CD14, induzindo a produção de IL-8 e TNF-α

(ATHMAN, WANG, MCDONALD et al., 2015).

No decorrer da infecção devido a liberação de citocinas pró-inflamatórias, outros

monócitos, assim como células dendríticas são recrutados da corrente sanguínea, sendo

responsáveis pela manutenção da infecção no hospedeiro (DANNENBERG, 1991;

STURGILL-KOSZYCKI, SCHLESINGER, CHAKRABORTY et al., 1994; PEDROZA-

GONZALEZ, GARCIA-ROMO, AGUILAR-LEON et al., 2004). Os monócitos recrutados

para os pulmões, aumentam a expressão de CD11c e tornam-se CD11b+/mid/CD11c+/mid, os

quais se diferenciam em macrófagos alveolares (CD11b-/mid CD11c+/high) e células

dendríticas (CD11b+/high/CD11c+/high) (GONZALEZ-JUARRERO, HATTLE, IZZO et al.,

2005). Após a ativação, as células dendríticas sofrem um processo de maturação que é

acompanhada por um aumento da síntese de MHC de classe I, pela expressão de moléculas

co-estimuladoras, como CD80 (B7.1) e CD86 e CD40, sendo observados nos primeiros dias

de infecção ao Mtb (GONZALEZ-JUARRERO, HATTLE, IZZO et al., 2005).

No modelo de infecção por Mtb no Zebrafish postulou-se que durante o processo de

infecção com os bacilos, ocorre a formação de granulomas epitelióides, antes de se

estabelecer a imunidade adaptativa (DAVIS e RAMAKRISHNAN, 2009). Também

chamada de infecção primária ou primo-infecção, esta fase é caracterizada por apresentar

lesões exsudativas com reação inflamatória aguda, contendo leucócitos circundando os

bacilos. Este tipo de exsudato é absorvido em 90% dos casos, com cicatrização. Com o

estabelecimento da infecção dos macrófagos estes recrutam outras células do sistema imune

5

e formam uma estrutura organizada chamada de granuloma (DANNENBERG, 1991).

Trabalhos utilizando camundongos isogênicos demonstram que a expansão das

micobactérias nos granulomas se dá por ciclos de morte de macrófagos infectados e a

fagocitose por múltiplos macrófagos que são constantemente recrutados (DAVIS e

RAMAKRISHNAN, 2009).

Nos primeiros estágios da TB, a necrose é o evento celular responsável pela morte

dos macrófagos (DANNENBERG, 1989). Entretanto, essas células também podem morrer

por apoptose, sendo causado pela indução de TNF-α, stress oxidativo, ou pela presença de

Mtb (WYLLIE, KERR e CURRIE, 1980). O fenômeno da apoptose se caracteriza por

condensação citoplasmática e nuclear com formação de fragmentos celulares ligados à

membrana. Esses fragmentos são chamados de corpos apoptóticos sendo fagocitados por

outras células e degradados dentro de fagossomas (FINK e COOKSON, 2005). Os corpos

apoptóticos formados carreiam antígenos micobacterianos que podem ser absorvidos e

apresentados de maneira cruzada por células dendríticas para as células TCD8+

(SCHAIBLE, WINAU, SIELING et al., 2003). Durante o processo de maturação das células

dendríticas, os antígenos de Mtb são apresentados aos linfócitos T auxiliares (CD4+), T

citotóxicas (CD8+), células Th17 e linfócitos B nos nódulos linfáticos.

A geração das populações celulares Th1 e Th17 pode ocorrer quando bacilos de Mtb

induzem os fagócitos a produzirem IL-12 ou IL-23, as quais contribuem para a diferenciação

das duas populações celulares, respectivamente, de acordo com a persistência de Mtb

(VELDHOEN, HOCKING, ATKINS et al., 2006; GEROSA, BALDANI-GUERRA,

LYAKH et al., 2008; GORIELY, NEURATH e GOLDMAN, 2008). Porém, após infecção

por Mtb, os linfócitos Tγδ são as primeiras células a induzirem produção da citocina IL-17

(LOCKHART, GREEN e FLYNN, 2006). Inicialmente a IL-17 promove o aumento de

recrutamento de neutrófilos e formação do granuloma (GRODE, SEILER, BAUMANN et

al., 2005). Com a persistência do estímulo por Mtb, a elevada produção de IL-17 por células

Th17, promove aumento nos níveis de infiltrado neutrofílico no pulmão, induzindo dano

tecidual e contribuindo para a imunopatologia da TB (CRUZ, FRAGA, FOUNTAIN et al.,

2010). Enquanto isso, os linfócitos Th1 atuam na produção e secreção de IFN-γ que

aumentam atividade microbicida dos fagócitos e inibem o crescimento de Mtb (NORTH e

JUNG, 2004; TORRADO e COOPER, 2010).

Os linfócitos B são um dos principais componentes do sistema imune, sendo

responsáveis pela imunidade humoral. Em se tratando de proteção para TB, os anticorpos

poderiam aumentar a imunidade por meio da neutralização de toxinas, opsonização, ativação

6

do complemento, promoção da liberação de citocinas, citotoxicidade dependente de

anticorpos, e apresentação de antígenos reforçada (IGIETSEME, EKO, HE et al., 2004;

RELJIC e IVANYI, 2006). Por meio da opsonização, os anticorpos podem aumentar a

internalização dos bacilos do Mtb pelos neutrófilos, monócitos ou macrófagos, por meio dos

receptores Fcγ presentes nos fagócitos. Esta internalização permite o processamento e

apresentação de antígenos pelos macrófagos e células dendríticas, desencadeando atividade

microbicida dessas células e permitindo assim maior estimulação das células TCD4+ e

TCD8+ (SCHUURHUIS, VAN MONTFOORT, IOAN-FACSINAY et al., 2006;

GEISSMANN, MANZ, JUNG et al., 2010). As vacinas para TB podem focar estratégias

que atinjam a imunidade das mucosas, agindo nos linfócitos B, permitindo melhorar a

proteção durante a infecção pelo M. tuberculosis (OTTENHOFF, 2012).

No modelo de infecção por Mtb em Zebrafish, com o estabelecimento da resposta

imune adaptativa, a formação do granuloma coincide com a expansão bacteriana, sugerindo

que a aceleração do estabelecimento da resposta imune adaptativa é impulsionada pelo

crescimento bacteriano (VOLKMAN, CLAY, BEERY et al., 2004; DAVIS e

RAMAKRISHNAN, 2009). É então estabelecida a tuberculose ativa com formação de lesão

tecidual caracterizada por uma inflamação granulomatosa de três zonas: uma com células

gigantes multinucleadas contendo os bacilos, uma camada média de células epitelióides e

uma camada periférica de fibroblastos, células mononucleares e linfócitos T e B dispersos.

A primeira zona apresenta necrose caseosa central, sendo denominados tubérculos.

Posteriormente ocorre a cicatrização por tecido fibroso ou calcificação (COSMA,

SHERMAN e RAMAKRISHNAN, 2003). Os granulomas parecem ser benéficos para o

indivíduo por conter e restringir a micobactéria (ULRICHS e KAUFMANN, 2006). No

entanto, resultados sugerem que a formação do granuloma é uma ferramenta para a

expansão da infecção pelo Mtb (DAVIS e RAMAKRISHNAN, 2009).

1.3. Modulação da Resposta Imune Inata por Antígenos de Mycobacterium tuberculosis

e produtos micobacterianos

Sabe-se que Mtb pode ser reconhecido por receptores em macrófagos como CR3,

TLR-2 e TLR-4, dentre outros (KIM, SOHN, KIM et al., 2012; TIWARI, SOORY e

RAGHUNAND, 2014). Durante muito tempo acreditou-se que proteínas isoladamente não

poderiam ser reconhecidas por TLRs em macrófagos ou células dendríticas. No entanto,

recentemente vem sendo demonstrado que algumas proteínas de Mtb são reconhecidas por

7

TLRs por meio dos quais modulam a resposta de macrófagos (VOSKUIL, VISCONTI e

SCHOOLNIK, 2004; CHATTERJEE, DWIVEDI, SINGH et al., 2011). Algumas dessas

proteínas já foram descritas na literatura, dentre elas HSP60, DNAK, PstS2, DATIN, ESAT-

6, PE35, PPE68 e Rv0652.

1.3.1. Proteínas de M. tuberculosis reconhecidas por TLR-2

Algumas das proteínas de Mtb que interagem com o TLR-2 parecem estar

relacionadas com regiões de virulência desta micobactéria. Dentre estas proteínas, algumas

foram destacadas neste contexto da revisão, sendo essas DATIN, ESAT-6, PE/PPE e

HSP60.

DATIN, também conhecida como Rv0079, é codificada pela ORF Rv0079, e

regulada pelo gene DosR de Mtb (VOSKUIL, VISCONTI e SCHOOLNIK, 2004). Seus

mecanismos de atuação nos macrófagos se dá por meio de sua interação com TLR-2 nessas

células, e indução de processos inibitórios associados a dormência. Estes fatores favorecem

uma atividade pró-inflamatória, ao induzir a produção das citocinas IL-1β, TNF-α, IL-8 e

IFN-γ nos macrófagos estimulados (KUMAR, LEWIN, RANI et al., 2013).

ESAT-6 (do inglês Early Secreted Antigen Targed 6) é uma proteína produzidas por

Mtb durante a fase ativa deste bacilo, a qual é codificada pela região RD-1 do gene (do

inglês Regions of Difference 1) de várias espécies do complexo M. tuberculosis, exceto nos

subtipos do M. bovis BCG (SORENSEN, NAGAI, HOUEN et al., 1995). Esta proteína

interage com TLR-2 e inibe a produção de citocinas pró-inflamatórias, como IL-12p40, IL-6

e TNF-α. Desta mandeira, modula os macrófagos a desempenharem funções anti-

inflamatórias (PATHAK, BASU, BASU et al., 2007). Porém, foi demonstrado que ao

interagir com TLR-2 em células dendríticas, ESAT-6 induz produção de IL-6 e TGF-β, bem

como a indução de células Th17, desempenhando um importante papel na virulência de Mtb

(CHATTERJEE, DWIVEDI, SINGH et al., 2011).

Proteínas do complexo PE/PPE fazem parte do sistema de secreção ESX, o qual é

composto por PE26, PE35, PE68, Rv 3425, dentre outras (FISHBEIN, VAN WYK,

WARREN et al., 2015). Duas importantes proteínas que participam formação do ESAT-6

são as proteínas PE35 e PPE68. Elas fazem parte do sistema de secreção ESX-1 e interagem

in vivo para a expressão de ESAT-6. Foi demonstrado recentemente que essas duas

proteínas, só ou combinadas, interagem com TLR-2 e induzem resposta anti-inflamatória em

macrófagos, por meio do aumento da produção de IL-10 e MCP-1 e redução da produção de

8

IL-12 em macrófagos (TIWARI, SOORY e RAGHUNAND, 2014). Uma outra proteína

pertencente a família PPE é a Rv3425. Esta proteína pertence a RD11 de Mtb (ZHANG,

WANG e LEI et al. 2007). A proteína Rv3425 tem sido intensamente estudada para ser

utilizada em modelo de vacina, tanto recombinanda com BCG, quanto como vacina de

subunidade proteica. Recentemente, a proteína Rv3425 foi fusionada com o Ag85B e

recombinada com a vacina BCG-Danish (BCG:Ag85B-Rv3425). A vacina BCG

expressando a proteína fusionada não melhora a proteção em relação a vacina BCG

(WANG, QIE E LIU et al. 2012). Porém, quando esta vacina é utilizada no sistema de prime

boost (rBCG:Ag85B-Rv3425 + Rv3425), seguido de desafio com Mtb, observou-se melhora

na proteção em relação ao BCG, tanto no baço quanto no pulmão dos camundongos, mesmo

após 22 semanas de infecção Mtb (YANG, GU, WANG, et al. 2016).

1.3.2. Proteínas de M. tuberculosis reconhecidas por TLR-4

Algumas proteínas de Mtb, não pertencentes às regiões de virulência, interagem com

macrófagos por meio da ligação com receptores do tipo TLR-4. Dentre estas proteínas

encontram-se Rv0652, DNAK e as proteínas que se encontram no complexo RpfA-E.

Uma dessas proteínas é o Rv0652 a qual pertence a cepa K de Mycobacterium

tuberculosis da família Beijing. Esta proteína desempenha atividades pró inflamatória com

indução de citocinas IL-12p40, IL-6, TNF-α e IL-1β por uma via dependente de Myd88 e

TRIF (KIM, SOHN, KIM et al., 2012; LEE, SHIN, LEE et al., 2014). Ela possue a

capacidade de promover o recrutamento e o amadurecimento de macrófagos e células

dendríticas, induzindo essas células a expressarem CD80, CD86 e MHC de classe I e MHC

de classe II (LEE, SHIN, LEE et al., 2014).

DNAK (Rv0350) é uma proteína extracelular de Mtb, também conhecida como

proteína de choque térmico 70 (HSP70) (HARTL, BRACHER e HAYER-HARTL, 2011).

Esta proteína ativa o macrófago a expressar molécula CD206 (MR) ao induzir para um perfil

anti-inflamatório (M2) produtor de arginase e citocina IL-10 (LOPES, BORGES, ARAUJO

et al., 2014). Ao interagir com TLR-4 ela induz aumento do processamento e apresentação

via MHC-II em macrófagos (TOBIAN, CANADAY e HARDING, 2004).

Mtb codifica 5 importantes proteínas associadas a sua ressuscitação do estado de

dormência, denomidadas Rpf (do inglês Ressucitation prototing fator). Compreendem as

RpfA-E (Rv0867c, Rv1009, Rv1884c, Rv2389c e Rv2450c) (Mavrici et al. 2014), as quais

são codificadas pela Região DosR (VOSKUIL, VISCONTI e SCHOOLNIK, 2004),

9

Recentemente foi demonstrado que a proteína RpfE de Mtb induz maturação de células

dendríticas, as quais passam a expressar CD80, CD86, MHCI e MHCII, bem como a

secretar IL-6, IL-1 beta, TNF-alfa. Neste estudo foi constadado que ao induzir maturação de

células dendríticas, esta proteína favorece o desenvolvimento de resposta imune adaptativa

Th1 e Th17 por via dependente de TLR-4, mas não de TLR-2 (CHOI, KIM E BACK et al.

2015).

Outras proteínas envolvidas na virulência de Mtb são o Ag85c (Rv 0129c) e o

MPT51 (Rv3803c), as quais possuem peso molecular que varia entre 27 a 32-kDa. Estas

proteínas fazem parte do mesmo complexo, porém com funções diferentes (OHARA,

OHARA-WADA, KITAURA et al., 1997). Enquanto o MPT51 garante a virulência de Mtb,

o Ag85C participa da síntese de mais de 40% do ácido micólico de Mtb, contribuindo para

manutenção da sua integridade e patogênese (KITAURA, OHARA, NAITO et al., 2000;

HARTH, HORWITZ, TABATADZE et al., 2002; SANKI, BOUCAU, RONNING et al.,

2009). O antígeno HspX (Rv2031c), que é uma proteína de choque térmico, provavelmente

participa na conformação final das proteínas de Mtb e favorece a adaptação do crescimento

do Mtb dentro dos macrófagos (CHANG, PRIMM, JAKANA et al., 1996; YUAN, CRANE,

SIMPSON et al., 1998; QAMRA, MANDE, COATES et al., 2005).

1.3.3. Proteínas de M. tuberculosis que interagem com TLR-2, TLR-4 e outros

receptores

HSP60 (do inglês Heat Shock Protein of 60 kDa) é uma proteína de choque térmico

de Mtb (Mtbhsp60), promove o aumento de fagocitose por macrófagos ao interagir com

TLR-2. Após interação, esta proteína induz a produção de IL-10, culminando na modulação

desses macrófagos a um perfil anti inflamatório. Porém, esta proteína também interage com

TLR-4 em macrófagos, induzindo a produção de TNF-α por essas células (PARVEEN,

VARMAN, NAIR et al., 2013). Por meio dos dois receptores a proteína Mtb hsp60 parece

induzir um perfil misto de macrófagos.

O Rv0934 é uma lipoproteína de 38-KDa, também conhecida como PstS2 (do inglês

Phosfatase transporte protein) (MALEN, SOFTELAND e WIKER, 2008). Esta lipoproteína

é uma adesina, atuando no processo de adesão ao se ligar ao receptor de manose (MR-

CD206) em macrófagos, por meio do qual ela induz a fagocitose por essas células

(ESPARZA, PALOMARES, GARCIA et al., 2015). Esta proteína é capaz de induzir Stress

do Retículo endoplasmático (ER stress) e apoptose de macrófagos. Ao interagir com os

10

receptores TLR-4 e TLR-2, ela induz macrófagos a produzirem MCP, TNF-α e IL-6

apresentando uma potente função pró-inflamatória (LIM, CHOI, LEE et al., 2015).

Recentemente tem sido demonstrado que produtos de Mycobacterium bovis BCG

também podem ser reconhecido por receptores PRRs em macrófagos. RNA de BCG pode

interagir com TLR-3 em macrófagos e induzir produção de IL-10, desempenhando um papel

anti-inflamatório (BAI, LIU, JI et al., 2014). O reconhecimento de RNA de BCG por TLR3

está relacionado com a regulação da resposta imune, uma vez que será induzido aumento de

infiltrado inflamatório, lesão tecidual e replicação bacteriana, devido ao desbalanço da

resposta pró-inflamatória (BAI, LIU, JI et al., 2014). Além disso, BCG induz aumento na

expressão de microRNAs (miR), especificamente o miR-124, em macrófagos alveolares.

Esta inibição parece estar relacionada com redução da expressão de TLR-6 e MyD88 e

TRAF6 por miR-124 (MA, LI, LI et al., 2014).

1.3.4. Proteína de Fusão CMX e resultados preliminares que comprovam sua

imunogenicidade.

Após observar a capacidade imunogência e antigênica do Ag85c, MPT51 e HspX,

foi escolhido as sequências de nucleotídeos correspondentes aos epítopos 85 a 159 do

Ag85C, 91 a 112 de MPT51 e o gene HspX, os quais amplificados por PCR a partir do

genoma de M. tuberculosis H37Rv usando primers específicos. Da fusão desses epítopos

construiu-se a proteína recombinante de fusão CMX (Ag85c_MPT51_HspX) (de Sousa et

al. 2012). Posteriormente, esta proteína foi utilizada em modelo de diagnóstico sorológico

da Tb, em modelo de vacina de subunidade protéica e em modelo vivo de vacina.

A proteína CMX foi utilizada em modelo de diagnóstico sorológico da Tuberculose,

utilizando uma pequena corte de 53 pacientes com TB comparando-os com 43 controles

saudáveis. Neste teste foi observado se a proteína CMX é capaz de distinguir pacientes com

TB de indivíduos saudáveis, tanto pela dosagem de IgM quanto pela dosagem de IgG, em

amostras de soros de indivíduos. Os indivíduos TB ativa apresentaram níveis de anticorpos

IgG superiores aos dos controles saudáveis (TB=0,407±0,141; C=0,167±0,072; p< 0,0001).

Ao se fixar uma sensibilidade de 100% com intervalo de confiança de 95% variando de

89,7% a 100% obteve-se uma especificidade de 71,4% (IC 95% de 53,7% a 85,3%). Para

IgM, os indivíduos com TB pulmonar ativa apresentaram média de leituras de anticorpos

IgM específicos para a proteína de fusão superiores (TB=0,305±0,09;C= 0,212±0,057;

p<0,0001). Ao se fixar uma sensibilidade de 80,0% com intervalo de confiança de 95%

11

variando de 63,06% a 91,56% obteve-se uma especificidade de 61,54% (IC 95% de 44,62%

a 76,64%) (De Souza et al. 2012).

Na tentativa de desenvolver uma vacina de subunidade utilizando a proteína de fusão

(CMX), nosso grupo de pesquisa formulou uma vacina contendo lipossoma e CpG DNA

como adjuvante e verificou a indução de resposta imune celular e humoral em camundongos

BALB/c, após receberem 3 imunizações subcutâneas com CMX e CpG DNA. Os grupos

controles foram vacinados somente com CpG DNA encapsulado com lipossoma, somente

lipossoma ou salina. Os resultados mostraram que a vacina CMX é eficaz na indução da

resposta imune humoral específica, pois foi capaz de induzir elevados níveis de IgG1 e

IgG2a no grupo imunizado com CMX encapsulado com lipossoma e CpG DNA. Para

avaliação da resposta imune celular foi verificada se a vacina CMX seria capaz de induzir

resposta imune específica aos linfócitos TCD4+. Resultados mostraram que a percentagem

de linfócitos TCD4+ expressando IFN-γ foram maiores no grupo imunizado com CMX que

os outros grupos. Similarmente, a percentagem de linfócitos TCD4+ expressando TNF-α

foram maiores no grupo imunizado com CMX que os outros grupos (De Souza et al. 2012).

Com o objetivo utilizar a proteína CMX em modelo de vacina viva, foi realizada

uma construção utilizando o Mycobacterium smegmatis recombinante expressando a

proteína CMX (mc2-CMX). Ao imunizar camundongos com esta vacina, ela manteve as

características imunogênicas da CMX, sendo boa indutora de anticorpos do tipo IgG1 e

IgG2a, bem como de linfócitos TCD4+IL-17+ em pulmão dos camundongos imunizados.

Esta resposta pareceu favorecer a capacidade protetora da vacina, quando os animais foram

desafiados com Mtb (Junqueira-Kipnis et al. 2013).

A proteína CMX também foi expressa em outro modelo vacinal, IKE-CMX. Neste

modelo, a vacina IKE-CMX induziu altos níveis de anticorpos IgG2a específicos, bem como

linfócitos TCD4+IL-17+ em pulmão de camundongos imunizados. Análise de populações de

macrófagos em pulmão desses camundongos revelou que esta vacina induz macrófagos

ativados (F4/80+CD11bmid CD11clow), demontrando uma possível modulação da resposta

imune inata (Junqueira-Kipnis et al. 2013).

12

2 JUSTIFICATIVA

Apesar do avanço da ciência desde a sua descoberta, a Tuberculose continua

sendo um dos principais problemas de saúde pública. Uma das medidas que podem

melhorar a prevenção e bloquear a transmissão do Mtb é o desenvolvimento de novas

vacinas que previnam o estabelecimento e a progressão da TB em humanos. Embora

exista a vacina BCG que é eficiente contra formas graves de TB na infância existe a

necessidade do desenvolvimento de novas vacinas para controlar a disseminação da TB,

uma vez que a BCG não proteje indivíduos na fase adulta contra a TB ativa.

Neste sentido, nosso grupo realizou a construção da rCMX, uma proteína de

fusão composta por epítopos imunodominantes dos antígenos Ag85c, MPT51 e HspX

inteiro de Mtb. Foi demonstrado que essa construção manteve a imunogenicidade dos

epítopos em camundongos e se mostrou antigênica em indivíduos com TB ativa.

Quando a proteína rCMX foi expressa por vetor vivo Mycobacterium smegmatis (mc2-

CMX) mostrou-se boa indutora de resposta imune do tipo Th1 e Th17 em pulmão de

camundongos imunizados, com proteção similar a BCG Moreau. Esta construção

também foi boa indutora da produção de anticorpos IgG1 e IgG2a importantes no

controle da TB.

No contexto atual da TB e da BCG, é necessário o desenvolvimento de uma

vacina que tenha melhor desempenho que a BCG usada atualmente, principalmente

devido à insuficiente indução de memória imunológica capaz de proteger jovens

adultos. Dentre as estratégias utilizadas para se desenvolver uma nova vacina está a

construção de uma rBCG e a associação desta com vacinas de subunidade proteica,

capaz de promover melhor desempenho que a BCG, quanto a indução de proteção e

memória.

Neste sentido, este trabalho visou apresentar o desenvolvimento de uma vacina

BCG recombinante expressando a proteína rCMX (rBCG-CMX) e avaliação da

capacidade da proteína rCMX em ativar macrófagos.

13

3. OBJETIVOS

3.1. OBJETIVO GERAL

Avaliar a proteção e a modulação da resposta imune induzida por BCG recombinante

expressando epítopos imunodominantes Ag85C, MPT-51 e HspX do Mycobacterium

tuberculosis induzida em modelo murino.

3.2. OBJETIVOS ESPECÍFICOS

- Realizar uma revisão de literatura sobre vacinas BCGs recombinantes, abordando os

artigos publicados entre os anos de 2008 e 2013, apresentado no artigo intitulado:

Recombinant BCG: Innovations on an old vaccine. Scope in BCG strains and

strategies to improve long lasting memory

- Estudar a indução de resposta imune adaptativa pela vacina rBCG-CMX, apresentada

no artigo intitulado: A New Recombinant BCG Vaccine Induces Specific Th17 and

Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis

- Entender os mecanismos inatos induzidos pela proteína rCMX, apresentada no artigo

intitulado: Modulation of the immune response induced by the recombinant fusion

protein CMX involves IL-6 and TGF-β production and TLR-4 stimulation

14

4 ARTIGOS

Artigo 1 – Recombinant BCG: Innovations on an old vaccine. Scope in BCG

strains and strategies to improve long lasting memory

Autores: Adeliane Castro da Costa, Sarah Veloso Nogueira, André Kipnis and

Ana Paula Junqueira-Kipnis.

Frontiers in Immunology (Publicado)

Artigo 2 – A New Recombinant BCG Vaccine Induces Specific Th17 and Th1

Effector Cells with Higher Protective Efficacy against Tuberculosis

Autores: Adeliane Castro da Costa, Abadio de Oliveira da Costa Júnior, Fábio

Muniz de Oliveira, Sarah Veloso Nogueira, Joseane Damaceno Rosa, Danilo

Pires Resende, André Kipnis e Ana Paula Junqueira-Kipnis.

PLOS one (Publicado)

Manuscrito – Modulation of the immune response induced by the recombinant

fusion protein CMX involves IL-6 and TGF-β production and TLR-4 stimulation

Autores: Adeliane Castro da Costa, Danilo Pires de Rezende, Bruno de Paula

Oliveira Santos, Karina Furlani Zoccal, Lúcia Helena Faccioli, André Kipnis e

Ana Paula Junqueira-Kipnis.

PLOS One (Submetido)

15

Artigo 1

Recombinant BCG: Innovations on an old vaccine. Scope in BCG strains and

strategies to improve long lasting memory

Adeliane Castro da Costa1, Sarah Veloso Nogueira1, André Kipnis1 and Ana Paula

Junqueira-Kipnis1*

1Microbiology, Immunology, Parasitology and Pathology Department. Federal

University of Goias.

*Dr. Ana Paula Junqueira-Kipnis

apkipnis@gmail.com

Laboratório de Imunopatologia das Doenças Infecciosas

Instituto de Patologia Tropical e Saúde Pública

Universidade Federal de Goiás

Rua 235 esquina com Primeira Avenida

Setor Universitário. Goiânia GO Brazil

74605-050

Abstract

BCG (Bacille Calmette Guérin), an attenuated vaccine derived from Mycobacterium

bovis, is the current vaccine against tuberculosis (TB). Despite its protection of active

TB in children, BCG has failed to protect adults against TB infection and active disease

development, especially in developing countries where the disease is endemic. There is

significant effort towards the development of a new TB vaccine. This review article

aims to address publications on recombinant BCG (rBCG) developed in the last five

years and highlight the strategies used to build an rBCG, trying to understand the

criteria used to improve their immunological memory and protection compared to BCG.

The literature review was done on April 2013, using the key words tuberculosis, rBCG

vaccine and memory. This review discusses the BCG strains and the strategies currently

used for the modification of BCG, including: overexpression of M. tuberculosis (Mtb)

immunodominant antigens already present in BCG; gene insertion of immunodominant

16

antigens from Mtb absent in the BCG vaccine; combination of introduction and over

expression of genes that were lost during the attenuation process of BCG; BCG

modifications for induction of T CD8+ immune response and cytokines expressing

rBCG. Among the vaccines visited, VPM1002, also called rBCGΔureC::hly is in human

clinical trials. Much progress has been made in the effort to improve BCG, with some

promising candidates, but considerable work is still needed to address functional long

last memory.

Key words: rBCG, tuberculosis, vaccine

Introduction

Tuberculosis (TB) is an infectious disease caused by Mycobacterium

tuberculosis (Mtb), an intracellular pathogen that, after infecting a host, can cause

disease or latency. TB continues to kill some 1.3 million people annually and 2 billion

people worldwide are infected with Mtb (Kamath et al., 2005; WHO, 2009). The

attenuated Mycobacterium bovis strain, known as BCG (Bacille Calmette-Guérin), is

currently the only TB vaccine approved for human use, but its protective efficacy

remains doubtful (WHO, 1998; Partnership WST, 2010). BCG was initially obtained

from a virulent strain and was developed in France between 1908 and 1921 by Albert

Calmette (1863-1933) and Camille Guérin (1872-1961). Although BCG is efficient in

some regions of the world, such as in Alaskan American Indians (Aronson et al, 2004;

Mangtani et al, 2014), the protection conferred by BCG varies between 0 and 80%

(WHO, 1979; Colditz et al., 1994; Trunz et al., 2006), protecting children from severe

forms.

To achieve BCG attenuation, more than 10 years of research with more than 230

serial passages were done in vitro (Calmette et al., 1926). This attenuation promoted

genomic deletions, that together with the evolution of M. bovis, resulted in 16 genomic

regions of differentiation (RD1-RD16, plus nRD18), when compared to Mtb genome

(Brosch et al., 2000; Joung & Ryoo, 2014). Of the region of differentiation lost during

attenuation, RD1 is a DNA segment comprising 9.5 kb, which was deleted in all other

BCG strains and it encodes epitopes, such as ESAT-6, CFP-10, Rv3873, PPE protein,

among others, that can be recognized by T lymphocytes (Cole et al., 1998); RD2 is a

10.7 kb DNA segment which encodes for the proteins Mpt-64, CFP-21, to name a few

17

(Joung & Ryoo, 2014); RD14 is a 9.1 kb section of DNA encoding proteins of the PE-

PGRS and Rv1771 families (gulonolactone dehydrogenase) (Behr et al.,1999); RD16 is

a 7.6 kb DNA section encoding Rv3405 which is responsible for colony morphology

characteristic, and formation of cell membrane constituent (Honda et al., 2006); nRD18

is a 1.5 kb segment containing the genes encoding SigI, an alternative RNA polymerase

sigma factor, that was only lost in the strains BCG Pasteur, Phipps, Frappier, Connaught

and Tice (Joung & Ryoo, 2014). During BCG attenuation process and the years that

followed, more than 14 sub-strains emerged: BCG-Russia (ATCC 35740), BCG-

Moreau/Rio de Janeiro, BCG-Tokyo, BCG-Sweden, BCG-Birkhaug (ATCC 35731),

BCG-Denmark 1331 (ATCC 35733), BCG-China, BCG-Prague, BCG-Glaxo (ATCC

35741), BCG-Tice (ATCC 35743), BCG-Frappier (ATCC 35735), BCG-Connaught,

BCG-Phipps (ATCC 35744), and BCG-Pasteur 1173 (Leung et al., 2008). They are

distributed throughout the world and they have been used for vaccine development to

prevent TB. The main concern is that BCG administration does not provide a reliable

protection for adults in the developing world, protecting just against the main causes of

infants TB, tuberculosis meningitis and miliary tuberculosis (WHO, 2009).

In order to address the evolution of new recombinant BCG vaccines, one must

have a defined immunological status goal desired for such vaccine. This is a

controversial issue, as there are no consensuses as to what is the ideal immune memory

phenotype that can confer protection. For instance, in animal models such as mouse,

both Mtb infection or BCG vaccine induce increased levels of lung CD4+ effector T

cells presenting the phenotype CD44hi CD62Llo CCR7lo, as well as memory cells. The

current memory cell phenotypes accepted are effector memory T cells (TEM) and

central memory T cells (TCM), characterized by CD44hi CD62Llo CCR7lo and CD44hi

CD62Lhi CCR7hi expression, respectively (Henao-Tamayo, et al, 2010; Junqueira-

Kipnis, et al, 2004; Kipnis et al, 2005). A cornerstone set for tuberculosis protection is

the importance of IFN-γ production by T cells (Flynn, et al, 1993; Cooper et al, 1997), a

cytokine crucial to stimulate the microbicide functions of macrophages. More recently,

some authors have proposed that the desired protective memory against TB infection

should have a central memory characteristic, with polyfunctional ability to produce

IFN-γ, TNF-α, and IL-2 cytokines (Ottenhoff, 2012) or a balance between IFN-γ and

IL-17 levels in order to avoid excessive pathology (Desel, et al, 2011).

The ultimate goal of a vaccine is its use among humans; consequently the

characterization of memory T cells in humans is also crucial. The major surface

18

biomarkers for human memory T cell population with effector phenotype are

CD45RAhi, CD45ROneg, CCR7neg, while central memory T cell populations present

CD45RAhi, CD45ROneg, CCR7pos. A follow up study conducted among children

vaccinated with BCG showed that specific memory T cells where stimulated and

present in the peripheral blood of those individuals for at least 52 weeks following

vaccination (Soares et al., 2013). It is interesting to observe that those induced memory

cells where polyfunctional (IFN-γ, TNF-α, and IL-2). Although several studies have

characterized the memory phenotypes induced by BCG, it is still not well established

the direct association of those populations with TB protection. Nowadays a long lasting

T cell memory population expressing CD127 has been associated with Mtb infection

and maybe also correlated to the protection shown by some exposed individuals (Jeong,

YH, et al, 2014). For the proposal of this review, it was considered as memory T cell

population the phenotype CD4+ CD44hi CD62Llo or specific CD4+ IFN-γ producing

cells.

A significant limitation in TB vaccine development and testing is the lack of an

optimal animal model that truly reflects the TB disease and immunity progress. While

there are several new vaccines being made in different laboratories, there are a diversity

of animal models (mice, rabbits, guinea pigs, non-human primates) and disease

outcomes being used by different laboratories, impairing an adequate comparison

between them. In addition there is no consensus on the protocol to be used for

vaccination and challenge, with different routes of immunization/infection, doses, BCG

and Mtb strains, and time periods being used. Short period of time between vaccination

and challenge does not allow full memory development, thus generating a bias toward

the correlation between memory T cell phenotype and protection. The most accepted

method for evaluating protection is the determination of the bacterial load following the

challenge of vaccinated animals compared to non-vaccinated infected controls.

Although a widely used method, the organs assessed to determine the bacterial load

varies among researchers and make it difficult to establish comparisons. Given all these

different parameters, in this review protection conferred by the different recombinant

BCG vaccines was considered when an overall significant reduction of the bacterial

load when compared to wild type BCG was achieved.

The factors that determine the induction of memory related to BCG are not well

understood. Some assumptions are directed to the characteristics of the BCG sub-

strains, which exhibit genotypic and phenotypic differences after attenuation process as

19

well as distinct residual virulence levels, the number of epitopes of each BCG strain or

the recombination strategy used for the development of a new vaccine (Behr et al.,

1997; Zhang et al, 2013). According to the research tools employed in this study, from

all sub-strains originated after this process, the strain most frequently tested over five

years were BCG Tokyo (BCG Japan), BCG Tice, BCG Danish (BCG Danmark/BCG-

SSI 1331), BCG Pasteur, BCG China (BCG Shanghai) and BCG Prague. It is also

hypothesized that the generation of the immune response and eventually the outcome of

a vaccination could be influenced by the type of strain background used. On the other

hand, there are preclinical animal data and human data demonstrating that different

strains of BCG confer the same level of protection (Castillo-Rodal et al, 2006; Davids et

al, 2006).

The main strategies used to develop new vaccines are based on the formulation

of subunit vaccines; on the production of non-recombinant viral vector vaccines that can

be used as BCG prime boost; and the construction of a recombinant BCG (rBCG),

which would confer the same protection with better induction of memory than BCG.

Some ways to construct a rBCG include over expression of promising Mtb

immunodominant antigens which are expressed by BCG, such as α-crystallin HspX

protein and complex 85 proteins (Ag85A, Ag85B and Ag85C) (DasGupta et al., 1998);

insertion of Mtb immunodominant antigens absent on BCG, such as those codified by

genes from RD1, RD2, RD3, RD14, RD15, RD16 and nRD18 (Zhang et al., 2013); the

combination of over expression with reintroduction of genes lost during BCG

attenuation; and BCG modification in order to induce CD8+ T immune response

proteins and cytokines (Tables 1 and 2).

Therefore, the aim of this review was to analyze which factors associated with

recombinant BCG could be able to induce long lasting memory and promote better

protection than the conventional BCG.

Does BCG epitope number influence in the induction of memory and protection of

rBCG vaccines?

As stated by Zhang et al. (2013), the number of epitopes in a particular strain

can be important for the development of a better vaccine that could replace BCG. If that

was the case, the strain more capable of inducing a good immune response would be

BCG Tokyo, since it comprises 359 epitopes suitable for being recognized by

20

lymphocytes (Zhang et al., 2013). To verify if there is sufficient data to support this

hypothesis in the last five years, we selected some criteria as summarized on Table 1.

The different rBCG vaccines were compared according to their ability to improve

protection by reducing the bacterial load relative to wild type BCG and to generate

specific memory CD4+ T cells. Among three different published studies using

recombinant BCG Tokyo, which exhibits greater number of epitopes, all showed better

protection than BCG that was associated to the recombinant generation strategy: the

over expression with re-introduction of lost genes, as well as with the presence of

cytokines (Table 1). Nevertheless, the strain that has been most widely used is BCG

Danish (BCG Danmark/BCG-SSI 1331), which have an average number of epitopes

(329 epitopes), and also provides improved protection and long lasting memory when

associated with the overexpression of Mtb antigens. Two rBCG Tice (328 epitopes)

vaccine constructions also obtained good results for induction of protection but only one

induced memory. Based on those publications it appears that the genetic background of

the BCG strains (number of epitopes) does not have a major role in inducing/improving

protection and memory.

Contrary to Zhang et al. (2013), other studies reported here, support the idea that

recombinant antigen selection to be expressed by BCG, and not the BCG strain

background, would be the significant aspect to be considered in the construction of an

improved vaccine, which would be a better inducer of memory and protection (Tang et

al., 2008; Dey et al., 2009; Sali et al., 2010). Moreover, it appears that overexpression of

certain antigens have been the key to make the most promising rBCG for induction of

memory and better protection than BCG.

When analyzing only publications of the last five years the conclusions

withdrawn may have been biased because we could be missing important work

addressing whether the BCG strain background (epitopes numbers) or the selected

antigen were important or not to improve memory and protection when compared to

BCG.

21

Table 1. BCG sub strains genetic background used for recombinant BCG vaccines development and ability to induce memory and

protection against tuberculosis.

Sub strains RDs * Epitopes Nº** Protection

better

than BCG?***

Memory?**

**

References

Tokyo/Japan RD1

359 3 Yes

Yes

Yes

No

Lin et al., 2011; Tang et al., 2008

Sugawara et al., 2009

Pasteur RD1,

RD2,RD14

, nRD18,

331 6 Yes

No

No

No

Yes

No

Sali et al., 2010

Tang et al., 2009

Chapman et al., 2012; Christy et al., 2012;Kong et al.,

2011

Danish/

Denmark

RD1, RD2, 329 11 Yes

No

Yes

No

Yes

Yes

No

No

Shi et al., 2010

Rahman et al., 2012

Dey et al., 2011; Sun et al., 2009; Jain et al., 2008; Qie et

al., 2009

Dey et al., 2010; Lu et al., 2012; Magalhães et al., 2008

Tice RD1, RD2,

nRD18

328 2 Yes

Yes

Yes

No

Hoft et al., 2008

Tullius et al., 2008

China /

Shanghai

RD1, RD2 321 6 Yes

No

No

No

Wang et al., 2012

Deng et al., 2010; Deng et al., 2012; Xu et al., 2009; Xu

et al., 2010;Yang et al., 2011

Prague RD1, RD2 318 3 Yes

No

No

No

Desel et al., 2011

Farinacci et al., 2012; Reece et al., 2011

* RD Region of difference;

** Number of publications in the last 5 years; *** Protection was evaluated by CFU analyses and considered when the bacterial load of challenged animals where lower than wild type BCG vaccinated animals;

**** Memory was defined as CD4+ CD44hi CD62Llo or CD4+ IFN- producing T cells specific immune responses.

22

Does the quantity of Mtb antigens incorporated in BCG result in greater

protection and memory development?

rBCG vaccines superexpressing Mtb immunodominant antigens

An important strategy used for the construction of a new TB vaccine is the

development of a rBCG super expressing Mtb immunodominant antigens, such as

proteins from the antigen 85 Complex, HspX protein, and also the association of both

proteins in one vaccine construction, which represents one of the favored approaches for

TB vaccine construction (Jain et al., 2008).

Some of the most important antigens used to construct BCG recombinant

vaccines are those from the Antigen 85 Complex that consists of Ag85A (Rv3804c),

Ag85B (Rv1886c) and Ag85C (Rv0129c), being encoded by fbpA, fbpB and fbpC2

genes, respectively, and presenting molecular weights between 30 and 32-kDa (Ohara et

al., 1997). Proteins of Ag85 complex have mycolyltransferase activity, thus they play a

role in the construction of Mtb cell wall and they are responsible for the mycolate

production of the cell wall, maintaining Mtb integrity and pathogenesis (Belisle et al.,

1997).

The protein Ag85B, used in the construction of the vaccine rBCG:30 (r30-

Ag85B), was able to generate protection in guinea pigs after challenge with Mtb

(Horwitz et al., 2000; Horwitz et al., 2003). This vaccine was in clinical trial I, and

when tested in human volunteers, it induced central and effector memory CD4 and CD8

T cells specific to Ag85B (Hoft et al., 2008). Currently, this vaccine is no longer being

tested on humans. The same antigen was used by Tullius et al. (2008) who developed a

mutant rBCG, rBCG (mbt) 30, resulting in a strain unable to synthesize mycobactin and

exoquelin molecules, which are essential for iron acquisition in deprivation of this

nutrient. The vaccine presented greater protection than conventional BCG. Another

approach was to design a rBCG pantothenate auxotroph, rBCG (panCD) 30. Both

vaccines rBCG (mbt) 30 and rBCG (panCD) 30 were more attenuated than BCG and

induced potent protective immunity, and cell mediated immunity in guinea pigs (Tullius

et al., 2008). These vaccines may have the potential to provide a safe alternative to HIV

positive individuals since BCG is not indicated to immunocompromised individuals.

This vaccine and others over expressing Ag85B were better in conferring

protection and memory than BCG. The 30 kDa antigen 85B is the most abundant

23

protein of the Ag85 complex, and is the most abundant extracellular protein of Mtb,

responsible for nearly one-quarter of the total extracellular protein in broth culture

(Harth et al., 1996). In addition, Ag85B is strongly recognized by T cells, can induce a

type Th1 immune response with IFN-γ production and was shown to have a good

protective capacity when used in DNA vaccine strategies (Palma et al., 2008).

Ag85C is also a major secretory protein and an immunodominant antigen, being

strongly recognized by sera from TB patients. In fact, it is responsible for almost 40%

of the mycolate content of the Mtb and its mycolyltransferase activity cannot be

substituted by Ag85A or Ag85B (Jackson et al., 1999). For this reason, Jain et al.

(2008) developed an rBCG expressing Ag85C under the transcriptional control of

mycobacteria promoters. Again, less granulomatous infiltration and less granuloma

formation were observed when compared to the group immunized with BCG and the

protection (reduced bacterial load in lungs and spleen better than ancestor BCG) was

associated with reduced levels of the mRNA codifying for the cytokines IFN-γ, TNF-α,

IL-12, and TGF-β, when compared to BCG, however, high levels of iNOS were

observed in comparison to BCG. On the other hand, previous studies with DNA

vaccines using Ag85C have demonstrated reduced production of IL-2 and IFN-γ,

displaying insufficient protection when animals were challenged with M. bovis BCG

(Lozes et al., 1997). Hence it is important to stress that not only the antigen can allow a

good protection status, but the association of the antigen expression plus a good vector

(e.g. BCG itself). Here, the strain used was BCG Danish, which may have contributed

for the results. Unfortunately those studies did not provide information regarding the

ability to generate memory cells.

Ag85A is also strongly recognized by T lymphocytes, inducing IL-2 and IFN-γ

production (Lozes et al., 1997). Immunization of mice and guinea pigs with

rBCG::Ag85A promoted reduction of pulmonary pathology severity and increase in

protection (lungs and spleen) (Sugawara et al., 2007). Consequently this vaccine was

also tested in Macaca mulatta, and, after challenge, the group immunized with

rBCG::Ag85A presented light to moderate pneumonia, while the non-vaccinated group

developed multilobar pneumonia, lymphadenopathy and atelectasis. Also, its protective

capacity was already appraised in a DNA vaccine system with Ag85A (Sugawara et al,

2003). Besides, they found that rBCG-Ag85A induced higher protective efficacy than

the parental BCG Tokyo (Sugawara et al., 2009). In that study a strategy of over

expressing the antigen in addition to the use of a BCG strain containing more natural

24

epitopes was employed (Table 1). Hence, this could justify the potential of this vaccine

for further studies such as memory induction.

Construction of recombinant BCG expressing single proteins resulted in

promising results. Following those studies significant progress started to be made with

the construction and testing of fusion recombinant proteins, combining two or more

protein coding regions, of one or more Mtb proteins because it is believed that the use

of combination of antigens may result in improved protective efficacy than rBCG

expressing only one antigen.

In order to analyze this hypothesis, Wang et al. developed three vaccine

constructions: rBCG::Ag85A (A), rBCG::Ag85B (B) and rBCG::AB, which were used

to immunize mice. The vaccine with fusion antigens: rBCG::AB showed better

protection after challenge with Mtb, when compared to BCG or rBCG expressing

Ag85A or Ag85B alone. Six and 24 weeks after vaccination, splenocytes of mice

immunized with rBCG::AB when stimulated with specific antigen secreted more IFN-γ

than splenocytes from mice immunized with the other rBCG (Wang et al., 2012).

Regrettably, no memory was evaluated in that study, but probably it was because the

memory induction is already known for rBCG::Ag85B (Hoft et al., 2008). On the other

hand, no studies with rBCG expressing Ag85A assessed memory response, so it would

be meaningful to verify if Ag85A could contribute or not for that. Although the

approach of recombinant fusion proteins have been shown of valuable use in the

protection against challenge with M. tuberculosis, the real potential to be used as a new

vaccine requires additional studies regarding the development of functional long lasting

memory.

Another antigen frequently used for recombinant expression in BCG is the

HspX protein. HspX (Rv2031c) is a heat shock protein encoded by the gene acr, with

molecular weight of 16 kDa, also known as α-cristalin (Chang et al., 1996). This protein

is one of the most abundant proteins that are produced during the latent or persistent

Mtb phase. Shi et al. developed a rBCG over expressing the immunodominant Mtb

antigen, HspX (rBCG::X) showing that rBCG::X provided better and long lasting

protection against Mtb infection than BCG, as evidenced by high levels of IFN-γ

production, low bacterial load in tissues and reduced lung pathology associated with

elevated levels of anti-HspX antibodies during week 6 and 24 (168 days) after rBCG::X

immunization, indicating that maybe, BCG::X can persist longer in vivo than BCG (Shi

et al, 2010).

25

Additionally, results obtained by Shi et al. advocate that expression of HspX by

BCG could improve the biological effects of this molecule which would explain the

higher expression of the protein Ag85B on the supernatant of cells as well as on the

lysate after infection with rBCG::X when compared to BCG (Shi et al., 2010). This

theory was also corroborated by Kong et al, (2011) who constructed an rBCG

expressing Mtb Ag85B under the control of hspX promoter. In that case, the expression

and immune response to Ag85B were modulated by hspX promoter. For example,

rBCG::PhspX-85B was able to induce intense specific Ag85B T cell proliferation, IFN-

γ production three weeks after infection with a greater increase after 12 weeks,

demonstrating long lasting cell mediated immunity. Despite its intense induction of

immune cell response, the protection induced by this vaccine, in lungs and spleen, was

similar to the BCG, indicating that in that model of Ag85B expression under the control

of a different promoter there was no improvement in the protective efficacy (Kong et

al., 2011).

Despite the fact that Ag85C is responsible for more than 40% of the mycolate

present in the mycobacteria cell wall (Belisle et al., 1997), evidences have shown that

the antigen Ag85B is the one that stood out in recombinant BCGs (rBCG) when it

comes to induction of memory and better protection than BCG (Tullius et al., 2008;

Hoft et al., 2008).

Although the use of fusion proteins have generated great expectations in the

scientific community, the use of combined proteins yielded no better memory than

BCG, according to the present accepted parameters, generating only better protection.

The increased protection observed among those recombinant vaccines can not be the

only improvement desired for the development of a new vaccine, once vaccination

using the available animal models to study new vaccines to TB do not eliminate all Mtb

bacteria from the tissues of challenged animals. Therefore new definitive protection

parameters are needed.

Association of over expression and reintroduction of antigens lost during

attenuation process

Some virulence regions, such as RD1, were lost during BCG attenuation

process. RD1 is absent in all BCG sub-strains, but present on virulent strains and

clinical isolates of M. bovis and M. tuberculosis. The association of Mtb genes lost in

26

the M. bovis attenuation process within rBCG has been used to improve the vaccine

efficacy (Brosch et al., 2000). The collection of well-defined T cell antigen epitopes has

been a widely used strategy for the construction of new vaccines. This collection is

based on the reintroduction of proteins whose gene regions were deleted during the

attenuation process. Some of those proteins are the 10 kDa culture filtrate protein (CFP-

10, Rv3874), ESAT-6, PPE family protein (Rv3873), INV (Rv1474), MPT64, to name a

few.

When evaluating the induction of immune response, it has been noticed that the

vaccine constructions with epitopes has been the most auspicious, although most of the

studies did not evaluate the protection or memory induced by these vaccines. Vaccine

constructs using those antigens were good inducers of Th1 immune response and

production of IFN-γ and IgG2a, and were excellent DTH (delayed type hypersensitive)

response inducers, superior to BCG, indicating that vaccination prompted specific

immune response. Conversely, some recombinant vaccines (BCG:CFP, BCG:FBP,

BCG:PPE and BCG:INV) showed protection similar to that of BCG (Christy et al.,

2012). Only rBCG vaccine expressing MPT64 antigen fused to a PE antigen (HPE-

ΔMPT64-BCG) showed superior protection than those immunized with BCG. This

protection was related to CD4 and CD8 T cell induction and the emergence of a specific

MPT64 T cell clone (Sali et al., 2010). In spite of the use of the same BCG strain in

those two works, the protection differences observed could point to the importance of

antigen choice, being the fusion a crucial factor that made the difference in providing a

better vaccine in this case.

When using proteins of the Ag85 complex, Qie et al. (2009) compared the

protective efficacy of rBCG-AMM (BCG expressing Ag85B-MPT64190-198-Mtb8.4)

to BCG. Animals vaccinated with rBCG-AMM generated more antigen-specific CD4

and CD8 T cells than those vaccinated with BCG and showed a more efficient response

to protect mice challenged with H37Rv. Moreover, rBCG-AMM was superior to BCG

in reducing the severity of the disease in the target organs like lungs and spleen,

indicating that rBCG-AMM could be a potential vaccine candidate for further studies.

Here again, this vaccine was not evaluated for memory induction though.

Some rBCG vaccines designed over the past 5 years, combined the ability to

generate strong immune response of the Ag85 proteins with the antigen ESAT-6 (Lu et

al., 2012; Xu et al., 2010; Deng et al., 2010). From those studies only one addressed

protection and memory development. For instance, Deng et al. (2010) constructed an

27

rBCG expressing the fusion protein Ag85A-ESAT-6 (rBCG-AE) and this vaccine

showed more potent immunogenicity than native BCG in mice and induced a shift

towards a Th1 type immune response with the increase in the ratio of both CD4 and

CD8T subsets. Thus, rBCG-AE elicited long-lasting and stronger Th1 type cell-

mediated immune responses than BCG. That group further evaluated the protective

efficacy conferred by rBCG-AE against Mtb infection in BALB/c mice (Deng et al.,

2012). Once more, an rBCG vaccine expressing ESAT-6 alone did not exceed the

parental BCG vaccine in the protection from Mtb H37Rv infection. That vaccine was

developed with a BCG-China strain, while others used BCG-Tokyo, BCG-Danish, or

BCG-Pasteur. As we previously stated, vaccine constructions overexpressing proteins

of the Antigen 85 Complex seem to be better than BCG when it comes to protection

efficacy while recombinant BCG-ESAT-6 presented protection similar or even inferior

to BCG. Still, using combined epitopes from proteins of the Ag85 complex or other

proteins and ESAT-6 improved macrophage activation and antigen presentation (Xu et

al., 2010), and strong humoral and cellular immune responses were induced (Lu et al.,

2012), although protection or memory generation were not addressed.

The development of rBCG vaccines associating or re-introducing genes lost

during BCG attenuation appears to improve protection and memory most frequently

when proteins from the complex Ag85 were associated to the fusion protein. This

observation could be biased due to the fact that the Ag85 proteins were the ones most

frequently used in the development of those rBCG vaccines. The combined results

attested that those were probably genes evolutionary kept by Mtb and were able to

induce strong immune response in animal models or humans, no matter what type of

BCG strains were used. The best combination though, was the fusion protein composed

by Ag85 and MPT64. Most of the studies presented here have not evaluated functional

memory, a crucial step if one intends to develop a long lasting protective vaccine.

rBCG vaccine expressing mammalian cytokines and Mtb proteins

Interleukins (ILs) play a central role in the immune system and have multiple

effects on different immune cells. IL-2, for example, has been used for the treatment of

some diseases, including tuberculosis, but the toxicity correlated with high dose of this

interleukin restricted its use. So, the solution to overcome that problem pointed to the

expression of rIL-2 and other cytokines by BCG (Kong & Kunimoto, 1995). Another

28

example is IL-15, an important cytokine to maintain survival and proliferation of CD8+

T cells with memory phenotype (McShane et al., 2002). In order to develop new

vaccines capable of improving BCG, some research strategies included the use of

recombinant BCG expressing interleukin, such as IL-2, IL-12, IL-15, GM-CSF, among

others.

Recombinant vaccines expressing cytokines induced effector polyfunctional

CD8 T cells and CD4 T cells (producing IFN-γ, IL-2 and TNF-α), as well as humoral

immune response with increase of specific IgG2a/IgG1 levels (Tang et al., 2008- rBCG-

Ag85B-IL-15; Lin et al., 2011- BCG :: Ag85B-CFP10-IL-12; Yang et al., 2011-rBCG:

GMCSF-ESAT6). Among those types of vaccines, the ones that stood out were those

expressing IL-15 and IL-12, as they induced CD8+ T (CD8+ CD44hi CD62Llo) and CD4+

T (CD4+ CD44hi CD62Llo) memory cells (Tang et al., 2008; Lin et al., 2011).

Although those types of vaccines showed better protection than BCG, the rBCG-

Ag85B-IL-15 vaccine seemed more promising because it presented greater induction of

memory CD8 T cells than memory CD4 T cells, in support to the theory that CD8 T

cells rather than CD4 T cells are important in long lasting protection against TB (Tang

et al., 2008). It is important to note that despite the positive influence of IL-15 in

inducing memory cells, its in vivo administration after priming with rBCG and followed

by challenge with Mtb, does not induce increase of CD8 T memory cells, phenomena

seen only when IL-15 is expressed by rBCG (Tang et al., 2008).

It seems as the induction of CD8+ T cells and polyfunctional CD8+ and CD4+ T

cells (producing IFN-γ, IL-2 and TNF-α) are responsible for the improvement of the

protection generated by rBCG while secretion of interleukins might play an important

role in the proliferation and maintenance of memory T cells.

29

Table 2. Description of strains and antigens used in the papers visited for this review. References published and indexed inPubMed from 2008 April 2013.

Reference Model Strain Antigen Challenge Protection

Lin et al., 2011 Mice 1.1.1.1.1.1.1.1.1 BCG Tokyo

rBCG1::Ag85B-CFP10(rBCG1)/BCG2::Ag85B-

CFP10-IL-12 (rBCG2)

No Yes + (In vitro)

Hoft et al., 2008 Human BCG Tice rBCG30 (Ag 85B) No Yes + (In vitro)

Dey et al., 2010

Guinea pigs BCG Danish rBCG-E6 (ESAT-6)

50–100 bacilli of Mtb Yes +

Dey et al., 2011 Guinea pigs BCG Danish rBCGacr 50–100 bacilli of Mtb Yes +

Shi et al., 2010 Mice BCG Danish BCG::HspX/ rBCG::85B 106 CFU of Mtb Yes +

Deng et al., 2012 Mice BCG China rBCG-AE 106 CFU of Mtb Yes –

Reece et al., 2011 Mice BCG Prague rBCGureC::hly or rBCGureC::hly 102 CFU of Mtb

Yes

Qie et al., 2009 Mice BCG Danish BCG: rBCG-Ag85B-Mpt64-Mtb8.4 106 CFU of Mtb

Yes +

Lu et al., 2012 Mice BCG Danish rBCG::Ag85B-ESAT6-Rv2608 No NA

Sugawara et al., 2009 Monkey BCG Tokyo rBCG-Ag85A 3000 CFU of Mtb Yes +

Magalhaes et al., 2008 Monkey BCG Danish rBCG AFRO 1 No NA

Xu et al., 2010 In vitro BCG China rBCG:Quimera 85B+ESAT-6 No NA

Sali et al., 2010 Mice BCG

Pasteur

rBCG:PE-MPT64/rBCG/HSP60MPT64 ~200 CFU of Mtb Yes +

Desel et al., 2011 Mice BCG Prague rBCGΔureC::hly+ 200-400 CFU of Mtb Yes +

Wang et al., 2012 Mice BCG China rBCG : Ag85A/rBCG :Ag85B/ rBCG:Ag85A-Ag85B 106 CFU of Mtb

Yes

Deng et al., 2010 Mice BCG-China rBCG: Ag85A-ESAT-6/rBCG :: Ag85A/ rBCG :

ESAT-6

No NA

Rahman et al., 2012 Monkey BCG Danish AFRO-1,Ag85A,Ag85B e TB10.4. 500 CFU of Mtb Yes +

Christy et al, 2012 Mice BCG

Pasteur

rBCGs: BCG::Ag85c, BCG::INV, BCG::PPE,

BCG::FBP e BCG::CFP

20 bacilli of Mtb Yes =

Farinacci et al., 2012 Mice BCG Prague rBCGΔureC::hly+ No NA

Kong et al., 2011 Mice BCG

Pasteur

rBCG: pHspX-Ag85B 100 bacilli of Mtb/lung Yes =

Yang et al., 2011 Mice BCG China rBCGs:BCG::GM-

CSF/BCG::ESAT6/BCG::GMCSF-ESAT6

No NA

Tang et al., 2008 Mice BCG Tokyo rBCG-85B-IL15/rBCG-85B 2 ×105 CFU of Mtb Yes =

Jain et al., 2008 Guinea pigs BCG Danish rBCG-85C

500 bacilli of Mtb Yes +

Grode et al., 2013 Human BCG Danish BCG ΔureC::hly HmR No NA

+ Protection superior than BCG; = similar to BCG; – less protection than BCG; NA not aplicable

30

BCG modification - induction of CD8 T immune response

M. tuberculosis and BCG preferentially localize inside antigen presenting cells

(APC) phagosomes, mainly within macrophages and dendritic cells. This localization

dictates antigen traffic via MHC-II, which results in preferential stimulation of CD4 T cells.

CD8 T cytolytic lymphocytes (CTLs) are essential for the clearance of intracellular Mtb

infection since CTLs aim to kill cells and bacteria through secretion of cytolytic and

antimicrobial effector molecules (perforin and granulysin). It is known that Mtb induce

apoptosis in infected cells, resulting in vesicles that transport mycobacteria antigens, which

can be captured by local dendritic cells, culminating with cross presentation of MHC-I and

MHC-II, stimulating CD8 and CD4 T cells, respectively (Schaible et al., 2003). It is also

acknowledged that BCG is a weak inducer of apoptosis and thus activates CD8 T cells to a

lesser extent (Schaible et al., 2003; Farinacci et al., 2012). With this regard, in an attempt to

improve BCG, rBCG vaccines have been developed to express listeriolysin (Hly) from

Listeria monocytogenes (Mandal et al., 2002) in its membrane, in combination with

deletion of ureC gene (rBCGΔureC::hly). One of the mechanisms BCG employs to survive

phagosome is pH neutralization, through Urease C (ureC) activity. To induce apoptosis Hly

needs an acidic pH, which is why an ureC mutant rBCG was developed, so phagolysosome

pH acidification occurs naturally (Mandal et al., 2002). By using this vaccine protocol,

Reece et al (2011) selected antigens based on their expression in response to nutrients

deprivation (Rv2659c), hypoxia (Rv1733c) or disease reactivation (Rv3407) and

transformed rBCGΔureC::hly with plasmid containing those antigens, rBCGΔureC::hly

(pMPIIB01). The improved performance of this vaccine implied in lower bacterial load in

spleen of mice (Reece et al., 2011). In addition, it induced Th-17, CD4+ and CD8+ T

responses promoting more protection than BCG (Desel et al., 2011; Farinacci et al., 2012).

This vaccine is the most promising rBCG vaccine generated and it finished phase I clinical

trial for safety with great success, and is being tested in newborns in a phase II clinical trial

(Grode et al., 2013). Although this vaccine aimed to improve T CD8 responses, it was

observed in vaccinated healthy humans induction of specific CD4 T cells secreting IFN- as

well as polyfunctional T CD4 responses.

31

In a similar approach, with the goal of obtaining a vaccine capable of inducing

increased CD8 T cell response, a recombinant BCG, rBCG AFRO-1 (BCG expressing

Ag85A, Ag85B and TB10.4) was used followed by two boosts with AERAS-402

(adenovirus vaccine 35 (rAd35) expressing Ag85A, Ag85B and TB10.4). AFRO-1 BCG

expresses perfingolysin O, which allows BCG scape to cell cytosol, promoting antigen

processing and presentation via MHC-I. After priming with rBCG AFRO-1, there was

delayed but strong IFN-γ production one week after boost with AERAS-402, as well as

strong proliferation of CD4 and CD8 T cells (Magalhães et al., 2008). This vaccine

promoted longer survival and IFN-γ production, however no differences in lungs and spleen

bacterial load between groups vaccinated with BCG or AFRO-1 (also known as AERAS-

422) were observed (Sun et al., 2009). Although a promising vaccine, AERAS-422 had to

be terminated because of development of shingles in some study participants that occurred

during phase I clinical trial (Kupferschmidt, 2011).

The strategy of BCG modification for induction of CD8 T specific immune response

has shown great impact, as the recombinant vaccine rBCGΔureC::hly is in clinical trial

(www.clinicaltrials.com).

Conclusions and Future Perspectives

BCG is used for almost 100 years, with more than eight million doses used.

Tuberculosis incidence on the other hand showed only a slow decrease during the last

decade, mainly due to the increase of the multi drug resistant strains and the HIV co-

infection (Mangtani et al, 2014). Two main cautions of BCG vaccine are associated to its

variable efficacy and immunity against M. tuberculosis infection resulting in a large pool of

latently/persistently-infected individuals. It’s also been discussed that BCG have better

protection among individuals from regions with lower environmental mycobacterial

contaminations and lower tuberculosis rates. Development of a new vaccine or

improvement of BCG to protect against TB is not an easy task, once the natural infection

per se does not induce protection or long lasting T or B functional memory cells since it

does not avoid re-infection. It appears that the coevolution between mycobacteria and the

human being favors the mycobacteria. Over the past five years, several attempts were

32

conducted to develop recombinant BCGs (Table 2). Improvement of BCG remains among

the best choices for the rational design of a TB vaccine. This review sought to discuss

recent TB studies advancing the BCG recombination strategy. The main purpose for

developing an rBCG is to design a vaccine capable of inducing long lasting functional

memory with protection similar or superior to that of BCG. Also, BCG is a strong inducer

of CD4+ T cells but it is an insufficient stimulator of CD8 T cells. The most effective rBCG

vaccination strategies in animal models and in human clinical trials to date were those that

stimulate both CD4+ T and CD8+ T cells to produce Th1-associated cytokines and cytotoxic

functions (Hanekom, 2005, Soares et al, 2011, www.clinicaltrials.com).

It is recognized that protein combinations, such as fusion proteins, as well as

expression of these proteins by different expression vectors have been used as important

strategies in the development of an rBCG vaccine with better efficacy than BCG.

Nevertheless, the vaccine approach super expressing Mtb proteins in BCG with better

performance was rBCG::30, which express only the protein Ag85B, being able to induce

central memory and more desirable protection than BCG (Hoft et al., 2008). This plasmid

based vaccine passed clinical trial phase I and currently is on hold awaiting the

development of auxotrophic BCG strains to avoid the use of antibiotic resistance gene

(www.clinicaltrials.com).

Intriguing is that the rBCG vaccine currently on phase II of human clinical trial is a

vaccine that did not introduce Mtb antigens or antigens lost by BCG during the attenuation

process. Reasonably, the rBCGΔureC::hly vaccine improved BCG antigen presentation by

dendritic cells (DC) improving its processing with the ultimate goal of activating CD8+ T

cells. Maybe this approach overcame some of the evolutionary mycobacteria

immunological scape and will allow a protective long lasting functional memory. Shortly in

time we will know if this vaccine induced better protection to tuberculosis (WHO, Global

report on Tuberculosis, 2013).

Further, the choice of parental BCG strain appears not to interfere with the

recombinant vaccine outcome, because some vaccines using the same parental BCG strains

have shown different outcome depending on the selected antigen or fusion protein used.

Likewise, the immune response profile of those vaccine candidates that showed better

protection than BCG was based upon CD4 and CD8 T cells with polyfunctional activities.

33

From all studies reviewed here, only six of them successfully evaluated memory in animal

models.

The animal models available to study vaccine for tuberculosis (mice, guinea pig or

no human primates) most of the time cannot predict the outcome among vaccinated

humans. It is well known that mice and guinea pigs are infected by BCG vaccination and

the duration of the vaccination and the time until challenge are crucial to address the

persistence of memory T cells. This premise could be used to justify the lower numbers of

work that addressed this issue over the past five years.

The real impact of these new vaccine using rBCG or other strategies that are

currently in clinical trials only will be observed in five to ten years from now, therefore

studies addressing new strategies to improve BCG needs to be continued.

Material and Methods

Study selection and data collection process. The search for this review was

conducted on April 2013 and was based on articles published in the last five years (2008-

2013). Articles were searched from PubMed Database using the key words: tuberculosis

protection and rBCG vaccine with the intention to address publications showing studies on

rBCG vaccine for tuberculosis, then further selecting manuscripts using the key words:

tuberculosis protection; rBCG vaccine and memory. Manuscripts without information of

BCG wild type strain and those that used the boost strategy without evaluating rBCG

responses alone were not included in this review.

Funding

This research was supported by CNPq.

Declaration of Interest sections:

The authors declare no conflict of interest.

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43

Artigo 2

A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with

Higher Protective Efficacy against Tuberculosis

Adeliane Castro da Costa1, Abadio de Oliveira Costa-Júnior1, Fábio Muniz de Oliveira2,

Sarah Veloso Nogueira1, Joseane Damaceno Rosa1, Danilo Pires Resende1, André Kipnis2,

Ana Paula Junqueira-Kipnis1*

1 Laboratório de Imunopatologia das Doenças Infecciosas, Instituto de Patologia Tropical e

Saúde Pública, Universidade Federal de Goiás, Goiânia, Goiás, Brazil

2 Laboratoório de Bacteriologia Molecular, Instituto de Patologia Tropical e Saúde Pública,

Universidade Federal de Goiás, Goiânia, Goiás, Brazil

Abstract

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb)

that is a major public health problem. The vaccine used for TB prevention is

Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in

protecting against pulmonary TB among adults. Consequently, several groups have pursued

the development of a new vaccine with a superior protective capacity to that of BCG. Here

we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein

(CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and

evaluated its immunogenicity and protection in a murine model of infection. The stability of

the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was

efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO)

production. Following mouse immunization, this vaccine induced a specific immune

response in cells from lungs and spleen to the fusion protein and to each of the component

recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1,

Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of

lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition,

44

when this vaccine was used in a prime-boost strategy together with rCMX, the lung

bacterial load was lower than the result observed by BCG vaccination. This study describes

the creation of a new promising vaccine for TB that we hope will be used in further studies

to address its safety before proceeding to clinical trials.

Citation: Costa ACd, Costa-Júnior AdO, Oliveira FMd, Nogueira SV, Rosa JD, et al.

(2014) A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells

with Higher Protective Efficacy against Tuberculosis. PLoS ONE 9 (11): e112848.

doi:10.1371/journal.pone.0112848

Editor: Delphi Chatterjee, Colorado State University, United States of America.

Received August 20, 2014; Accepted October 15, 2014; Published November 14, 2014

Copyright: 2014 Costa et al. This is an open-access article distributed under the terms of

the Creative Commons Attribution License, which permits unrestricted use, distribution,

and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully

available without restriction. All relevant data are within the paper and its Supporting

Information files.

Funding: This study was financed by the National Council for Scientific and Technological

Development (CNPq, Project #301976/2011-2, 472906/2011-9, 301198/2009-8,

472909/2011-8) and by Fundaçãode Amparo a Pesquisa do Estado de Goiás (FAPEG-

PRONEX). ACC received a PhD fellowship from CNPq. AOCJ, DPR, and FMO each

received a MSc fellowship from CNPq. SVN received a Post Doc fellowship from CNPq.

JDR received an undergraduate fellowship from PIBIC-CNPq. The funders had no role in

study design, data collection and analysis, decision to publish, or preparation of the

manuscript.

45

Competing Interests: The authors have declared that no competing interests exist.

* Email: apkipnis@gmail.com

Introduction

Tuberculosis (TB) is a public health problem causing 8.6 million new cases and 1.3

million deaths annually [1]. The causative agent of TB is Mycobacterium tuberculosis

(Mtb), an intracellular pathogen that after infecting the host can either cause active disease

or remain latent. In this context, it is estimated that one third of the world population is

latently infected with Mtb, of which approximately 10% will develop active disease [2,1].

Currently, the vaccine used for TB prevention is Bacillus Calmette-Guérin (BCG), an

attenuated Mycobacterium bovis strain used since 1921 [3]. Despite being the only

approved vaccine for human use and conferring protection against tuberculous meningitis

and miliary TB in children, its protective efficacy remains questionable, as it does not

protect young adults against pulmonary TB [4,5,6].

The factors determining the variable protective mechanisms induced by BCG are not

well understood. Some suppositions point towards the BCG sub-strain characteristics. It has

acquired genotypic and phenotypic differences, such as residual virulence and epitope

number variation, after the attenuation process and the several sub-culturing passages made

through the years [7–8]. In addition, BCG has limited capacity to induce long lasting

memory and, in humans, the vaccine induces an immune response with Th1 effector cells

producing IFN-γ [9,10,11]. Although IFN-γ is crucial for the immune response to Mtb,

studies have shown this cytokine is not a surrogate marker of the protection conferred by

BCG [12,11]. To address this matter, several groups have been working on the

development of protein subunit vaccines, new adjuvants, attenuated/auxotrophic Mtb

strains, and recombinant BCG (rBCG) vaccines, among other approaches [13–14].

Different strategies are being used by the groups modifying BCG, such as the expression of

immunodominant Mtb antigens [15], the association of re-introduction and super-

expression of antigens lost during the process of BCG attenuation [16], the development of

rBCG expressing cytokines and Mtb proteins [17], and the heterologous expression of

proteins in rBCG to induce CD8+ T lymphocytes [18].

46

While evaluating the rBCG vaccines produced in the last five years, it was observed

that the selection of Mtb antigens used in the construction of the rBCG was more important

for vaccine efficacy than the BCG subtypes used to make them [19]. However, comparing

the BCG subtypes used to construct recombinant vaccines, sub strains BCG Tokyo and

BCG Moreau presented more immune dominant epitopes than the other sub strains, and all

rBCG produced using the Tokyo strain protected better than the wild type BCG [20].

Sequencing of the complete genome and an evaluation of the proteome profile of BCG

Moreau were performed, but this strain was poorly used to build a TB recombinant vaccine

[21–22]. Some studies have shown that BCG Moreau is a good carrier and efficiently

induces a specific immune response to other diseases, such as pertussis, entero- pathogenic

Escherichia coli, or bladder cancer [23–25]. BCG Moreau has been used for more than 80

years in Brazil, attesting to its safety. This strain is currently being tested again as oral

vaccine and is showing better performance than BCG Danish [26]. This prompted us to

develop a recombinant TB vaccine using the BCG subtype Moreau.

Most of the time, the choice of the antigens used to develop an rBCG is based on the

different phases of Mtb infection. Active and latent TB are distinct phases of the disease

that can be characterized by their antigen expression, and these antigens are effective at

inducing an immune response [27]. Our group and others have demonstrated that patients

with active pulmonary TB and latently infected individuals respond differently to several

Mtb antigens, such as antigen 85 complex proteins, MPT51 and HspX [28–31]. In our

previous work, we developed a fusion protein (CMX) composed of the immunodominant

antigens from Mtb: Ag85C, MPT51 and the entire HspX protein, [32] which are expressed

in different stages of TB (active and latent phases of the disease). This construction retained

the immunogenicity of the original proteins in vaccinated mice and was also specifically

recognized by individuals with active TB [32]. To determine if this fusion protein could be

expressed by a live vector, and consequently be used as a vaccine for TB, a recombinant

Mycobacterium smegmatis (mc2) was designed to express CMX (mc2-CMX). This vaccine

induced a specific immune response to CMX that culminated with protection similar to that

observed following vaccination with BCG Moreau [33]. In this scenario, the fusion protein

CMX was capable of adding important immunogenic properties to mycobacterium vaccine

vectors, inducing an effective response to control Mtb infection in mice.

47

Based on the previous studies, both ours and others, we hypothesize that using BCG

subtype Moreau to develop a new rBCG expressing CMX will add immunological

characteristics that are missing in conventional BCG and therefore induce an specific

immune response better able to control the infection by Mtb. Our data here show that the

expression of CMX protein by the rBCG Moreau vaccine (rBCG-CMX) is a determining

factor for inducing specific Th1 and Th17 responses, in addition to polyfunctional T cells.

These responses may be responsible for the reduction in the inflammatory lung lesions

induced by Mtb challenge in BALB/c mice and the reduction in the bacterial load.

Moreover, prime vaccination with rBCG-CMX followed by boosting with rCMX further

reduced the lung bacterial load as compared to the reduction caused by BCG Moreau.

Materials and Methods

Bacterial strains, growth conditions, and plasmid and vaccine preparations

The M. bovis BCG Moreau strain, kindly provided by the Butantan Institute, was

grown in 7H9 media (Becton and Dickinson, Le pont de Claix-France) supplemented with

10 oleic acid, albumin, dextrose and catalase (OADC-Becton and Dick- inson, Le pont de

Claix- France), 0.5% glycerol and 0.05% Tween 80, at 37 oC in a humid atmosphere and

5% CO2 for approximately three weeks. The recombinant BCG strains were obtained after

electroporation of BCG Moreau with one of the three expression plasmids (pLA71, pLA72,

and pMIP12). These plasmids have mycobacteria and Escherichia coli replication origins

and use the gene for kanamycin resistance as a selection marker, as described by Varaldo et

al. (2004) [34]. The gene coding for the fusion CMX protein (Ag85C, MPT51, and HspX)

[32] was obtained from Mtb (H37Rv) DNA and inserted in the pLA71, pLA73 and pMIP12

mycobacteria expression vectors as described by Junqueira-Kipnis et al. (2013) [33]. The

employed expression plasmids enable the recombinant gene to be expressed with either the

signal peptide of the β-lactamase from M. fortuitum (pLA71) or the entire β-lactamase

protein (pLA73), or, alternatively, the protein can be highly expressed intracellularly

(pMIP12). Transformants with empty plasmids were used as controls. The recombinant

vaccines obtained were cultured under the same conditions as the BCG Moreau described

above, with the addition of 20 mg/mL of kanamycin. The vaccines were grown in a single

48

lot in 7H9 supplemented with OADC, and the concentration of the lots were determined by

plating serial dilutions of each vaccine onto 7H11 agar plates with or without kanamycin

(20 mg/mL).

Animals

BALB/c female mice, 4 to 8 weeks of age, from the Instituto de Patologia Tropical e

Saúde Pública/UFG animal housing were maintained in micro-isolators equipped with

HEPA filters for air intake and exhaustion, and provided with water and a chow diet ad

libitum. The room temperature was kept at 20–24 °C with a relative humidity of 40–70%

and light/dark cycles of 12 hours. Mice were handled according to the Sociedade Brasileira

de Ciência em Animais de Laboratório (SBCAL/COBEA) guidelines. The study was

approved by the Ethical Committee for Animal use (CEUA: Comite de Ética no uso de

animais; #229/11) of the Universidade Federal de Goiás.

PCR and Western blotting

To confirm the presence of the CMX fusion gene (˜860 base pairs), a PCR reaction

using Ag85C forward (5’ggtctgcgggcccaggatg 3’) and HspX reverse (5’

tcagttggtggaccggatctgaatgtg 3’) primers (10 nmol of each) in the same conditions as

described previously [32]. The expression of CMX (˜35 kDa) by the different vectors was

assessed by Western blot, as described previously [33].

Assessment of in vivo plasmid stability

BALB/c mice were immunized with 106 colony forming units (CFU) of rBCG-

pLA71-CMX or rBCG-pLA71 subcutaneously in the dorsal region. Animals were

euthanized at different time points (3 mice/group/time point) and the dorsal tissue at the

injection site was cut out and macerated. The homogenized tissue was plated onto 7H11

agar supplemented with OADC, 0.5% glycerol and 20 mg/mL of kanamycin. After

incubation at 37°C with 5% CO2 for approximately 30 days, the plates were analyzed for

49

bacterial growth and the numbers of CFU were determined. The DNA from a representative

colony was extracted by boiling the entire colony, and the supernatant was submitted to

PCR for detection of the CMX gene. This experiment was repeated three times.

Mouse peritoneal macrophage preparation, culture, and infection

Peritoneal macrophages were obtained after injection of 1 mL of thioglycolate into the

peritoneal cavity of BALB/c mice four days prior to macrophage collection. Mice were

euthanized by cervical dislocation and 5 mL of ice cold phosphate buffered saline (PBS)

was injected into the peritoneal cavity, followed by vigorous massage. The recovered cells

were distributed in a 24 well plate at a concentration of 1x106 cells per mL and incubated

with 5% CO2 for 24 hours to allow for adherence. In some of the cultured wells, circular

glass cover slides were introduced to allow for microscopic evaluation. Macrophages were

infected with BCG or rBCG-CMX at a multiplicity of infection (MOI=10) or incubated

with LPS (5 mg/mL), as a control. Infected macrophages were incubated at 37°C with 5%

CO2. After 3 hours, the supernatant was discarded, the cells were washed, and new media

was added to the wells. After 18 hours, the supernatant was collected and plated on 7H11

agar plates to determine the number of bacteria that were not phagocytosed. Infected

macrophages were washed three times with RPMI medium (HIMEDIA, Mumbai-India) and

then lysed with water and plated on 7H11 agar to determine the level of CFU of the

intracellular bacteria. Some of the infected macrophages were kept for an additional

incubation of 48 hours and used for nitric oxide (NO) quantification of the supernatant by

the Griess method, as described below. The cover slides from macrophages infected for 3

hours were washed three times with PBS at 37°C, fixed with methanol and stained with

Ziehl Neelsen, for acid fast bacilli visualization, or Instant Prov (Newprov, Pinhais- Brazil),

for cell visualization.

Nitric oxide determination

Supernatants (100 mL) from macrophage cultures that had been stimulated or not

(control) with BCG, rBCG, or LPS were stored in a 96 well plate at –20°C until use. Fifty

50

microliters of the supernatant was transferred to another 96 well plate and 50 mL Griess

reagent (1% sulphanilamide, 2% phosphoric acid, and 0.1% naphthylethylene diamine

dihydrochloride) was added, followed by 15 minutes of incubation at room temperature,

protected from the light. A serial dilution of nitrite was included in additional wells to

provide a standard curve for comparison. The absorbance was measured in a

spectrophotometer (Thermo LabSystems Multiskan RC/MS/EX Microplate Reader) at 595

nm.

BCG and rBCG-CMX immunizations

BALB/c mice were separated into three groups: Control, BCG Moreau, and rBCG-

CMX. Five to six animals were used in each group. Prior to use, the vaccines were thawed

and the concentrations adjusted with PBS/0.05% Tween 80, so that each animal would

receive 106 CFU in 100 L by subcutaneous injection in the dorsal region. The vaccine

concentrations were confirmed by plating the remaining inocula on 7H11 agar

supplemented with OADC. An additional group of animals, previously vaccinated with

rBCG-CMX (N = 5) was given a booster, 30 days later with 20 mg/mL of rCMX/CPG

DNA prepared as described at de Souza et al, 2012 [32]. This experiment was repeated two

times in BALB/c mice and one time in C57BL/6 mice.

Cell preparation for immune response evaluation

Thirty days after immunization, six animals from each group of the BALB/c mice

were euthanized and the spleens and left lung lobes were collected. Spleens were prepared

into single cell suspensions using 70 mm cell strainers (BD Biosciences, Lincoln Park, NJ)

and the cells were resuspended with RPMI medium. Erythrocytes were lysed with lysis

solution (0.15 M NH4Cl, 10 mM KHCO3) and the cells were washed and resuspended

with RPMI supplemented with 20% fetal calf serum, 0.15% sodium bicarbonate, 1% L-

glutamine (200 mM; Sigma-Aldrich-Brazil, São Paulo), 1% non-essential amino acids

(Sigma-Aldrich). Cells were counted in a Neubauer chamber and the concentration was

adjusted to 1x106 cells/mL. Prior to collection, the lungs were perfused with ice-cold PBS

51

containing 45 U/mL of heparin (Sigma-Aldrich-Brazil, São Paulo) and processed as

described previously [33]. The lungs were digested with DNAse IV (30 mg/mL; Sigma-

Aldrich) and collagenase III (0.7 mg/mL; Sigma- Aldrich-Brazil, São Paulo) for 30 min

at 37°C. The digested lungs were prepared into single cell suspensions using 70 mm cell

strainers and submitted to erythrocyte lysis. The cells were washed and resuspended with

RPMI, and the concentrations were adjusted to 1x106 cells/mL.

Ag85 (Rv0129c), MPT51 (Rv3803c), HspX (Rv2031c) and CMX specific

cytokine evaluation by lung and spleen lymphocytes

In a 96 well cell culture plate (CellWells TM), 200 mL of spleen or lung cell

suspensions were cultivated without (media alone) or with recombinant CMX or

with only one of the component recombinant proteins, Ag85, MPT51 or HspX

(single proteins were used at a concentration of 10 mg/mL) or ConA (positive

control) in a 5% CO2 incubator at 37°C for 4 hours. Monensin (3 mM;

eBioscience) was then added to the wells and the cultures were further incubated for

4 hours. Cells were treated with 0.1% sodium azide in PBS for 30 min at room

temperature. After centrifuging, the cells were stained with anti-CD4 Percp

(eBioscience, clone RM4-5) or anti-CD4 FITC (BD PharMingen, clone RM4-5) for

30 min. Cells were then, permeabilized with Perm Fix/Perm Wash (BD

PharMingen), washed with 0.1% sodium azide in PBS, and then stained with the

following antibodies to access the expressions of a panel of Th1 cytokines: anti-

TNF-α FITC (BD PharMingen-MP6; clone: XT22), anti-IL-2PE (eBioscience-JES6;

clone: 5H4), and anti-IFN-γ APC (eBioscience; clone: XMG1.2). To access the

expression of a panel of Th17 cytokines, cells were stained with: anti-IL-2 PE

(eBioscience, clone: JES6-5H4), anti-IL-17A Percp (eBioscience, clone: eBio17B7),

and anti-IFN-γ APC (eBioscience, clone: XMG1.2) for 30 min. Cell acquisition of

100,000 events per sample was performed in a BD FACS Verse (Universidade de

Brasília-UNB) flow cytometer and the acquired data were analyzed using FlowJo

8.7 software. Lymphocytes were selected based on their size (Forward scatter, FSC)

and granularity (side scatter, SSC). The specific immune responses were determined

52

by subtracting the result of the media alone stimulation from the responses to each

of the antigens.

Mycobacterium tuberculosis intravenous infection

The Mycobaterium tuberculosis (H37Rv) strain was maintained as described

previously [33]. A vial from a constant lot was thawed and the inoculum was

adjusted to the concentration of 106 CFU/mL by diluting with PBS containing

0.05% Tween 80. Ninety days after immunization with rBCG-CMX or BCG

Moreau, 100 mL of the inoculum was injected into the retro-orbital plexus. The

bacterial load of infection was determined by plating the lung homogenates from

one mouse from each group on the day following infection on 7H11 agar

supplemented with OADC. Forty-five days after infection, mice were euthanized

and the anterior and mediastinal right lung lobes were collected, homogenized, and

plated on 7H11 agar supplemented with OADC. The bacterial load was determined

by counting the CFU after 21 days of incubation at 37°C.

Histopathology

The lungs of mice euthanized 45 days after the Mtb challenge were perfused

with 0.05% heparin by injection in the heart right ventricle. The posterior right lobes

were collected, conditioned in histological cassettes, and fixed with 10% buffered

formaldehyde. Samples were sectioned into 5 mm thick slices and stained with

hematoxylin and eosin (HE) for analysis via microscopy (Axio scope.A1- Carl

Zeiss). Scores for the observed lesions were determined based on the area with

lesions relative to the area of the total visual field. The results are presented as the

percentage of area with lesions. Three different fields were evaluated per slide for

each animal of each group.

Statistical analysis

53

The data were analyzed using Microsoft Office Excel 2011 and Prism (version 5.0c,

GraphPad) software. The results represent the mean and standard deviation for each

experimental group. The results from rBCG-CMX and BCG groups were compared using

One-Way Anova followed by Dunnett’s post-hoc test. Values of p<0.05 were considered

statistically significant. All experiments repetition showed similar responses.

Results

1. Recombinant vaccine construction, rCMX expression analysis and in vivo plasmid

stability

The expression of heterologous proteins in mycobacteria can be influenced by several

factors such as administration dose, cellular localization, and expression stability, among

others [34–35]. To obtain the best possible expression, we tested three different plasmid

constructions to express CMX: pLA71/CMX, pLA73/ CMX, and pMIP12/CMX. As shown

in Figure 1A, all three constructions contained the CMX fused gene and were successfully

transformed into BCG Moreau. Western blot analysis of recombinant BCG cultures

revealed that only the plasmid pLA71/ CMX was capable of inducing the expression of

CMX protein (Fig. 1B, Figure S1). Thus we performed the following analysis only with the

recombinant vaccine rBCG-pLA71/CMX, hence- forward referred to as rBCG-CMX.

54

Figure 1. Plasmid construction and CMX expression for three different rBCG-CMX

vaccines. (A) PCR products corresponding to the CMX fusion gene, CMX (˜860 bp), from

all three plasmid constructions and their respective empty controls: pLA71, pLA73 and

pMIP12. M: molecular weight marker; NC: negative control reaction, using water; BCG:

55

DNA from BCG-Moreau; rBCG transformed with pLA71, pLA73 and pMIP12, with

(CMX) or without (empty) the fusion gene; PC: positive control reaction. (B) Analysis of

CMX expression in rBCG-pLA71/CMX. Western blot of BCG transformants containing

pLA71/CMX or empty vector using polyclonal antibody produced against rCMX. M:

molecular mass marker; CMX: purified recombinant CMX; pLA71/CMX: rBCG with

plasmid pLA71/CMX; pLA71: rBCG with plasmid pLA71.

doi:10.1371/journal.pone.0112848.g001

In order to verify the stability of the plasmid within the recombinant vaccine rBCG-

CMX in vivo without antibiotic selective pressure, mice were vaccinated subcutaneously

and the tissue of the site of infection was macerated at different time points and plated on

media with or without the selective antibiotic kanamycin. As shown in Figure 2, the

number of CFU recovered from media with or without antibiotic was similar, indicating

that the recombinant vaccine recovered from mice retained the plasmids up to 15 days after

immunization (Figs. 2A and B). The presence of plasmid was further confirmed by

performing PCR specific to the CMX gene (Fig. 2C).

56

Figure 2. Stability of rBCG-CMX in vivo. (A) Images of plates showing the

mycobacterial growth of rBCG-CMX recovered from the dorsal region of mice 5, 10 and

15 days after subcutaneous immunization, and plated on media with kanamycin (kan) or

without (W/o). (B) CFU counts recovered at different time points from the dorsal region of

57

mice after immunization. (C) CMX gene detection by PCR for three isolated colonies from

plates W/o kanamycin (Lanes 3–5). Lanes 1: M: molecular weight marker; 2: Negative

control: NC: water. doi:10.1371/journal.pone.0112848.g002

2. rBCG-CMX is phagocytosed by peritoneal macrophages at higher levels than BCG

Moreau but induces similar levels of NO

Vaccine phagocytosis and processing to present antigens has been shown to be an

important factor responsible for the capacity to induce a protective immune response [36].

Thus the tendency of peritoneal macrophages to phagocytose rBCG-CMX was analyzed

(Fig. 3). After 18 hours of infection, the recovered CFU from rBCG-CMX infected

macrophages was higher than that from BCG Moreau infected macrophages (Fig. 3A,

p<0.01). Acid fast staining of infected macrophage cultures confirmed that peritoneal

macrophages had higher numbers of rBCG-CMX than of BCG (Fig. 3C). The analysis of

CFU from culture supernatants confirmed that the groups were equally infected by the

different vaccines (Fig. 3A).

Phagocytosis induces respiratory burst activation with its consequent production of

NO. As another way to evaluate phagocytosis rates, the production of nitric oxide (NO) was

evaluated in the culture supernatant of infected peritoneal macrophages. No difference in

NO production was observed between the two vaccines evaluated (Fig. 3B). Despite the

increased number of bacilli inside the peritoneal macrophages infected with rBCG-CMX,

similar levels of NO were induced by both vaccines.

58

Figure 3. Levels of phagocytosis by peritoneal macrophages of BCG and rBCG-CMX

59

after infection (MOI = 10). (A) Macrophages were infected with BCG or rBCG-CMX and

the bacterial load in both the supernatant (sup) and inside the macrophages (MΦ) were

determined. The amount of viable bacteria was determined by plating supernatant or cell

lysates onto 7H11 agar supplemented with OADC and counting the CFU 28 days after

incubation at 37°C. *(p<0.01) significant difference between the compared groups (log10

scale). (B) Nitric oxide (NO) production by macrophages infected with BCG or rBCG-

CMX was determined. Uninfected media (Control) and LPS-stimulated (LPS) macrophages

were included as controls. (C) Microscopic evaluation of peritoneal macrophages, 3 hours

after infection with BCG or rBCG-CMX stained with Instant Prov or Ziehl Neelsen.

Uninfected macrophages (Control) were included as a negative control. The results shown

are representative of three different experiments. doi:10.1371/journal.pone.0112848.g003

3. rBCG-CMX vaccine induces a specific cellular immune response

Since rBCG-CMX was stable in vivo and phagocytosis of it induced macrophage

activation (as measured by NO production), we questioned whether this vaccine would be

able to induce a specific response to CMX and/or to the recombinant antigens alone (Fig.

4A). Immunization with rBCG-CMX vaccine induced higher numbers of CD4+ T

lymphocytes positive for IFN-y specific for CMX in cells from the spleen and lungs of

BALB/c immunized mice 30 days after vaccination than did immunization with BCG

Moreau (Figs. 4B and C, p<0.05; Th1 representative dot plots in Figure S2A). Similarly,

rBCG-CMX induced higher levels of specific Th17 cells, an important group of cells for

protection from Mtb and development of memory, in the cells from the spleen and lungs of

immunized mice (Figs. 4D and E, p<0.05; Th17 representative dot plots in Fig. S2B).

60

Figure 4. Immunogenicity of rBCG-CMX in BALB/c mice. (A) Experimental time line.

BALB/c mice were immunized with rBCG-CMX or BCG Moreau. Thirty days later, 6 mice

per group were euthanized for evaluation of vaccine-induced immunogenicity. Ninety days

after immunization, mice were intravenously (i.v.) challenged with 105 CFU of H37Rv.

Forty-five days after i.v. challenge, the lung bacterial load (CFU) and lesions (H&E) were

assessed. (B–E) Specific cellular immune responses induced with rCMX stimulation ex

61

vivo. Spleen (B and D) and lung (C and E) cell suspensions from vaccinated and

unvaccinated (Control) mice were stimulated with rCMX. Cells positive for both CD4 and

IFN-y (B and C) or CD4 and IL-17 (D and E) were determined by flow cytometry.

Lymphocytes were selected based on size and granularity. Flow cytometry gates were set to

analyze CD4+ T cells, and then the fluorescence of antibodies detecting IFN-y+ or IL-17+

cells was recorded. These data are representative of two independent experiments

(N=6,*p<0.05). doi:10.1371/journal.pone.0112848.g004

We next questioned which protein(s) of the recombinant CMX fusion protein could

contribute to the induction of IFN-y (Fig. 5) and/or IL-17 (Fig. 6) by CD4+ T lymphocytes.

As depicted in Figure 5, ex vivo stimulation of spleen and lung cells from rBCG- CMX

vaccinated mice with Ag85, MPT51, or HspX all specifically induced CD4+IFN-y+ cells

(Figs. 5A and C, p<0.05). A significantly higher number of spleen cells were observed

responding to MPT51 than to Ag85 or HspX. In mice vaccinated with BCG, cells that were

CD4+IFN-y+ were only induced in response to Ag85 and MPT51 stimulation, but not in

response to HspX. Additionally, these CD4+IFN-y+ cells were induced to a lesser extent

than in the spleen or lung cells from mice vaccinated with rBCG-CMX (Figs. 5B and D,

p<0.05).

62

Figure 5. Levels of CD4+IFN-y+ T cells induced by ex vivo stimulation with

recombinant Ag85, MPT51, and HspX. Thirty days after vaccination, lung and spleen

suspensions were stimulated ex vivo with Ag85, MPT51, HspX, or medium alone. The

number of cells positive for CD4 and IFN-y was determined by flow cytometry.

Lymphocytes were selected based on size and granularity. Gates were set to analyze CD4+

T cells, and then the fluorescence of antibodies detecting IFN-y+ cells was recorded. (A–B)

Spleen cells from mice vaccinated with (A) rBCG-CMX or (B) BCG. (C–D) Lung cells

from mice vaccinated with (C) rBCG-CMX or (D) BCG. In A and C, all results were

different from the medium stimulation. These data are representative of two independent

experiments (N=6,*p<0.05). doi:10.1371/journal.pone.0112848.g005

Upon antigen stimulation in cells from rBCG-CMX-immunized mice, high numbers

of Th17 lymphocytes were induced. In spleen cells, the highest response was to MPT51

63

(Fig. 6A), while in the lungs, all antigens stimulated the production of IL-17 to a similar

degree (Fig. 6C). The number of CD4+IL-17+ spleen cells responding to MPT51 was

significantly higher than the amount of cells responding to Ag85 antigen (Fig. 6A, p<0.05).

Th17 expression was not induced in spleen or lung cells from mice vaccinated with BCG

when stimulated with any of the recombi- nant proteins (Ag85, MPT51 and HspX) (Figs.

6B and D).

Figure 6. Levels of CD4+IL-17+ T cells induced by ex vivo stimulation with

recombinant Ag85, MPT51, and HspX. Thirty days after vaccination, lung and spleen

suspensions were stimulated ex vivo with Ag85, MPT51, HspX, or medium alone. The

number of cells positive for CD4 and IL-17 was determined by flow cytometry.

Lymphocytes were selected based on size and granularity. Gates were set to analyze CD4+

T cells, and then the fluorescence of antibodies detecting IL-17+ cells was recorded. (A–B)

Spleen cells from mice vaccinated with (A) rBCG-CMX or (B) BCG. (C–D) Lung cells

64

from mice vaccinated with (C) rBCG-CMX or (D) BCG. In A and C, all results were

different from the medium stimulation. These data are representative of two independent

experiments (N = 6, *p<0.05). doi:10.1371/journal.pone.0112848.g006

Although the above experiments determined that the rBCG-CMX vaccine generates

Th1 (IFN-y) and Th17 specific responses, it remained important to verify the induction of

polyfunctional CD4+ T cells, since several publications have associated these cells with

protection against Mtb [33,37]. Ex vivo stimulation of spleen and lung cells with CMX

increased the numbers of CD4+ T cells positive for both IL-2 and IFN-y (Figs. 7A and C) as

well as for both TNF-α and IFN-y (Figs. 7B and D) in cells from rBCG-CMX vaccinated

mice as compared to the levels in cells from BCG Moreau vaccinated mice.

65

Figure 7. Levels of polyfunctional CD4+ T cells induced by BCG and rBCG-CMX

vaccines. Spleen (A and C) and lung (B and D) cell suspensions from vaccinated and

control mice stimulated with rCMX. (A–B) CD4+IL-2+IFN-y+ cells or (C–D) CD4+TNF-

α+IFN-y+ cells were analyzed by flow cytometry. Lymphocytes were selected based on size

and granularity. Gates were set to analyze CD4+ T cells, and then the fluorescence of

antibodies detecting IL-2+ and IFN-y+ or TNF-α+ and IFN-y+ cells was recorded. These data

are representative of two independent experiments (N = 6, *p<0.05).

doi:10.1371/journal.pone.0112848.g007.

66

4. rBCG-CMX reduces the lung bacterial load

Although we found that immunization with rBCG-CMX was capable of inducing a

specific immune response to CMX in BALB/c mice, this response alone is not sufficient to

predict the protection properties of a vaccine. Thus, immunized mice were challenged with

Mtb and the protective capacity was evaluated by assessing the bacterial load 45 days later.

As observed in Figure 8, mice immunized with rBCG-CMX had a significantly lower

bacterial load in the lungs than the unimmunized mice. To test if the protection could be

improved in a prime-boost strategy, rBCG-CMX immunized mice were boosted 30 days

later with rCMX/CPG DNA vaccine formulation and challenged with Mtb. Surprisingly, a

boost with rCMX subunit vaccine showed the lowest lung bacterial load at 45 days post

Mtb infection (Fig. 8).

67

Figure 8. Bacterial load in the lungs of BALB/c mice 45 days after Mycobacterium

tuberculosis challenge. Ninety days after immunization, three mice from each group

(control, BCG and rBCG-CMX) were challenged with 105 CFU of Mycobacterium

tuberculosis H37Rv intravenously into the orbital sinus plexus. One additional group of

animals received a booster of rCMX/CPG DNA, 30 days after rBCG-CMX vaccination and

challenged with Mtb 30 days post the immunization (rBCG-CMX+CMX). Forty-five days

after challenge, mice were euthanized and the anterior and mediastinal right lung lobes

were collected, homogenized, and plated on Middlebrook 7H11agar supplemented with

OADC to determine the bacterial load by counting the number of CFU. *Significant

differences between infected (control) and vaccinated groups. #Significant differences

between rBCG-CMX and rBCG-CMX+CMX groups. ★Significant differences between

rBCG-CMX+CMX and BCG groups analyzed by t test (p<0.05).

doi:10.1371/journal.pone.0112848.g008

68

5. The immune response induced by the rBCG-CMX vaccine reduces TB

pulmonary lesions

Lung architecture preservation is yet another important aspect of a successful

vaccine against TB. Histological analysis of the lungs of vaccinated mice challenged with

Mtb showed that 45 days after challenge, unimmunized mice had intensive lymphocytic

and neutrophilic infiltrates, significantly compromising the lung tissue architecture,

together with the presence of a few hemorrhagic foci and foamy macrophages (Fig. 9A).

BCG-vaccinated mice, instead, showed significantly fewer lung lesions, with a

preservation of alveolar spaces and very limited lymphocytic infiltrate foci (Fig. 9B).

The recombinant vaccine greatly preserved the lung architecture, showing very few

inflammatory infiltrates (Fig. 9C). Similar results were obtained for animals immunized

with rBCG-CMX and boosted with rCMX (Data not shown). The differences in

inflammatory responses upon Mtb challenge between all three groups are summarized in

the scores of their lung lesions, which are presented in Figure 9D.

69

Figure 9. Representative lung pathology of Balb/c mice after challenge. Vaccinated

mice were challenged i.v. with 105 CFU of virulent M. tuberculosis H37Rv strain. Forty-

five days after infection, lung tissue sections from different vaccine groups were harvested.

Images are representative of two distinct experiments. HE staining is shown with 20X

magnification. (A) Unvaccinated group. Black arrowheads: Foamy macrophages. (B) BCG-

vaccinated group. (C) rBCG-CMX vaccinated group. (D) Histological score of the lesion

area from three representative fields obtained by AxioVision 4.9.1 software, through ratio

of lesioned and total field area. Data are presented as percentages (%).

doi:10.1371/journal.pone.0112848.g009

70

Discussion

In this study, a recombinant vaccine expressing the fusion protein CMX (rBCG-

CMX) was used to immunize BALB/c mice and was shown to be efficient in protecting

mice against Mtb challenge. The recombinant vaccine induced higher levels of CD4+IFN-y+

and CD4+IL-17+ T cells, as well as higher levels of CD4+TNF-α+IFN-y+ and CD4+IL-

2+IFN-y+ polyfunctional T lymphocytes specific for CMX in BALB/c mice.

During the attenuation process of BCG, some antigens important for the induction of

a protective immune response were lost [38]. This is thought to be one reason that BCG

does not provide long lasting protection in humans. In the pursuit of a new TB vaccine,

several groups have tried to insert heterologous genes into BCG and in doing so many

different expression systems have been tested [35]. Our approach was to test three different

plasmid constructions to express the fusion protein CMX. Of the systems we tested, only

the one that expressed the recombinant fusion protein together with the signal peptide b-

lactamase (pLA71) was successful and stable in vivo. Other antigens have been expressed

with the same three plasmids used in this study, but with different results. For example, the

Schistosoma mansoni antigen Sm14 [34] and the pertussis toxin subunit S1 [39] were only

successfully expressed in plasmid pLA73, which expresses the recombinant gene with the

entire b-lactamase protein.

Macrophages infected with wild type BCG or rBCG produced similar amounts of

NO. In the murine model, the production of NO has been shown to be critical for the

control of mycobacterial growth [40]. Although NO production helps to control the

progression of infection, its effects are concentration dependent. In low doses, NO acts as a

signaling molecule to promote vascular integrity, mediate neurotransmission, and help

regulate cellular respiration. In high concentrations, NO inhibits respiration and can cause

protein and DNA damage [41–42]. In M. bovis BCG, NO seems to limit inflammatory

responses, in part by down- regulating the accumulation of activated T cells [43]. We found

that a significant amount of rBCG-CMX was phagocytosed, and that it can reside and

survive within the macrophages (Fig. 3). Our data show that rBCG-CMX was phagocytosed

in higher amounts than BCG Moreau (Figs. 3A and B). However, the induction of NO by

71

the recombinant vaccine was similar to that induced by BCG Moreau. These data suggest

that our recombinant vaccine is viable, since it has not lost its ability to induce an immune

response.

After finding that rBCG-CMX was efficiently phagocytosed, we anticipated that

antigen processing and presentation to naive T lymphocytes in vivo would be favored, and

data from our next experiment support this idea. In cells from the lungs and spleen of

rBCG-CMX vaccinated mice, stimulation with CMX induced high levels of T cells that

were CD4+IFN-y+ (Figs. 4B and C). The importance of IFN-y in protection against TB is

well established, as it induces an increase in phagocytosis and Mtb destruction,

consequently reducing the bacterial load [44]. In spite of this, there is controversy about the

role of IFN-y in vaccine models. In high concentrations, IFN-y induces apoptosis of CD4+

effector T lymphocytes, lowering the potential to generate memory cells [11].

Th17 cells are thought to be responsible for TB protection, as they have an early

memory cell signature [45]. Our recombinant vaccine was shown to induce CMX-specific

CD4+IL-17+ T cells in the spleen and lungs (Figs. 4D and E). The expression of CMX by

Mycobacterium smegmatis (mc2-CMX) was also shown to induce high levels of CD4+IL-

17+ T lymphocytes in the spleen and lungs [33] that directly correlated to protection. The

importance of Th17 in vaccine models and in TB is controversial, but it is known that in

chronic infections, such as TB, constitutive or late IL-17 production is related to the degree

of interstitial inflammatory involvement and tissue lesion [46]. Instead, when produced

early as is the case for vaccination, IL-17 is important for the induction of protective

memory cells for TB [47,45].

Our vaccine, rBCG-CMX, induced Th1 and Th17 immune responses that were

specific to CMX. Furthermore, we demonstrated that this recombinant vaccine induced Th1

and Th17 immune responses to each of the CMX component proteins, rAg85, rMPT51, and

rHspX, alone (Figs. 5A and C; Figs. 6A and C). The induction of immune responses to

these proteins suggests that the construction of the CMX protein retained the

immunodominant characteristics of its components. It is important to note that the use of

recombinant BCG vaccines described in the literature, most of the times did not test the

specific immune response to the heterologous antigens [48–49]. Most studies only

72

evaluated the specific response to PPD (Purified Protein derivative) as a stimulus, and here

we showed that an immune response was generated to the heterologous protein [49].

It has already been shown that rBCG expressing HspX or Ag85 complex proteins

(Ag85A, Ag85B, Ag85C) induce superior protection to wild type BCG [50–52].

Interestingly, we found a pronounced response to stimulation with MPT51. This protein

belongs to new family of non-catalytic alfa/beta hydrolases (Fbpc1) which act in binding

the fibronectin extracellular matrix [53]. As demonstrated by another study, MPT51

effectively induces Th1 immune responses, promoting protection in mice challenged with

Mtb [54]. The characteristics of these proteins were retained in CMX, which contributed to

the ability of rBCG-CMX to promote important immune responses and protection.

In spleen and lung cells from mice immunized with the BCG vaccine, stimulation

with rAg85 and rMPT51 induced a Th1 response, but not stimulation with HspX or CMX

(Figs. 5A and B). This may be related to the poor ability of BCG to induce specific

responses to certain proteins, such as HspX which is expressed in low levels by BCG

[55,50,56]. Interestingly, despite containing the same original proteins as those composing

the CMX protein, immunization with BCG Moreau did not induce a specific response

against CMX. Additionally, BCG was not able to induce a Th17 immune response to any of

the component recombinant proteins (Figs. 6A and B). Although it has previously been

shown that Th17 responses generated by BCG vaccination induce TB infection control in

non-human primates [47], we did not observe similar results with our recombinant proteins.

We found an increased number of CD4+ polyfunctional T cells among mice

immunized with rBCG-CMX relative to the number in those who received the BCG

Moreau vaccine. The recombinant vaccine induced high levels of polyfunctional T cells

expressing both IL-2 and IFN-y (Figs. 7A and B) and high levels of these cells expressing

both TNF-α and IFN-y (Figs. 7C and D). It has been demonstrated that polyfunctional cells

are important for protection against intracellular bacteria, as well as viral, parasitic, and

chronic bacterial infections, such as TB [57,37]. Additionally, it has been shown that

polyfunctional cells are involved in providing protection against TB [37]. Consequently, we

believe that the cellular profile induced by rBCG-CMX is likely the result of our addition of

CMX to BCG [32].

73

The ability of rBCG-CMX to induce protection against Mtb challenge showed a

tendency to improve the protection conferred by BCG Moreau. Vaccination with rBCG-

CMX significantly reduced the lung bacterial load of BALB/c mice (Fig. 8). Because the

only difference between the vaccines were the presence of CMX, we decided to address if

using a booster with rCMX would increase the immune response to CMX, and

consequently the protection to Mtb. The improved protection observed (rBCG-CMX +

rCMX) must be due to the extra presence of the recombinant fusion protein CMX, as only

the recombinant vaccine induced a significant increase in the proliferation and migration of

specific CD4+ T cells in the spleen and lungs (Figs. 4 and 5) of immunized mice. Like in

here, not all recombinant BCG vaccines expressing fusion proteins that have been tested

were able to induce superior protection when compared with BCG [58]. Thus we believe

that the recombinant CMX protein, composed of Mtb immunodominant antigens (Ag85C,

MPT-51, and HspX) that relate to different infection phases, added significant

immunogenic properties to BCG which were crucial to the observed protection. This is the

first study using limited number of animals (3–6) to demonstrate the efficacy of the fusion

protein CMX. We are now setting up collaborations to test the CMX in a more appropriate

guinea pig model. Other studies from our group have characterized those properties by

investigating rCMX in the context of M. smegmatis mc2 155 (mc2-CMX). Additionally, we

observed this phenomenon with the IKE vaccine (IKE-CMX), which also induced a

significant reduction in bacterial load in comparison to vaccination with IKE lacking the

recombinant antigen [33]. Taken together, the data demonstrate that CMX can play an

important role in the enhancement of protective immune responses induced by vaccines

against Mtb [32–33].

Achieving the correct balance between the induction of Th1 and Th17 cells is an

important goal for an effective vaccine against Mtb. While the induced IFN-y will act on

the activation of infected cells, IL-17 will regulate the resulting inflammatory response by

inducing protective cells [49]. As shown in Figure 9, the lungs of mice vaccinated with

rBCG-CMX had a larger preserved area of the lungs compared to the lungs of BCG Moreau

immunized mice. In addition the group receiving the recombinant vaccine showed little

inflammatory infiltration and very few necrotic foci and coalescent alveoli, all of which are

known for being favorable areas for bacilli replication [59–60]. The reduced bacterial load

74

of the lungs found in rBCG-CMX challenged mice corroborates those observations (Fig. 8).

In addition, no foamy macrophages were found in the lungs of mice vaccinated with the

recombinant vaccine (Fig. 9), which is important as those cells are known to be bacilli

reservoirs [61–62].

In conclusion, the addition of the recombinant fusion protein CMX to BCG Moreau

generated a recombinant vaccine with superior immunological properties. This vaccine

induced a balanced IFN-y and IL17 cytokine response from CD4+ T cells and was able to

protect mice from Mtb.

75

Supporting Information

Figure S1. CMX expression analysis from rBCG transformed with recombinant

plasmids pLA73/CMX and pMIP12/CMX. Western blot analysis of whole cell lysates

from rBCG transformants using polyclonal antibodies raised against rCMX. (A) rBCG

containing pLA73/CMX or empty vector. M: molecular mass marker; CMX: purified

recombinant CMX; pLA73/CMX: rBCG with plasmid pLA73/CMX; pLA73: rBCG with

plasmid pLA73. (B) rBCG containing pMIP12/CMX or empty vector. M: molecular mass

marker; CMX: purified recombinant CMX; pMIP12/CMX: rBCG with plasmid

pMIP12/CMX; pMIP12: rBCG with plasmid pMIP12.

doi:10.1371/journal.pone.0112848.s001. (TIF)

76

Figure S2. Representative dot plots of TCD4+IFN-γ+ and TCD4+IL-17+ cells. Splenic

cells from non-immunized mice (Control) or mice immunized with BCG or with rBCG-

CMX were stimulated with medium or one of the following recombinant proteins: rAg85,

rMPT51, rHspX or rCMX. Lymphocytes were selected based on their size and granulocity

77

and antigen specific TCD4+IFN-γ+ (A) and TCD4+IL-17+ (B) cells were analyzed based on

their fluorescence. doi:10.1371/journal.pone.0112848.s002. (TIF)

Acknowledgments

We are thankful to Associação de Combate ao Câncer de Goiás and Universidade de

Brasília (UnB) for allowing access to their flow cytometry core facilities, to Dr. Aline

Carvalho Batista, from Faculdade de Odontologia – UFG, for collaborating in the

processing and analysis of histological preparations, and to Drs. Alexander Augusto da

Silveira and Lorena Cristina Santos for technical assistance in constructing the recombinant

vaccines.

Author Contributions

Conceived and designed the experiments: APJK AK. Performed the experiments: APJK

AK ACC AOCJ FMO SVN JDR DPR. Analyzed the data: APJK AK ACC AOCJ FMO

SVN JDR DPR. Contributed reagents/materials/analysis tools: APJK AK. Contributed to

the writing of the manuscript: APJK AK ACC AOCJ FMO SVN JDR DPR. Cytometry

experiments and analysis: ACC.

78

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Manuscrito

Modulation of the immune response induced by the recombinant fusion protein

CMX involves IL-6 and TGF-β production and TLR-4 stimulation

Adeliane Castro da Costa1, Danilo Pires de Resende1, Bruno de Paula Oliveira Santos1,

Karina Furlani Zoccal2, Lúcia Helena Faccioli2, André Kipnis1,3 and Ana Paula

Junqueira-Kipnis1,3*

1. Laboratório de Imunopatologia das Doenças Infecciosas, Instituto de

Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, Goiânia,

Goiás, Brazil.

2. Laboratório de Inflamação e Imunologia das Parasitoses, Departamento de

Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências

Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto,

Brazil

3. Laboratório de Bacteriologia Molecular, Instituto de Patologia Tropical e

Saúde Pública, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.

*Corresponding address: Rua 235 esquina com Primeira Avenida, S/N.

Laboratório de Imunopatologia das Doenças Infecciosas, sala 325. Instituto de

Patologia Tropical e Saúde Pública. Universidade Federal de Goiás. Setor

Universitário, Goiânia- Goiás, Brazil. CEP 74605-050. Phone: +55 62

32096174; Fax: +55 62 32096363; E-mail: apkipnis@gmail.com.

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Abstract

Tuberculosis (TB) is an infectious disease that can be prevented by the application of

the Mycobacterium bovis Bacillus Calmette–Guérin (BCG) vaccine. Due to the low

protection offered by this vaccine in adults, new, more effective formulations have been

developed with better potential for Th1 and Th17 response induction. A recombinant

BCG vaccine expressing the CMX fusion protein (rBCG-CMX) induced Th1 and Th17

responses and provided better protection than BCG. The aim of the present study was to

evaluate the innate immune response that promotes the induction of Th17 responses by

rBCG-CMX and that differentiates this formulation from the BCG vaccine. BALB/c

mice were intranasally infected with the BCG and rBCG-CMX vaccines, and after 4

days, flow cytometry of lung macrophages and ex vivo testing to determine IL-6, IL-1α

and TGF- levels were performed. RAW 264 and BMM cells as well as peritoneal and

alveolar macrophages from C57BL/6, BALB/c, TLR-2-/- and TLR-4-/- mice were also

used for in vitro tests. Compared to the BCG vaccine, the rBCG-CMX vaccine induced

more migration of F4/80+CD11bhigh macrophages to the lung. Pulmonary macrophages

from both groups expressed CD86 and CD206 and induced IL-1α and TGF-β. BMM

infected with rBCG-CMX underwent apoptosis and necrosis, whereas those infected

with BCG underwent necrosis. In addition, macrophages infected with rBCG-CMX

expressed more major histocompatibility complex class II (MHC-II) molecules. The

recombinant CMX (rCMX) protein activated the transcription factor NF-κB,

culminating in the production of IL-6, IL-1α and TGF-β in the BMM and peritoneal

and alveolar macrophages of BALB/c and C57BL/6 mice. Upon stimulation of the

BMM of TLR-2-/- and TLR-4-/- mice, we found that this activation is dependent on

TLR-4. We concluded that both the rBCG-CMX vaccine and the rCMX recombinant

fusion protein are capable of modulating and activating the innate immune response in a

TLR-4–dependent manner.

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Introduction

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that interacts with

macrophages and dendritic cells via receptors such as toll-like receptors (TLRs),

complement receptors (CR) and mannose receptors (CD206) [1-6]. Macrophages are

important innate immunity components in tuberculosis (TB) because they directly

participate in the response to this microorganism [7]. When recognized by phagocytes

through TLRs, Mtb induces the production of TNF-, IL-6, IL-12, IL-23 and IL-1α,

which contribute to the differentiation of Th1, Th17 and other lymphocytes, as well as

to the self-renewal processes of alveolar macrophages [8-12, 4, 13, 14]. Some Mtb

proteins, such as ESAT-6, PPE57, Ag85c and Rv0652, are recognized by TLR-2, CR3

and TLR-4, modulating macrophage responses [15, 2, 6]. ESAT-6, for example, has

been shown to modulate macrophages and dendritic cells in vitro, producing cytokines

that inhibit Th1 responses and that facilitate Th17 responses [16]. However, vaccine

models have attempted to find antigens capable of inducing a balance between Th1 and

Th17 cells, as it is postulated that a balance must exist between these cell populations to

control Mtb infection during development of the granuloma [17]. For this reason, it is of

the utmost importance to develop new vaccines using proteins that promote this

balanced response.

The BCG (Bacillus Calmette-Guérin) vaccine, which is the current vaccine used

to control TB, is believed to induce a strong Th1 response but a much lower Th17

response [18] . BCG is an attenuated strain of Mycobacterium bovis, which, during the

attenuation process, lost important virulence regions [19]. Despite being the only

vaccine approved for human use and providing protection against TB meningitis and

miliary TB in children, the protective effect of BCG remains questionable, as it does not

protect adults against pulmonary TB [20-22]. This lack of protection in the adult phase

may be due to its poor ability to induce a good vaccine response, with a balance

between Th1 and Th17 responses [23]. It has been demonstrated that the induction of

vaccine responses in mucosa may promote the induction of Th17 because mucosal

macrophages have an anti-inflammatory profile, with the ability to produce both TGF-β

and IL-6 [24]. Another mechanism that may promote the induction of Th17 is the

induction of apoptosis. The induction of cell death by apoptosis in macrophages can

promote the release of apoptotic bodies that induce the cross-presentation of vaccine

peptides by major histocompatibility complex class I (MHC-I) and class II (MHC-II)

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molecules by dendritic cells, promoting the induction of mixed TCD4+, TCD8+ and

Th17 responses. These responses have been demonstrated by some vaccines that have

shown superior protection to that induced by BCG [23].

The BCG expressing the CMX fusion protein (rBCG-CMX) vaccine and the

mc2-CMX vaccine have been able to induce Th1 responses and potentiate the induction

of Th17, thereby promoting an equilibrium in the induction of these cellular responses.

Regardless of the vehicle used, whether BCG or Mycobacterium smegmatis, vaccines

expressing the recombinant CMX protein (rCMX) have contributed to protection

against Mtb. Moreover, when used in another vector, IKE-CMX, rCMX has been able

to activate more macrophages than the vector alone [25, 26]. Thus, macrophages appear

to be involved in the protection conferred by the vaccine, indicating that rCMX may be

modulating the innate immune response and promoting the vaccine immune response.

The rCMX fusion protein is composed of immunodominant epitopes of the

antigens rAg85c, rMPT51 and full-length rHspX [27]. Ag85c (Rv0129c) and MPT51

(Rv3803c) are part of the same complex and are important virulence factors [28].

Ag85C, for example, participates in the synthesis of mycolic acid, a component of the

Mtb cell wall, which represents more than 40% of the dry weight of Mtb [29-31]. The

HspX antigen (Rv2031c) is a protein that promotes the growth of Mtb within

macrophages [32-34].

Based on this evidence, the aim of the present study is to evaluate whether the

rCMX protein acts on macrophages to promote the vaccine immune response. The

results demonstrate that the rCMX protein, when expressed by BCG (rBCG-CMX),

induces the activation of pulmonary and alveolar macrophages, with an increase in the

expression of activation molecules, as well as the induction of apoptosis and increased

MHC-II expression. The interaction of rCMX activates the transcription factor NF-κB

and induces the production of the cytokines TGF- and IL-6 via TLR-4.

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Material and Methods

Animals

Specific pathogen-free BALB/c and C57BL/6 mice were obtained from the Tropical

Institute of Pathology and Public Health, Federal University of Goiás (Universidade

Federal de Goiás – UFG). TLR-4 and TLR-2 mice (KO or -/-) from the School of

Pharmacy, Federal University of São Paulo (Universidade Federal de São Paulo – USP),

4-8 weeks old, were donated by S. Akira (Osaka University, Osaka, Japan). The animals

were kept at a constant temperature (24 ± 1°C) and humidity (50% ± 5%) in isolators

with HEPA filters during all experimental procedures. The animals were fed a sterile

diet specific to mice and were provided water ad libitum under controlled light

conditions (12-h light and dark period). Animals were monitored daily, and none of the

mice exhibited any symptoms of clinical disease, as assessed by the attending

veterinarian. Euthanasia was performed by cervical dislocation by a trained researcher.

The animals were handled according to the guidelines of the Brazilian Scientific Society

for the Use of Laboratory Animals (Sociedade Brasileira de Ciência em Animais de

Laboratório - SBCAL/COBEA). The study was approved by the Ethics Committee for

Animal Use (Comitê de Ética no Uso de Animais - CEUA; #229/11) of the UFG.

Antigens and vaccines

The recombinant antigens of Mycobacterium tuberculosis, rAg85c, rMPT51, rHspX and

rCMX, were produced in Escherichia coli in the Immunopathology and Infectious

Diseases Laboratory at the Institute of Tropical Pathology and Public Health (Instituto

de Patologia Tropical e Saúde Pública - IPTSP), UFG. All antigens were prepared as

previously described by de Souza et al. (2012) [27]. After purification of the rAg85c,

rMPT51, rHspX and rCMX proteins, they were subjected to an LPS-removal process

using a ToxinEraserTM Endotoxin Removal Kit. The procedure was performed

according to the manufacturer's instructions (GenScript - 860 Centennial Ave.,

Piscataway, NJ 08854, USA).

The strain of M. bovis BCG-Moreau, kindly donated by the Butantan Institute (Becton

and Dickinson, Le Pont de Claix-France), was grown in 7H9 liquid culture medium

supplemented with 10% oleic acid, dextrose and catalase (OADC), 0.5% glycerol and

0.05% Tween 80 and incubated at 37°C in a humidified atmosphere with 5% CO2 for

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approximately 21 days. The rBCG strains were obtained via electroporation of the

BCG-Moreau strain with the pLA71 expression plasmid as described previously [25].

Intranasal infection of BALB/c mice with BCG Moreau or rBCG-CMX

The BALB/c mice were divided into three groups: Control, BCG Moreau and rBCG-

CMX. Aliquots of the rBCG-CMX and BCG Moreau vaccines were removed from the -

80°C freezer and diluted in 0.05% PBS Tween 80 at a concentration of 1x108 CFU/mL.

A volume of 100 μL of the vaccine was intranasally administered, with small doses of

20 μL given at a time, allowing the animal to breathe between doses, until 100 μL of

vaccine had been administered. The saline group received 100 μL of PBS/0.05%

Tween-80. The immunizations were performed in a single dose. After preparation of the

vaccines, a sample was plated to confirm the concentration. After immunization, the

animals were observed for 3 h to check for signs of apathy, wheezing or any change in

behavior that showed extreme discomfort. If an animal produced signs and symptoms

that were incompatible with animal welfare, then a trained veterinarian proceeded to

humanely euthanize the animal. No animals presented such symptoms during the

experiment.

Macrophages

Peritoneal and alveolar macrophages were obtained by peritoneal or bronchial alveolar

lavage [25, 35]. The alveolar lavages were centrifuged at 1000 x g and 4°C for 10 min.

The supernatant was discarded, and the cells were resuspended in 1 mL of complete

RPMI medium (cRPMI - HIMEDIA, Mumbai, India) containing 2 mM-glutamine, 100

U/mL penicillin, 1000 U/mL GIBCO, 10 nM pyruvate and 10% FBS. The cells were

counted using Trypan blue (Code 1263C061, Amresco Solon, Ohio USA 44139-

4300) in a hemocytometer. The peritoneal macrophage cultures were adjusted to a

concentration of 106 cells per well (24-well plates), while the alveolar macrophages

were used at a concentration of 2x105 cells per well (96-well plates). The plates were

cultured for 24 h until stimulation with antigens. The recombinant antigens rAg85c,

rMPT51, rHspX and rCMX were used at a concentration of 20 µg/mL and, after

standardization, were added to the macrophage cultures. After 24 h of stimulation, the

culture supernatants were collected for cytokine dosage.

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Lung homogenates

Four days after intranasal infection, the BALB/c mice were euthanized by cervical

dislocation. The left lung lobes were collected and prepared as described by da Costa et

al. (2014) [25]. Once obtained, the lung was treated with a solution of DNAse IV (30

μg/mL; Sigma-Aldrich) and collagenase III (0.7 mg/ml; Sigma-Aldrich) for 1 h at 37°C.

To obtain a cell suspension, the tissue was passed through a 70-m cell filter (BD

BioSciences, Lincoln Park, NJ). The erythrocytes were lysed with lysis solution (0.15

M NH4Cl, 10 mM KHCO3), and the cells were then washed and resuspended in cRPMI

medium and adjusted to 1x106 cells/mL. The cell suspensions were divided for culture

or flow cytometry. For culture, the cells were maintained for 24 h without stimulus and

incubated at 37ºC in a humidified 5% CO2 atmosphere. After this period, the culture

supernatants were collected and stored at -20°C until the time of cytokine dosage. For

flow cytometry, the cell suspensions were labeled immediately after isolation.

Cytokine dosage

The supernatants of the cell cultures stimulated with the rAg85c rMPT51, rHspX or

rCMX proteins and supernatants of the cells infected with BCG, rBCG-CMX or empty

rBCG-PLA71 vaccines were used for IL-6, IL -1α and TGF-β dosage using ELISA

(Enzyme-Linked Immunosorbent Assay) according to the manufacturer's instructions,

using the kits Mouse IL-6, IL-1α and TGF-β ELISA Ready-SET-Go (eBioscience,

Inc.). Optical density readings were taken at 450 nm on an ELISA reader (THERMO

PLATE- TP-READER). The results were obtained after the determination of the

standard curve calculated from the readings of different concentrations of recombinant

cytokines provided by the commercial kits.

Bone Marrow-Derived Macrophages (BMM)

Two mice from each strain (BALB/c, C57BL/6, TLR-2-/- and TLR-4-/-) were used in this

experiment. The mice were euthanized by cervical dislocation. The bone marrow cells

were washed, processed and resuspended in cRPMI, adjusted to 106 per mL and plated

in a 24-well plate containing 10 g/mL recombinant murine granulocyte-macrophage

colony stimulating factor (GM-CSF) (eBioscience). After differentiation, the

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macrophages were resuspended and cultured in a 96-well plate at a concentration of

2x105 per well for 24 hours before stimulation with the antigens.

BMM infection with BCG, rBCG-CMX and empty rBCG-PLA71

BMM were infected with 5x106 CFU of the BCG, rBCG-CMX aor empty rBCG-

PLA71 vaccines [MOI 5:1]. The cultures followed the conditions described above.

After 3 h, the supernatants containing non-phagocytosed bacteria were discarded, and

the cells were washed twice with PBS at 37°C. After the addition of 500 L of complete

RPMI medium without antibiotic, the plate was incubated for 24 h under the same

conditions. Subsequently, the culture supernatants were obtained and stored at -20°C for

cytokine dosage, and the cells were used for apoptosis testing and immunoblotting.

Apoptosis test

BMM infected with BCG, rBCG-CMX or empty rBCG-PLA71 were incubated with

PBS for 1 h on ice. After this period, the cells were centrifuged and adjusted to a

concentration of 1x105 cells/mL. A 100-μL aliquot of this suspension was added to 100

μL of annexin-binding buffer and incubated with 5 μL of FITC-Annexin V and 10 μL of

propidium iodide (PI) for 15 min at room temperature. Finally, 400 μL of annexin-

binding buffer was added, and data were acquired using a BD FACS Verse flow

cytometer (UFG). Macrophages stimulated with medium alone were used as negative

controls, and macrophages treated with 3% paraformaldehyde served as positive

controls.

Immunoblotting

For immunoblotting, macrophages from cultures infected with the BCG, rBCG-CMX or

empty rBCG-PLA71 vaccines were lysed with 50 µL of sterile water after 24 h of

culture. A 20-μL aliquot of each lysate was spotted on a nitrocellulose membrane

(Trans-Blot-Bio-Rad Laboratories), and the membrane was then blocked with 25 mL

of PBS/5% milk. After incubation at 4°C for 18 h under agitation, the membrane was

treated with anti-CMX antibody (de Souza et al. 2012). After 2 h of incubation, the

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membrane was washed with PBS/0.05% Tween 20 and incubated for 1 h at 37°C with

biotinylated anti-IgG1 and anti-IgG2a antibody (1:15,000-Southern Biotechnology

Associates, Inc.). The membrane was then washed again and incubated with Avidin-

peroxidase (1:500) in PBS/2% milk for 1 h at room temperature with agitation. After an

additional washing step, the membrane was treated with developer buffer containing

0.015% diaminobenzidine (DAB) and 0.03% H2O2 in PBS, and it was gently shaken

while protected from light.

Indirect Dosage of NF-B/AP-1 activity

To evaluate NF-B/AP-1 activation, RAW-Blue cells (macrophages) that express the

SEAP (secreted embryonic alkaline phosphatase) gene under the control of NF-B/AP-

1 were used, as described by Zoccal et al. (2014) [36]. The dosage was performed after

24 h of stimulation with rAg85c, rMPT51, rHspX R and CMX (20 g/ml).

Flow Cytometry

Macrophages derived from the bone marrow and lung homogenates of BALB/c mice

were evaluated using flow cytometry. The lung cells and BMM were treated with 10%

mouse serum for 30 min. After treatment, the cells were washed with 200 µL of

PBS/azide. After centrifugation, the lung macrophages were incubated for 30 min with

FITC anti-CD206 (Clone MR5D3 - Santa Cruz Biotechnology), anti-CD86 PE (Clone

GL1-eBioscience Inc. San Diego, CA), anti-CD11b PERCP (Clone M1/70 - BD

Biosciences Pharmingen, San Jose, CA) and anti-F4/80 APC (Clone BM8 -

eBioscience, Inc. San Diego, CA) antibodies. Meanwhile, the BMM were labeled with

anti-CD206 FITC (MR5D3 - Santa Cruz Biotechnology), anti-CD86 PE (Clone GL1 -

eBioscience Inc. San Diego, CA), anti-MHCII PERCP (Clone M5/114.15.2 -

BioLegend ,)and anti-F4/80 APC (Clone BM8 - eBioscience, Inc. San Diego, CA)

antibodies. After the addition of 200 μL of PBS/azide and further centrifugation, the

cells were treated with PERM FIX (BD Cytofix/CytopermTM) for 20 min at 2-8°C. They

were then washed and resuspended in 200 μL of PBS/azide. A total of 50,000 events

were acquired using the BD FACS Verse flow cytometer (UFG). Data were analyzed

using FlowJo software, version 8.7.

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Statistical analyses

Data were tabulated and analyzed using Microsoft Office Excel 2011 and Prism

software (version 5.0c, GraphPad). The results are presented as the mean and standard

deviation for each experimental group. The results using recombinant proteins as

stimuli were evaluated by multimetric tests using a one-way ANOVA followed by

comparison of each experimental group and the negative control group (medium) using

Dunnett's test. p<0.05 was considered statistically significant. All experiments were

repeated three times.

Results

The rBCG-CMX vaccine modulates innate immunity and induces cytokines

that promote protection

In vaccination models, a balanced Th1 and Th17 response may be responsible

for protection after infection with Mtb. Among the cytokines that are involved in

protecting against TB, TGF- is involved in the induction of Th17 after infection or

vaccination [37], and IL-1α is involved in well-structured granuloma formation [12].

We therefore asked whether the rBCG-CMX vaccine was able to induce the production

of TGF- and IL-1α [25]. To evaluate the induction of TGF-β and IL-1α, an ex vivo

test was performed using lung homogenates from intranasally immunized BALB/c mice

[38, 39], and another in vitro test was performed using BMM from BALB/c mice.

Four days after infection, the rBCG-CMX vaccine was found to induce higher

levels of IL-1α compared with BCG in the homogenates. Paradoxically, when

macrophages were infected in vitro, the rBCG-CMX vaccine induced lower IL-1α

levels than the BCG vaccination did (Fig 1 A and B, p<0.05). Similar results were

observed for TGF-β levels (Fig 1 C and D, p<0.05).

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Fig 1. The ex vivo and in vitro induction of cytokines involved in Th17

differentiation. Mice were intranasally vaccinated with BCG-Moreau and rBCG-CMX.

Immunization was performed using 107 CFU of vaccine per mouse. The animals were

euthanized 4 days after immunization, and the production of TGF-β and IL-1α was

subsequently carried out ex vivo. For in vitro BMM testing, the mice were infected

with 5 MOI of BCG or rBCG-CMX for 3 h, and after 24 hours A) the in vitro

production of IL-1α, B) the ex vivo production of IL-1α, C) the in vitro production of

TGF-β, and D) the ex vivo production of TGF-β were performed. *p<0.05 differece

between the rBCG-CMX group and the saline group; #p<0.05 difference between the

rBCG-CMX group and the BCG-Moreau group. A total of 5 mice were used per group.

rBCG-CMX vaccine promotes increased pulmonary macrophage population

IL-1α is responsible for the proliferation of CD11blow alveolar macrophages and

the induction of CD11b expression [12, 40]. Because IL-1α induction was achieved by

intranasal inoculation of the vaccine, we asked whether the rBCG-CMX vaccine

participated in the activation of lung macrophages in vaccinated mice. BALB/c mice

were intranasally infected/vaccinated [38, 39], and after 4 days, the lung macrophages

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were evaluated. The lungs of mice vaccinated with rBCG-CMX had a higher number of

activated F4/80+CD11bhigh macrophages than did mice vaccinated with BCG (Fig 2;

p<0.05).

The F4/80+CD11bhigh macrophages induced by rBCG-CMX or BCG expressed

similar levels of CD86 (Fig 2C; p=0.05). The rBCG-CMX vaccine induced increased

expression of CD86-activating molecules in F4/80+CD11blow macrophages (Fig 2D;

p<0.05) compared with lung macrophages from animals vaccinated with BCG.

However, the rBCG-CMX vaccine induced a greater number of F4/80+CD11bhigh and

F4/80+CD11blow macrophages expressing CD206 compared with either the BCG

vaccine or saline alone (Fig 2E and F: p<0.05).

The rBCG-CMX vaccine thus promoted an increase in macrophages with high

CD86 and CD206 expression in the lungs of infected animals, indicating a difference in

activation from the immune response induced by BCG.

Fig 2. In vivo induction of macrophage profile. Mice were intranasally vaccinated

with BCG-Moreau and rBCG-CMX. Immunization was performed using 107 CFU of

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vaccine per mouse. The animals were euthanized 4 days after immunization, and lung

flow cytometry was performed to observe macrophage activity. A) Dot plot of

cytometry for F480+CD11bhigh and F480+CD11blow macrophages. B) Percentage of

F480+CD11bhigh macrophages. C) Absolute numbers of F480+CD11bhigh macrophages.

D) F480+CD11bhigh macrophages expressing MIF-CD86. E) F480+CD11blow

macrophages expressing MIF-CD86. F) F480+CD11bhigh macrophages expressing

CD206-MIF. G) F480+CD11blow macrophages expressing CD206-MIF. *p<0.05. A

total of 5 mice were used per group.

The rCMX protein appears to enhance macrophage survival and rBCG-CMX

vaccine processing

Previously, the rBCG-CMX vaccine has been shown to undergo greater

phagocytosis by peritoneal macrophages [25] and induce better CD206 expression,

which has been implicated in macrophage phagocytosis and survival [41]. Therefore,

we asked whether the induction of TGF-β in vivo promotes the apoptosis of the infected

macrophages, as this cytokine is related to the induction of apoptosis [42].

After BMM infection, the BCG-Moreau vaccine was observed to induce more

necrosis than the rBCG-CMX vaccine (Fig 3A: #p<0.05). In contrast, rBCG-CMX-

infected macrophages preferentially died by apoptosis (Fig 3B and C: #p<0.05). The

rBCG-CMX vaccine appears to slow down the process of cell death, even with the

presence of PI and Annexin V double-positive cells, when compared to controls (Fig

3C; *p<0.05). In the present study, the rBCG-CMX vaccine promoted greater

expression of MHC-II in infected macrophages compared with BCG-Moreau, which

may reflect better processing and presentation of the rBCG-CMX vaccine (Fig 3D;

*p<0.05).

The apoptosis test revealed that the vaccine appeared to slow down the process

of cell death and may have caused an increase in the expression of molecules related to

vaccine processing.

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Fig 3. The rBCG-CMX vaccine induces more macrophage apoptosis and better

vaccine processing than does BCG-Moreau. Bone marrow-derived macrophages were

infected with BCG and rBCG-CMX (MOI 5) for 3 h. After this time, excess non-

phagocytosed bacteria were removed. After 24 h of culture, the macrophages were

subjected to flow cytometry (Fig A, B and F) and apoptosis testing (Fig C, D and E).

Analysis was performed using Student's t test. *p<0.05.

The CMX protein appears to be responsible for modifying the BCG response in

macrophages, promoting the expression of survival markers in macrophages and

improving rBCG-CMX vaccine processing. The expression of the CMX protein within

BMM has been confirmed (S1 Fig).

The rCMX protein induces the production of cytokines that promote

vaccine protection in BMM from BALB/c mice

Some Mtb proteins are, individually, able to activate the immune response in

macrophage models, inducing either a pro- or an anti-inflammatory response [43-45].

The present study proposed that the rCMX protein, or the set of proteins that comprise

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it, could activate inflammation, thereby promoting the induction of better vaccine

protection.

To this end, the activation of NF-κB and the production of IL-6 were evaluated

in macrophages stimulated with the recombinant fusion protein or the individual

proteins. RAW-Blue cells and BMM from BALB/c mice were stimulated with rAg85c,

rMPT51, rHspX and rCMX for 24 h (Fig 4). All proteins activated NF-κB (Fig 4A;

p<0.05). However, there was differential stimulation of the inflammatory cytokines.

When BMM were treated with these proteins, the induction of IL-6 production was

found to be restricted to rAg85c and rCMX (Fig 4B; p<0.05). Despite inducing NF-κB

activation, rMPT51 and rHspX did not activate IL-6 production in BMM from

BALB/c mice (Fig 4B; p<0.05).

As stated earlier, the rBCG-CMX vaccine induces increased TGF-β and IL-1α

production in the lungs of BALB/c mice. These cytokines are related to the induction of

Th17 responses and to a protective response [46, 12]. We therefore asked whether the

proteins that make-up rCMX were capable of inducing the production of these

cytokines. The results demonstrate that only rAg85c and rCMX induced TFG-β and IL-

1α in the BMM from BALB/c mice (Fig 4C and D; p<0.05).

The only protein present in rCMX that was capable of inducing IL-6, TFG-β and

IL-1α was rAg85c. The lack of induction by the rMPT51 and rHspX proteins did not

prevent rCMX from inducing the production of all cytokines, although this induction

was at lower levels. However, the rCMX protein, when expressed in vivo by BCG, may

modify the vaccine response.

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Fig 4. Production of cytokines by rAg85c, rMPT51, rHspX and rCMX proteins.

A) RAW-Blue cells were stimulated with 20 µg/mL of recombinant proteins for 24 h.

Supernatants were obtained for indirect dosage of NF-κB activity. Macrophages derived

from the bone marrow of BALB/c mice were cultured with 20 µg/mL of recombinant

proteins for 24 h. After this period, the supernatant collected was subjected to determine

the levels of IL-6 (B), TFG-β (C) and IL-1α (D) cytokines. *p<0.05 difference between

stimuli and the medium.

rCMX induces cytokines in the BMM of C57BL/6 mice that promote vaccine

protection

The mouse strains C57BL/6 and BALB/c are known to have genetic differences

that affect the induction of Mtb-protective responses and that the ability to induce

protection in these two models appears to be associated with different cellular profiles

[47]. Furthermore, following vaccination with BCG, protective immune response

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induction has been shown to differ between these two models [48, 47]. However, in

regard to vaccine protection, the vaccine in question must be able to produce a response

in any evaluated model. In the results observed earlier, only rAg85c and rCMX were

able to activate the immune response in BALB/c mouse macrophages. However, we

asked whether the rCMX protein could activate the immune response in the C57BL/6

model without being affected by the genetic background of this model.

For this purpose, BMM from C57BL/6 mice were stimulated with the rAg85c,

rMPT51, rHspX and rCMX proteins for 24 h. IL-6 induction was observed for all

analyzed proteins (Fig 5A; p<0.05). rAg85c, rHspX and rCMX induced the production

of IL-1α (Fig 5B; p<0.05). In contrast to what was observed in the BALB/c model, only

the rMPT51 protein was unable to induce the production of this cytokine (Fig 5B;

p=0.05). Surprisingly, all of the proteins were capable of inducing TFG-β production in

the BMM from C57BL/6 mice (Fig 5C, p<0.05).

Fig 5. Production of cytokines by rAg85c, rMPT51, rHspX and rCMX proteins.

A) Macrophages derived from the bone marrow of C57BL/6 mice were cultured with 20

µg/mL of recombinant proteins for 24 hours. After this period, the supernatant collected

was subjected to dosage of IL-6 (A), IL-1α (B) and TFG-β (C) cytokines, respectively.

*p<0.05 difference between stimuli and the medium.

BMM are a good model for eliminating natural activation bias, but they do not

reliably reflect infection or immunization. To better simulate the mucosal environment,

we used alveolar and peritoneal macrophages from both mouse strains, which could

allow us to evaluate the behavior of cells of the primary infection site and the peripheral

response.

The results show that all the proteins induced IL-6 production in the alveolar

macrophages of BALB/c mice (Fig 6A; p<0.05). However, only the rAg85c and rCMX

proteins stimulated these macrophages in C57BL/6 mice (Fig 6B; p<0.05). In the

101

peritoneal macrophages, only the rMPT51 protein was incapable of stimulating IL-6

production in the C57BL/6 model, with the other proteins achieving similar induction in

both models (Fig 6C and D; p<0.05).

Although the induction of IL-6 production by rCMX varied between the models,

IL-6 was always induced in the macrophages from the different genetic profiles.

Fig 6. Production of cytokine IL-6 by rAg85c, rMPT51, rHspX and rCMX

proteins. A) Alveolar and peritoneal macrophages from BALB/c and C57BL/6 mice

were obtained and cultured with 20 µg/mL of recombinant proteins for 24 h. After this

period, the supernatant collected was subjected to determine the level of IL-6. A and B)

Alveolar macrophages from BALB/c and C57BL/6 mice, respectively. C and D)

Peritoneal macrophages from BALB/c and C57BL/6 mice, respectively. *p<0.05

diffeence between stimuli and the medium.

TLR-4 appears to interact with rAg85c, rMPT51, rHspX and rCMX in IL-6

production

102

Given the context in which these proteins stimulate the immune response, we

asked whether they were recognized by innate immune response receptors. In the

context of TB, TLR-2 and TLR-4 are implicated in host cell interactions with Mtb [43,

45]. Due to the increased production of IL-6 inflammatory mediators of NF-κB activity,

we used BMM from TLR-2-/- and TLR-4-/- mice to explore whether TLR-2 and TLR-4

are involved in recognizing the rAg85c, rMPT51, rHspX and rCMX proteins.

At 24 h after stimulating the BMM from TLR-2-/- mice with rAg85c, rMPT51,

rHspX and rCMX proteins, IL-6 production was unchanged (Fig 7A, p<0.05). Rather,

IL-6 production was reduced in the BMM of TLR-4-/- mice, demonstrating that these

proteins depend on this receptor to induce IL-6 production (Fig 7B, p<0.05). These

results demonstrate that the rAg85c, rMPT51 and rCMX proteins appear to interact with

TLR-4 in IL-6 production.

Fig 7. TLR receptors related to the recognition of rAg85c, rMPT51, rHspX and

rCMX in BMM from TLR-2 KO and TLR-4 KO mice. A) Macrophages derived

from the bone marrow of TLR-2 KO and TLR-4 KO mice were cultured with 20 µg/mL

of recombinant proteins for 24 h. After this period, the supernatant was subjected to

dosage of IL-6 in BMM from TLR-2 KO mice (A) or in BMM from TLR-4 KO mice

(B). *p<0.05 difference between the stimuli and the meadium.

103

S1 Fig. rBCG-CMX vaccine expresses the rCMX protein 24 h after infection in

macrophages. Bone marrow-derived macrophages were infected with BCG and rBCG-

CMX (MOI 5) for 3 h. After this period, excess non-phagocytosed bacteria were

removed. After 24 h of culture, the macrophages were lysed, and dot blots were

performed to detect rCMX protein expression.

rCMX BCG-PLA71Φ rBCG-CMX BCG Moreau

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Discussion

Previous studies have shown that the rBCG-CMX vaccine induces both Th1 and

Th17 responses, both of which are important in controlling Mtb infection. In the present

study, we propose that the rCMX protein, when expressed by the rBCG vaccine (rBCG-

CMX), is able to activate the innate immune response and modulate the response of this

vaccine, promoting the induction of the Th1 and Th17 response. These results

demonstrate that macrophages activated by the rBCG-CMX vaccine are more numerous

and have higher CD86 and CD206 expression, accompanied by the production of TGF-

β and IL-1α. This vaccine induces more apoptosis and greater MHC-II expression in

infected macrophages than does the BCG vaccine. The rCMX protein was able to

induce IL-1α, IL-6 and TGF-β production via TLR-4 signaling, demonstrating that

TLR-4 is probably responsible for inducing the Th1 and Th17 response to the rBCG-

CMX vaccine.

The mucosa contains important cells, such as macrophages, which have been

established as essential in the production of innate immune memory after vaccination

[49]. In Mtb infection, the first induced response begins mainly on the surface of the

respiratory mucosa; therefore, it has been shown that the first line of defense should be

produced at the pathogen infection site to promote a better protective response [50]. An

alternative means of evaluating the ability of the rBCG-CMX vaccine to activate the

innate immune response would therefore be to evaluate pulmonary macrophages

following infection with the vaccine.

Macrophages are known to produce cytokines such as TGF-β and IL-1α, which

are involved in specific protective response induction [51, 52]. TGF- is related to the

formation of fibrous tissue and the differentiation of Th17 cells, important events in the

protection induced by Mtb vaccines [14]. Our results showed that 4 days after infection,

the rBCG-CMX vaccine induced higher levels of TGF-β than the BCG vaccine (Fig

1D). This result corroborates previous studies that have shown that when expressed by

rBCG (rBCG-CMX) or mc2 (mc2-CMX), the CMX protein is a strong inducer of the

Th17 response in immunized mice. Concurrently, it was shown that, after 4 days of

infection, the rBCG-CMX vaccine induced greater IL-1α production than the BCG

vaccine (Fig 1B). Huaux et al. (2015) observed that IL-1α is responsible for the

proliferation of CD11blow alveolar macrophages and the activation of these

105

macrophages in CD11bhigh [12]. This observation suggests that the rBCG-CMX vaccine

may be inducing higher proliferation of the macrophage population produced following

infection with the vaccine.

These activated macrophages migrate to the lung tissue and are active in

granuloma formation [12]. After infection, the rBCG-CMX vaccine was further shown

to induce a greater number of activated F4/80+CD11bhigh macrophages in the lung,

indicating that this vaccine has good potential to promote macrophage recruitment to

the infection site. In our results, F4/80+CD11bhigh macrophages had similar CD86

expression to and greater CD206 expression than that induced by the BCG-Moreau

vaccine (Fig 2B, D and F). These results corroborate the study by Mirza et al. (2011),

who showed that TGF-β is produced concomitant to the induction of CD206 expression.

These cells are important during the activation of the adaptive immune response, as the

co-stimulatory molecules CD86 and CD206 and phagocytosis receptors are strongly

associated with the presentation of antigens to T lymphocytes [53].

Another evaluated macrophage population was F4/80+CD11blow, which are

probably the alveolar macrophages [12]. This population showed greater expression of

both CD86 and CD206 after infection with the rBCG-CMX vaccine (Fig 2E and G).

CD206 is a type C lectin that is responsible for the endocytosis and phagocytosis of

microorganisms that contain manoglycoproteins, including Mtb [54]. This result

supports the phenomenon observed in previous studies of the rBCG-CMX vaccine,

which was shown to undergo higher phagocytosis than BCG-Moreau and may promote

the induction of a better protective response [25].

Another important specific defense mechanism that is promoted by rBCG-CMX

is the induction of apoptosis in the infected macrophages. Previously, macrophages

infected with rBCG::ΔureC::Hly (+) have been shown to induce apoptotic bodies that

are directly associated with the activation of TCD4+ and TCD8+ by increasing antigen

representation by MHC-II and MHC-I [23]. The results presented here showed that the

rBCG-CMX vaccine induced more apoptosis than the BCG vaccine (Fig 3A), and this

fact can be directly associated with the presentation of antigens via MHC-II (Fig. 3D),

which can enhance the TCD4+ lymphocyte response [55]. These results corroborate the

induction of TGF-β in vivo, as this cytokine is related to the induction of apoptosis [42],

and the two events promote a better protective response.

These findings lead us to hypothesize that the rCMX protein may be modulating

the response induced by the BCG vaccine, modifying and improving its ability to

106

activate the immune response. The rCMX protein consists of the immunodominant

epitopes of the rAg85c, rHspX and full-length rMPT51 proteins [27]. When expressed

by the rBCG vaccine (rBCG-CMX), the rCMX protein modifies the immune response

induced by BCG-Moreau. Therefore, we suggest that the rCMX protein may be

modulating the response of the BCG vaccine, as well as the macrophage response.

Several studies have shown that some Mtb proteins are able to activate the innate

immune response in macrophages and dendritic cells to interact with TLRs [42, 56, 43].

We therefore verified the pro-inflammatory ability of each individual protein, as well as

the rCMX protein, which allowed us to infer an immunomodulatory effect on the part of

these proteins. We observed that the rAg85c, rMPT51, rHspX and rCMX proteins

activated NF-κB, highlighting the ability of these proteins to induce a pro-inflammatory

response (Fig 4A) [57]. However, only rAg85c and rCMX were able to induce the

production of IL-6, IL-1α and TGF-β. Given the immunomodulatory ability of Ag85-

complex proteins [2], we believe that Ag85c is the main protein in rCMX capable of

inducing the IL-6, IL-1α and TGF-β cytokines in BMM from BALB/c mice (Fig 4).

This hypothesis is supported by the fact that rAg85c has been shown to exhibit powerful

pro-inflammatory activity. When expressed by a BCG vaccine (rBCG-Ag85c), Ag85c

promoted a better protective response than that promoted by BCG in the induction of

pro- and anti-inflammatory cytokines such as IL-12, TNF-α, IFN-γ and TGF-β in

immunized guinea pig lungs [58]. By contrast, only MPT51 was unable to induce the

production of cytokines in BMM from C57BL/6 mice (Fig 5). The C57BL/6 and

BALB/c strains are known to differ in their adaptive immune response induction

following BCG vaccination or Mtb infection. However, this difference does not affect

the control of infection by the two models [48, 47]. Regardless of the model utilized,

rAg85c in particular played an essential role in the response induced by rCMX,

suggesting that this protein is able to activate the immune response in BMM from both

models.

As the observed activation of inflammation may be related to the BMM

response profile, we evaluated this response in alveolar and peritoneal macrophages,

which best represent a mucosal microenvironment. Although the induction of IL-6

production by rCMX varied between the two models, IL-6 cytokine production was

maintained between the different macrophage profiles. The innate immune response

produced by the rCMX protein, with the induction of pro- and anti-inflammatory

107

cytokines such as IL-6, IL-1α and TGF-β, may have promoted the induction of Th17

responses in vivo and may have contributed to the greater protection induced by BCG

when rCMX was used as a booster [25].

Some studies have shown that Mtb antigens are capable of regulating the

immune response of the host by interacting with TLRs [43, 44]. In the present study, we

demonstrated that the rAg85c, rMPT51 and rCMX proteins all interacted with TLR-4 to

promote IL-6 induction (Fig 7). The Rv0652 protein of Mtb induces a TLR-4-dependent

pro-inflammatory immune response by stimulating BMM and RAW 264.7

macrophages [45]. A study by Yu demonstrated that 24 h after urothelial cancer cells

were infected with the BCG vaccine, cell death was induced and genes important for the

induction of apoptosis were activated via TLR-7 receptor activation [59]. This finding

led to the belief that the rCMX protein was expressed by the BCG vaccine in 24 h, as

demonstrated in S1 Fig. After expression, rCMX induces apoptosis by interacting with

TLR-4, as this receptor is associated with the induction of apoptosis in macrophages

[60]. Although we did not use a TLR-4 blocker to confirm the interaction, our results

support the hypothesis that this interaction occurs, as we detected a reduction in IL-6

production after LPS stimulation in TLR-4 KO mouse macrophages (Fig 7). Moreover,

through the induction of apoptosis and the release of apoptotic bodies, the vaccine may

promote the participation of other cells such as dendritic cells to increase the induction

of the adaptive immune response [23], thus potentially fostering a better protective

response.

Among the difficulties and limitations of the present work, we should mention

the potential contamination of the proteins with LPS; the effect of the plasmid on BCG

recombination; and the different strains of mice used in this study. To solve these

problems, LPS was removed from the recombinant proteins using LPS extraction kits

(Toxin Removal Kit) that are currently being used in Mtb protein testing [61].

Regarding the effects of the plasmid on BCG, some studies in the TB vaccine field have

used a BCG vaccine expressing an empty plasmid as a control group. No changes were

observed regarding the specific response compared with what would be expected from

BCG [15]. The other obstacle we faced was the use of two mouse models, BALB/c and

C57BL/6. Mtb infection in C57BL/6 mice is known to result in better induction of

balanced Th1 and Th17 cells than in the BALB/c model [48]. However, the BALB/c

model has more IL-10 producing Treg cells, which may negatively affect bacterial load

108

reduction, causing greater susceptibility in this model than in the C57BL/6 model [47].

Contrary to what is observed in Mtb infection after vaccination with BCG, BALB/c

mice showed better induction of the Th1 and Th17 response than C57BL/6 mice [62].

Although the response of the two models to Mtb infection or BCG vaccination differs,

protection in the two models does not change [62]. It would be important to discover

more about the innate immune response of these two models, and further study is

required in this area.

These results allow us to suggest that the rCMX protein modulates the innate

immune response by interacting with TLR-4 in macrophages and by inducing IL-6 and

TGF-β, which may explain the production of the Th17 response by the rBCG-CMX

vaccine.

Aknowledgements

This study was financed by the National Council for Scientific and

Technological Development (CNPq, Project#301976/2011-2, 472906/2011-9,

301198/2009-8, 472909/2011-8) and by Fundação de Amparo a Pesquisa do Estado de

Goiás (FAPEG-PRONEX).

We are thankful to Universidade de Brasília (UnB), for allowing access to

cytometry core facilities.

Experimental design and set up: APJK e AK. Experimental development and

data analyses: APJK, AK, ACC, DPR, Bruno, KFZ. Grant PIs that afforded reagents,

materials and analysis tools for all experiments APJK e AK, LHF. Critical discussion

and writing of the manuscript: APJK, AK, ACC, DPR, Bruno, KFZ, LHF. Cytometry

experiments and analysis: ACC.

ACC: Received a PhD fellowship from CNPq; DPR: Received an MSc

fellowship from CNPq; Karina, Bruno - Received an undergraduate fellowship from

CNPq.

109

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5 DISCUSSÃO

Neste trabalho foi realizado a avaliação da imunogenicidade de uma vacina

rBCG expressando a proteína de fusão CMX, a qual é composta por epítopos

imunodominantes de Ag85c_MPT51_HspX. Neste sentido, abordamos no primeiro

capítulo em um artigo de revisão algumas vacinas BCG recombinantes que foram

construídas nos últimos 5 anos, demonstranos que apesar de algumas induzirem

proteção superior a induzida por BCG a memória foi pouco explorada ou não havia

sua indução (DA COSTA, COSTA-JUNIOR ADE, DE OLIVEIRA et al., 2014). No

segundo capítulo desenvolvemos uma nova vacina rBCG-CMX que induziu boa

proteção provavelmente em virtude da população de células Th17 e Th1 específicas

para CMX (DA COSTA, NOGUEIRA, KIPNIS et al., 2014). No terceiro capítulo

demonstramos que a vacina recombinante e a proteína de fusão CMX que compõe a

BCG, modula a resposta imune favorecendo a indução de citocinas pró e anti-

inflamatórias.

Atualmente, a vacina utilizada para prevenção da TB é a BCG (Bacilo Calmette-

Guérin), uma cepa atenuada derivada de uma cepa virulenta do Mycobacterium bovis,

a qual foi atenuada após mais de 13 anos de cultura in vitro, sendo utilizada desde

1921 (Calmette et al., 1929). É uma das vacinas mais largamente administradas

mundialmente e a única vacina disponível que previne infecções contra M.

tuberculosis (RAPPUOLI & ADEREM, 2011), a qual é produzida em vários

laboratórios no mundo. Apesar de ser a única vacina aprovada para uso humano, e

conferir proteção em crianças contra meningite tuberculosa e TB miliar, seu efeito

protetor continua questionável, uma vez que não protege adultos contra TB pulmonar

(WHO, 1998; PARTNERSHIP PARTNERSHIP WST, 2010; LIENHARDT &

ZUMLA, 2005).

Apesar das cepas utilizadas mundialmente serem originadas do Mycobacterium

bovis atenuado, as mesmas podem não ser bacteriologicamente iguais, devido às

variações biológicas de cepas, que apresentam características genotípicas e fenotípicas

notavelmente diferentes, resultando em variações no que diz respeito à viabilidade,

imunogenicidade, reatogenicidade e virulência residual (BARRETO, PEREIRA,

FERREIRA, et al. 2006).

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Atualmente estudos genômicos demonstram que a vacina BCG difere em

algumas características genéticas, e diversos estudos realizados para avaliar o nível de

proteção da BCG contra TB pulmonar apontaram, dentre outros aspectos, enorme

variação na proteção conferida, decorrentes do desenho, ou áreas geográficas onde

foram realizados. Isso tem gerado incertezas quanto à capacidade de proteção desta

vacina (BARRETO, PEREIRA, FERREIRA, et al. 2006). Em crianças, a proteção é

estimada em 52 a 100%, para a prevenção de meningite tuberculosa, e de 2 a 80% na

prevenção de tuberculose pulmonar. As causas da falha na eficiência da vacina BCG

podem estar relacionadas com os seguintes fatores: exposição prévia a micobactérias

ambientais (Black et al. 2002), variações genéticas da população ou das cepas

utilizadas como vacinas (GRODE, SEILER, BAUMANN, et al. 2005), dentre outras.

Além disso, a vacina BCG é contra indicada para pacientes HIV positivos, recém-

nascidos com peso inferior a dois quilogramas, pacientes em situação de

imunocomprometimento, mulheres grávidas e pessoas com teste tuberculínico (TST)

positivo, ou que estejam submetidos a algum tratamento prolongado com esteróides ou

drogas imunossupressoras e doenças infecciosas como sarampo e varicela (PAUL &

FINE, 2001).

A profilaxia da TB utilizando a vacina BCG apresenta algumas variações quanto

ao esquema de vacinação, sendo baseada na taxa de incidência da TB. A vacina BCG

é considerada segura, sendo administrada em recém-nascidos, estimulando uma

memória imunológica protetora considerada de longa duração. Entretanto, estudos

demonstram que a proteção da vacina, em diferentes populações, apresenta variações

que pode ser devido à relativa eficácia da BCG em crianças, o que pode ser explicada

pela indução da memória somente na idade precoce (neonatal ou infância) na qual o

sistema imune não está totalmente maduro e aumento da suscetibilidade de jovens e

adultos a co-infecção com helmintos, vírus (como HIV) (ANDERSEN & DOHERT,

2005). No Brasil, estudos realizados concluem que aqui também há uma variabilidade

na proteção, principalmente após os dez anos de imunização. Apesar dos esforços do

Programa de Controle Nacional da Tuberculose, a cobertura vacinal em crianças

menores de um ano é em torno de 90% e a incidência da TB continua alta devido a

falhas na execução e acompanhamento do tratamento dos doentes.

Em humanos, esta vacina induz resposta imune com células efetoras do tipo Th1

produtoras de IFN-γ (SOARES, SCRIBA, JOSEPH, et al. 2008; STENGER, S.;

HANSEN, D.A.; TEITEL BAUM, et al., 1998). Já foi demonstrado que o IFN-γ

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induzido pela BCG pode contribuir para a redução na formação de células de memória

inicialmente induzida pela vacina (ABEBE, 2012). Além disso, estudos demonstram

que o IFN-γ não está diretamente relacionado com a proteção conferida pela BCG

(MITTRUCKER, STENHOOF, KOHLER, et al., 2007; ABEBE, 2012).

Apesar se acreditar que a inserção de antígenos de Mtb na BCG poderia

melhorar a memória induzida por ela, não foi o que se observou ao utilizar várias

estratégias de recombinação da vacina BCG, como demonstrado na literatura, em uma

revisão de literatura, entre os anos de 2008 a 2013. Das 24 vacinas avaliadas neste

período, apenas 10 apresentaram proteção superior a induzida por BCG. Destas, 4

eram vacinas BCGs super expressando antígenos simples não pertencentes a regiões

de virulência de Mtb, tais como, HspX, Ag85C, Ag85A e Ag85B (HOFT,

BLAZEVIC, ABATE et al., 2008; TANG, YAMADA, SHIBATA et al., 2008;

SUGAWARA, SUN, MIZUNO et al., 2009; SHI, CHEN, CHEN et al., 2010); e 1 de

região de virulência, ESAT-6 (DEY, JAIN, KHERA et al., 2009). Quanto aos demais,

3 vacinas expressando proteínas de fusão combinando região de virulência ou não

(rBCG1::Ag85B-CFP10/BCG2::Ag85B-CFP10-IL-12; rBCG-Ag85B-Mpt64-Mtb8.4;

AFRO-1, Ag85A, Ag85B e TB10.4.) (QIE, WANG, LIU et al., 2009; LIN, SU,

CHANG et al., 2012; RAHMAN, MAGALHAES, RAHMAN et al., 2012). Ademais,

1 vacina utilizou a fusão de proteínas de região de virulência (rBCG:PE-

MPT64/rBCG/HSP60MPT64) e 1 vacina apresentou expressão de listeriolisina

(rBCGΔureC::hly+) (DESEL, DORHOI, BANDERMANN et al., 2011). Dentre as

vacinas BCG que foram recombinadas com proteínas de Mtb, apesar de terem

apresentado proteção superior a induzida por BCG, apenas 4 dessas vacinas avaliaram

a indução de células de memória.

A resposta imune induzida por essas vacinas é do tipo Th1, com produção de

IFN- (HOFT, BLAZEVIC, ABATE et al., 2008; TANG, YAMADA, SHIBATA et

al., 2008; SHI, CHEN, CHEN et al., 2010; LIN, SU, CHANG et al., 2012). Não foi

avaliado exatamente qual mecanismo que as proteínas inseridas nessa vacina

proporcionaram para que houvesse a melhora na resposta imune induzida. Ao

contrário, a vacina rBCGΔureC::hly+ também apresentou indução de células de

memória, a qual induziu o balanço entre a resposta Th1 e Th17. Esta vacina foi capaz

de ativar e modular a resposta de macrófagos, nos quais induziu acentuado processo de

apoptose, como geração de piroptose, capaz de induzir resposta Th1, TCD8+ e Th17

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(DESEL, DORHOI, BANDERMANN et al., 2011; FARINACCI, WEBER e

KAUFMANN, 2012).

No segundo artigo demonstramos que a proteína de fusão CMX é capaz de

adicionar propriedades imunogênicas importantes em vetores vacinais, induzindo

resposta efetiva no controle da infecção por Mtb em camundongos. A inserção da

proteína CMX na vacina BCG pode ter acrescentado características imunológicas

ausentes na BCG convencional, podendo induzir populações celulares importantes no

controle da infecção por Mtb. Nossos resultados demonstram que a inserção da

proteína CMX na vacina BCG recombinante (rBCG-CMX) foi um fator determinante

para indução de resposta Th1 e Th17, além de células polifuncionais que

possivelmente foram responsáveis pela redução das lesões inflamatórias no pulmão de

camundongos BALB/c , reduzindo significantemente a carga bacilar em comparação

com imunização com BCG Moreau (DA COSTA, COSTA-JUNIOR ADE, DE

OLIVEIRA et al., 2014). Especula-se que uma boa resposta induzida por vacina contra

TB tenha que haver o balanço entre as populações Th17 e Th1 (KHADER, BELL,

PEARL et al., 2007; MATSUYAMA, ISHII, YAGETA et al., 2014; GARCIA-

PELAYO, BACHY, KAVEH et al., 2015). Porém a vacina BCG também induz

resposta Th1 e Th17, porém a indução de IL-17 é muito inferior a indução de IFN-

(ARTS, BLOK, AABY et al., 2015). Tem-se tentado justificar as falhas da vacina

BCG por ser forte indutora de IFN-, esta citocina pode resultar na apoptose das

células T efetoras, reduzindo o pool de células de memória (BEHR e SMALL, 1997).

Por outro lado, as células Th1 poderiam inibir diretamente a manutenção de células

Th17, fenômeno este que pode reduzir as células Th17 e quebrar o balanço entre as

duas respostas, bem como reduzindo as células de memória (TORRADO e COOPER,

2010).

Têm-se demonstrado recentemente que os macrófagos possuem um papel

essencial na geração de resposta imune de memória, e que a indução desta resposta

inicial é preponderante para a geração de uma boa resposta vacinal (YOSHIDA,

MAEKAWA, ZHU et al., 2015). Por muito tempo acreditaram que proteínas não

pudessem interagir com a imunidade inata, porém tem sido demonstrado que muitas

proteínas o fazem, dentre elas muitas de Mtb, as quais são capazes de interagir com

receptores em macrófagos e células dendríticas, tais como TLR-4, TLR-2, CR3, dentre

outros (HETLAND e WIKER, 1994; KIM, SOHN, KIM et al., 2012) (XU, ZHANG,

SHEN et al., 2015). Têm-se tentado associar a dependência entre a geração da resposta

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imune inata e adaptativa. A interação de proteínas com TLR-2 tem sido associado com

a geração de uma resposta Th2, como a proteína ESAT-6, justificando a indução de

uma resposta Th17 (CHATTERJEE, DWIVEDI, SINGH et al., 2011). Porém, a

interação com o TLR-2 parece estar associado a proteínas de região de virulência, tais

como ESAT-6, PE35, PPE68, CFP (CHATTERJEE, DWIVEDI, SINGH et al., 2011;

TIWARI, SOORY e RAGHUNAND, 2014). Conquanto, as demais proteínas que não

estão contidas em região de virulência perecem interagir com TLR-4, como observado

com a proteína Rv0652 (KIM, SOHN, KIM et al., 2012), bem como as proteínas

analisadas neste trabalho, sendo essas Ag85c, MPT51 e HspX. No modelo vacinal

apresentado neste trabalho, a proteína de fusão CMX (Ag85c_MPT51_HspX) parece

interagir com TLR-4 em macrófagos e induzir a produção de citocinas importantes na

geração de uma resposta imune pró e anti-inflamatória.

No terceiro artigo aqui avaliado, foi observado que estas proteínas são capazes de

induzir produção de citocinas TGF- e IL-6, as quais participam na geração de células

Th17 (MURANSKI, BORMAN, KERKAR et al., 2011). Recentemente foi

demonstrado que o TLR-4, bem como receptor de manose (MR) participam diretamente

na geração de células Th17 e Tc17 em infecção por Paracoccidioides brasiliensis

(LOURES, ARAUJO, FERIOTTI et al., 2015). Portanto, a interação da rCMX com

TLR-4, promovendo a geração de TGF- e IL-6, pode ter proporcionado a geração de

resposta imune Th17 específica.

Com o intuito de demonstrar se existe alguma relação entre a interação de CMX

com TLRs e a capacidade de indução de Th1 e Th17 por rBCG-CMX, realizamos

imunização de camundongos C57BL/6, TLR-2 KO e TLR-4 KO. Nossos resultados

demonstram que ao ser expressa pela vacina rBCG após imunização, a proteina CMX

induz linfócitos TCD4+IFN-+ e TCD4+IL-17 em baço de camundongos C57BL/6,

porém na ausência de TLR-2 e TLR-4, não há indução dessas populacõess celulares.

Anteriormente demonstramos que a proteína CMX interage com o TLR-4 mas não com

o TLR-2 para induzir IL-6, porém não havíamos observado se o TLR-4 e TLR-2 estava

relacionado com a indução de outras citocinas importantes para indução de Th1 e Th17,

como IL-12 e TGF-β (HASAN, M.; NEUMANN, B.; HAUPELTSHOFER et al. 2005).

Esse dado poderia justificar a importância dos receptors TLR-2 e TLR-4 na indução dos

linfócitos Th1 e Th17 após imunização com a vacina rBCG-CMX. Apesar de não

possuirmos esses dados, tem sido demonstrado na literature que Mtb depende do TLR-2

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e TLR-9 para induzir resposta Th1, sendo que esses receptors podem estar ralacionados

com a resistência de Mtb (BAFICA, SCANGA, FENG et al. 2005). Outra trabalho tem

enfatizado que ESAT-6 favorece a indução de Th17 e inibe a indução de Th1 por meio

da interação com TLR-2 (WANG, BARNES, HUANG et al. 2012). Sendo assim,

podemos inferir que a proteína CMX depende de TLR-2 e TLR-4 para promover a

indução de Th1 e Th17 quando expressa por rBCG-CMX.

Diante dos resultados obtidos neste trabalho, propomos que a proteina rCMX

interage com o TLR-4, podendo induzir a ativação do fator de transcrição NF-κβ,

culminando na produção das citocinas IL-6 e TFG-β. Estas duas citocinas promovem a

diferenciação de Th17, fenômeno este que ocorreu após imunização com a vacina

rBCG-CMX (da Costa et al. 2014). Quando rCMX é expressa por rBCG, ocorre a

interação desta com TLR-4, bem como a ativação de macrófagos, com aumento na

expressão de CD206, apesar de manter a expressão de CD86 (Figura 8). Além disso a

indução de apoptose com corpos apoptóticos pode melhorar a indução da resposta

adaptativa, favorecendo a indução de Th1 e Th17.

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Figure 8. Modelo de indução de Th17 por rBCG-CMX. Proteína rCMX interage

com TLR-4 em macrófagos e induz a ativação de NF-kB. Esta ativação promove a

indução de IL-1α, TGF-β e IL-6. A vacina rBCG-CMX induz mais expressão de CD206

e semelhante indução da expressão de CD86. A proteína rCMX expressa por BCG

promoveu a esta vacina a capacidade de induzir mais produção dessas citocinas in vivo

que a vacina BCG, promovendo um microambiente para indução de resposta imune

Th17. Apoptotics bodies ; CD206+ Molecules; CD86+ Molecules;

Interleukin 6 (IL-6); TGF-; Interleukin 1 alfa (IL-); rCMX

Protein.

No presente trabalho, tem-se observado uma indução tanto de células Th17 quanto

de células Th1. A proteção induzida pelas duas vacinas é semelhante, porém, quando se

utiliza o booster com a proteína de fusão rCMX, a proteção melhora consideravelmente

(DA COSTA, COSTA-JUNIOR ADE, DE OLIVEIRA et al., 2014). O booster com a

proteína de fusão pode ter induzido a proliferação de células Th1 e Th17,

proporcionando melhor eficácia da vacina, bem como o aumento no pool de células de

memória (LI CAUSI, PARIKH, CHUDLEY et al., 2015). Mais trabalhos seriam

necessários para a avaliação de células de memória induzido pela vacina rBCG-CMX.

Não sabemos como esta vacina se comportaria em humanos, uma vez que a

atuação das células Th17 em humanos e camundongos apresentam aspectos diferentes.

Nos camundongos, sabe-se que essas células apresentam um papel protetor contra

bactérias extracelulares e fungos, por induzirem neutrofilia. No caso da resposta ao

Mtb também parece ser crucial tanto na formação do granuloma quanto na adequação

da inflamação necessária para a eliminação do agente (OUYANG, W.; KOLI, JK.;

ZHENG Y., 2008). Em humanos, as células Th17 apresentam maior plasticidade,

podendo se diferenciar em vários tipos celulares, como Th1, Th2, Th17/2, Th17/1. As

células Th17/2, por exemplo, podem secretar IL-17 e IL-4, apresentando a capacidade

de sobreviver por mais tempo, como uma célula de memória. Neste sentido, enquanto

ensaios clínicos não sejam realizados para verificar se esta ou outras vacinas contra TB

geram células Th17 em humanos, as vantagens da indução destas células ainda deverá

ser melhor estudada (COSMI, MAGGI, SANTARLASCI et al. 2010).

IL-6 TGF-β IL-1a

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6. CONCLUSÕES

Neste trabalho foi avaliado a resposta imune induzida por uma vacina BCG

recombinante expressando a proteína CMX, a qual representa os epítopos

imunodominantes do Ag85C, MPT51 e HspX de Mycobacterium tuberculosis. Após

fazer um panorama das vacinas construídas mundialmente e avaliar a eficácia da vacina

rBCG-CMX em modelo murino, bem como seu mecanismo imune, concluiu-se que:

Uma nova vacina rBCG-CMX foi desenvolvida e esta foi boa indutora de resposta

imune Th1 e Th17, corroborando com uma proteção superior a induzida por BCG.

A proteína rCMX modula a resposta induzida pela vacina BCG, bem como seu

microambiente, favorecendo a indução de citocinas pró-inflamatórias (IL-6) e anti-

inflamatória (TGF-β), por uma via em que há a participação de TLR-4 .

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8. ANEXOS

Anexo 1 – Parecer do Comitê de Ética, TCLE

Anexo 2 – Comprovantes de submissão dos artigos/ aceite para publicação para

artigos ainda não publicados/ dói dos artigos publicados

Anexo 3 – Outros anexos

136

Anexo 1 – Parecer do Comitê de Ética, TCLE

137

138

139

140

141

Anexo 2 – Artigo 1: Recombinant BCG: Innovations on an old vaccine. Scope in

BCG strains and strategies to improve long lasting memory

Adeliane Castro da Costa1, Sarah Veloso Nogueira1, André Kipnis1 and Ana Paula

Junqueira-Kipnis1*

frontiers in IMMUNOLOGY(Publicado)

142

143

Anexo 3 – Artigo 2: Protection conferred by the recombinant vaccine BCG-CMX

is related to the induction of Th17 and polyfunctional cells in BALB/c mice.

Autores: Adeliane Castro da Costa1, Abadio de Oliveira da Costa Júnior1, Fábio Muniz

de Oliveira2, Sarah Veloso Nogueira1, Joseane Damaceno Rosa1, Danilo Pires Resende1,

André Kipnis2 e Ana Paula Junqueira-Kipnis1*.

PLOS one (Publicado)

144