UNIVERSIDADE FEDERAL DE PELOTAS
Programa de Pós
Produção de antígenos de
Pichia pastoris
Daiane Drawanz Hartwig
UNIVERSIDADE FEDERAL DE PELOTAS
Programa de Pós-Graduação em Biotecnologia
Tese
Produção de antígenos de Leptospira interrogans
e avaliação do potencial imunoprotetor
contra leptospirose
Daiane Drawanz Hartwig
Pelotas, 2010
UNIVERSIDADE FEDERAL DE PELOTAS
Graduação em Biotecnologia
Leptospira interrogans em
potencial imunoprotetor
DAIANE DRAWANZ HARTWIG
PRODUÇÃO DE ANTÍGENOS DE Leptospira interrogans EM Pichia pastoris E AVALIAÇÃO DO POTENCIAL IMUNOPROTETOR CONTRA LEPTOSPIROSE
Tese apresentada ao Programa de
Pós-Graduação em Biotecnologia da
Universidade Federal de Pelotas, como requisito parcial à obtenção do título de
Doutor em Ciências (área do
conhecimento: Biologia Molecular e Imunologia).
Orientador: Odir Antônio Dellagostin
Co-Orientador (a): Fabiana Kömmling Seixas
Dados de catalogação na fonte: Ubirajara Buddin Cruz – CRB-10/1032 Biblioteca de Ciência & Tecnologia - UFPel
H337p Hartwig, Daiane Drawanz
Produção de antígenos de Leptospira interrogans em Pichia
pastoris e avaliação do potencial imunoprotetor contra
leptospirose / Daiane Drawanz Hartwig. – 103f. – Tese (Doutorado). Programa de Pós-Graduação em Biotecnologia. Universidade Federal de Pelotas. Centro de Desenvolvimento Tecnológico. Pelotas, 2010. – Orientador Odir Antônio
Dellagostin ; co-orientador Fabiana Kömmling Seixas. 1.Biotecnologia. 2.Leptospirose. 3.Leptospira
interrogans. 4.Vacina recombinante. 5.Pichia pastoris.
I.Dellagostin, Odir Antônio. II.Seixas, Fabiana Kömmling. III.Título.
Banca examinadora:
Prof. Dr. Alan John Alexander McBride, Centro de Pesquisas Gonçalo Moniz Prof. Dr. Fabricio Rochedo Conceição, Universidade Federal de Pelotas Prof. Dra. Flávia Weykamp da Cruz McBride, Universidade Federal da Bahia Prof. Dr. Odir Antônio Dellagostin, Universidade Federal de Pelotas
Dedicatória
Aos meus amados pais, minha irmã Andréia e ao Élcio, por participarem
deste vínculo de amor, suporte e alegria que é a minha família.
Agradecimentos
À Universidade Federal de Pelotas pela oportunidade de realizar um Curso de Pós-Graduação de qualidade.
Ao meu orientador, Odir A. Dellagostin, pela orientação durante o doutorado, sem a qual não seria possível a realização deste trabalho.
A minha co-orientadora e amiga Fabiana K. Seixas, pela amizade, pela incansável ajuda e presença constante, mesmo durante sua licença maternidade.
A toda a minha família, principalmente meus pais, minha irmã Andréia e o Élcio, pela dedicação, laços de amor, amizade e respeito construídos durante toda a vida, pelo exemplo de caráter, por estarem do meu lado nos momentos de alegria e tristeza, vibrando com minhas vitórias e me consolando nas derrotas, também pelos momentos de descontração tão preciosos.
A todos os amigos e colegas do laboratório de Biologia Molecular, Amilton, André, Caroline, Clarisse, Daniela, Karen, Kátia Bacelo, Kátia, Michel, Michele, Samuel, Sérgio, Silvana, Thaís, Vanessa, Vanuza e em especial a Karine Forster, por toda a ajuda, pela amizade, convívio e pelo apoio quer fosse com palavras ou gestos de incentivo.
A minha estagiária Thaís, por todo o apoio e dedicação dispensados durante a execução dos experimentos.
Aos demais colegas da Pós-Graduação, professores, alunos e funcionários do Centro de Biotecnologia, pelos momentos de descontração, amizade, bom convívio e apoio durante todo o Doutorado.
Aos funcionários e amigos do Biotério Central da Universidade Federal de Pelotas pelos cuidados dispensados com os animais da experimentação e pela dedicação. Aos hamsters, sem os quais não seria possível a realização de etapas
fundamentais deste estudo.
A todos que contribuíram de alguma forma para a realização deste trabalho. Ao CNPq, pela concessão da bolsa de Doutorado.
A Deus por me dar a força espiritual necessária para conseguir seguir em frente e muitas vezes me guiar pelo melhor caminho, mesmo sem que eu percebesse, fazendo as coisas acontecerem no momento certo.
RESUMO
HARTWIG, Daiane Drawanz. Produção de antígenos de Leptospira interrogans
em Pichia pastoris e avaliação do potencial imunoprotetor contra leptospirose.
2010. 103 f. Tese (Doutorado) - Programa de Pós-Graduação em Biotecnologia. Universidade Federal de Pelotas, Pelotas.
Leptospirose é uma doença infecciosa grave causada por espiroquetas patogênicas do gênero Leptospira, sendo classificada como uma zoonose de ampla distribuição mundial. Esta doença resulta morbidade e mortalidade em humanos e animais, justificando a aplicação de estratégias profiláticas. As vacinas atuais contra a leptospirose são compostas por bactérias inativadas e não estimulam proteção cruzada. Assim, existe a necessidade de desenvolver uma vacina efetiva. No presente estudo, as proteínas de membrana externa LigANI e LipL32 foram utilizadas, pois são apontadas como potenciais vacinógenos. Estas, em sua forma recombinante, costumam ser expressas em Escherichia coli e, como vacina de subunidade tem apresentado eficiência variável. Nós descrevemos neste trabalho a utilização da levedura Pichia pastoris como sistema de expressão alternativo. Os
genes ligANI e lipL32 foram clonados no vetor pPICZαB, que permitiu a expressão
secretória das proteínas em P. pastoris. O rendimento das proteínas neste sistema foi de 276 mg/L para LigANI e 285 mg/L para LipL32. As proteínas recombinantes foram glicosiladas e mantiveram-se antigênicas. O potencial imunoprotetor das proteínas foi avaliado em modelo hamster desafiado com cepa virulenta de L. interrogans sorovar Copenhageni. Ambas as proteínas induziram altas taxas de anticorpos (P < 0,001). Os animais imunizados com LigANI e LipL32, utilizando hidróxido de alumínio como adjuvante, não apresentaram proteção contra o desafio, mas demonstraram um aumento significativo na sobrevida (P < 0,001). Em conclusão, a levedura P. pastoris demonstrou ser um eficiente sistema de expressão heterólogo das proteínas LigANI e LipL32 de L. interrogans. A proteína LigANI secretada e glicosilada pode ser utilizada no controle da leptospirose, embora estudos adicionais sejam necessários.
Palavras-chave: leptospirose; Leptospira interrogans; vacina recombinante; Pichia
ABSTRACT
HARTWIG, Daiane Drawanz. Production antigens from Leptospira interrogans in Pichia pastoris and evaluation of immunoprotective potential against
leptospirosis. 2010. 103 p. Tese (Doutorado) – Programa de Pós-Graduação em
Biotecnologia, Universidade Federal de Pelotas, Pelotas.
Leptospirosis is a serious infectious disease caused by pathogenic spirochetes of the genus Leptospira, it is classified as a zoonosis of worldwide distribution. This disease results morbidity and mortality in humans and animals, justifying the application of prophylactic strategies. Current vaccines against leptospirosis are composed of inactivated bacteria and do not stimulate cross-protection. Thus, there is need to develop a safe and effective vaccine. In this study, we used the outer membrane proteins LigANI e LipL32, because they have been identified as vaccinogens. These, in their recombinant form, are usually expressed in Escherichia coli and as subunit vaccines have shown variable efficacy. We describe in this work the use of Pichia pastoris as an alternative expression system. The genes ligANI and lipL32 were
cloned into vector pPICZαB, which allowed the secretory expression of proteins in P.
pastoris. The protein yield in this system was 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and remained antigenic. The immunoprotective potential was evaluated in the hamster model, challenged with virulent L. interrogans serovar Copenhageni. Both proteins induced high levels of antibodies (P < 0.001). The animals immunized with LigANI and LipL32 using aluminium hydroxide as adjuvant, showed no protection against challenge, but showed a significant increase in survival (P < 0.001). In conclusion, the yeast P. pastoris has proved an efficient heterologous expression system of LigANI and LipL32 L. interrogans proteins. The secreted and glycosylated LigANI protein may be used in the control of leptospirosis, although additional studies are needed.
Keywords: leptospirosis, Leptospira interrogans; recombinant vaccine; Pichia
SUMÁRIO
PRODUÇÃO DE ANTÍGENOS DE Leptospira interrogans EM Pichia pastoris E
AVALIAÇÃO DO POTENCIAL IMUNOPROTETOR CONTRA LEPTOSPIROSE ... 1
RESUMO... 6
ABSTRACT ... 7
1 INTRODUÇÃO GERAL... 10
2 ARTIGO 1 ... 15
LEPTOSPIROSIS: RECENT ADVANCES IN VACCINES AND IMMUNE PROFILE16 3 ARTIGO 2 ... 42
HIGH YIELD EXPRESSION OF LEPTOSPIROSIS VACCINE CANDIDATES LIGA AND LIPL32 IN THE METHYLOTROPHIC YEAST PICHIA PASTORIS……….43 ABSTRACT………...44 BACKGROUND………45 RESULTS………...………...46 DISCUSSION….………..48 CONCLUSIONS…...………50 METHODS……….……51 COMPETING INTERESTS……….55 AUTHORS’ CONTRIBUTIONS………..56 ACKNOWLEDGEMENTS…...56 REFERENCES...57 4 ARTIGO 3 ... 69
IMMUNOPROTECTION BY LIGA AND LIPL32 PRODUCED IN PICHIA PASTORIS AND EVALUATED IN THE HAMSTER MODEL OF LETHAL LEPTOSPIROSIS………...70
ABSTRACT………...71
INTRODUCTION………..72
MATERIAL AND METHODS…...…...………...73
DISCUSSION……...………79
REFERENCES...83
5 CONCLUSÕES...95
6 REFERÊNCIAS...96
1. INTRODUÇÃO GERAL
Leptospirose, causada por bactérias patogênicas do gênero Leptospira, é uma zoonose de importância global que afeta o homem e demais mamíferos (BHARTI,A.R. et al., 2003;FAINE,S.B. et al., 1999;VINETZ,J.M., 2001). A globalização e as desigualdades sociais produzem padrões epidemiológicos divergentes para a leptospirose (MCBRIDE,A.J. et al., 2005;REIS,R.B. et al., 2008). É caracterizada como uma doença re-emergente de maior ocorrência em regiões tropicais e subtropicais, que apresentam condições precárias de saneamento (BHARADWAJ,R., 2004), podendo estar associada ainda a atividades recreacionais, esportivas ou a desastres naturais (DESAI,S. et al., 2009).
Humanos podem infectar-se através do contato com urina de animais portadores de leptospiras patogênicas, principalmente roedores. No entanto, muitos outros animais podem estar envolvidos na transmissão, pois é uma doença comum
entre animais domésticos e silvestres (BHARTI,A.R. et al.,
2003;KOIZUMI,N.;WATANABE,H., 2005a;LEVETT,P.N., 2001). No homem, a apresentação clínica é altamente variável, sendo em sua fase inicial sugestiva de influenza, malária ou dengue, necessitando de um diagnóstico diferencial efetivo em áreas com epidemia ou alta incidência destas doenças (ELLIS,T. et al., 2008). Em sua forma aguda, a leptospirose pode desencadear uma série de sinais clínicos e afetar múltiplos órgãos, incluindo o fígado (icterícia), rins (nefrite), pulmões (hemorragia pulmonar) e cérebro (meningite), com taxas de mortalidade de 10-15%, associadas à doença de Weil, chegando a 70%, nos casos de síndrome hemorrágica pulmonar grave (FAINE,S.B. et al., 1999;GOUVEIA,E.L. et al., 2008;SEGURA,E.R. et al., 2005). Nestes casos graves mesmo com estratégias de intervenção agressivas, as taxas de mortalidade permanecem altas. A expressão gênica aumentada de efetores imunes pró e anti-inflamatórios, induzidos por uma grande carga infectante de leptospiras patogências parece ser a causa de quadros de leptospirose severa (VERNEL-PAUILLAC,F.;GOARANT,C., 2010)
Sendo considerado um problema de saúde pública, somado as perdas econômicas no setor agropecuário, o uso de vacinas contra a leptospirose se justifica em populações de risco. Ainda não existe uma vacina efetiva para uso humano, embora existam ensaios em fase pré-clinica e clínica neste sentido. Em Cuba, foram vacinadas mais de 10.000 pessoas com uma bacterina, obtendo-se
78% de proteção (MARTINEZ,R. et al., 2004). Já na China, o protótipo de vacina testado em humanos não protegeu crianças menores de 14 anos (ZHUO,J.T. et al., 1995). As vacinas em desenvolvimento para uso humano, assim como as disponíveis para uso animal, e que se baseiam na célula inteira inativada de isolados locais, caracterizam-se por induzir imunidade baixa e de curta duração, além de sorovar específica, pois induzem anticorpos contra o lipopolissacarídeo (LPS) destas bactérias, requerendo imunizações anuais (ANDRE-FONTAINE,G. et al., 2003;KOIZUMI,N.;WATANABE,H., 2005a;PETERSEN,A.M. et al., 2001;SONRIER,C. et al., 2000). Estas vacinas em alguns casos podem prevenir o desenvolvimento da doença, mas não a leptospirúria (ALT,D.P. et al., 2001). Existem mais de 270 sorovares patogênicos de Leptospira e esta diversidade antigênica tem sido atribuída a composição do LPS (BULACH,D.M. et al., 2000). Estas limitações dificultam a obtenção de uma vacina multivalente efetiva.
Dentre as leptospiras patogênicas que tiveram seu genoma seqüenciado, L. interrogans contém cerca de 3530 prováveis seqüências codificadoras (CDS) no
sorovar Copenhageni e 3613 no sorovar Lai, enquanto L. borgpetersenii sorovar
Hardjo apresenta 2909 e 2949 CDS para os isolados L550 e JB197, respectivamente (BULACH,D.M. et al., 2006). A análise da seqüência genômica dos isolados de Leptospira seqüenciados tem possibilitado a identificação de novos alvos candidatos ao desenvolvimento da vacina ou de novos testes para diagnóstico. Atualmente, estudos celulares e moleculares destes antígenos têm focado em fatores de mobilidade bacteriana, LPS, proteínas de membrana externa (outer membrane proteins_OMPs) e fatores de virulência (WANG,Z. et al., 2007). Dentre eles, nosso grupo de pesquisa tem avaliado o potencial de OMPs, como a lipoproteína LipL32 e as Leptospiral immunoglobulin-like proteins (Lig).
LipL32, também chamada de proteina-1 associada a hemolisina (Hap-1) (BRANGER,C. et al., 2001), é a OMP mais abundante exposta na superfície celular (CULLEN,P.A. et al., 2005), sendo conservada entre as espécies patogênicas e ausente nas saprófitas (HAAKE,D.A. et al., 2004). Esta proteína é altamente imunogênica e cerca de 95% dos pacientes com leptospirose produzem anticorpos anti-LipL32 durante a infecção (FLANNERY,B. et al., 2001). Além disso, foi demonstrado que ela é expressa durante a infecção em hamsters (HAAKE,D.A. et al., 2000), modelo clássico de estudo para a leptospirose (HAAKE,D.A., 2006). LipL32 é uma proteína ligante de componentes da matriz extracelular (EMC), como
colágeno, fibronectina e laminina (HAUK,P. et al., 2008). As proteínas Ligs também são expostas na superfície de leptospiras patogênicas e têm como característica repetições em tandem de 90 aminoácidos, que constituem domínios, os chamados Big (bacterial immunoglobulin-like repeat domains). Estes domínios foram originalmente identificados em moléculas de adesão de outras bactérias, como intiminas de Escherichia coli e invasinas de Yersinia pseudotuberculosis (HAMBURGER,Z.A. et al., 1999;LUO,Y. et al., 2000). Os genes lig deixam de ser transcritos em cepas de alta passagem, e estão ausentes nas saprófitas (MATSUNAGA,J. et al., 2003;PALANIAPPAN,R.U. et al., 2002;PALANIAPPAN,R.U. et al., 2004). As proteínas Lig medeiam interações com proteínas que compõem a ECM das células do hospedeiro, como fibronectina, fibrinogênio, colágeno, laminina, elastina e tropoelastina (CHOY,H.A. et al., 2007;LIN,Y.P. et al., 2009). O potencial imunoprotetor das proteínas LipL32 e LigA tem sido demonstrado e, para o antígeno LipL32, foi relatado que não há indução de resposta imune protetora quando a proteína recombinante é inoculada com adjuvante, mas este antígeno protege como vacina de DNA (BRANGER,C. et al., 2005) ou quando expresso por adenovírus (BRANGER,C. et al., 2001) ou Mycobacterium bovis BCG (SEIXAS,F.K. et al., 2007). Já para o antígeno LigA tanto sob a forma proteína recombinante (SILVA,E.F. et al., 2007), quanto como vacina de DNA (FAISAL,S.M. et al., 2008) ou utilizando micro-esferas e lipossomos (FAISAL,S.M. et al., 2009) demonstraram proteção em hamsters.
Dentre as vacinas recombinantes existentes: (i) vacinas de subunidade, (ii) vacinas de DNA e (iii) vacinas vetorizadas, as de subunidade recombinante apresentam a clara vantagem de serem licenciadas pelos órgãos de regulamentação competentes (CLARK,T.G.;CASSIDY-HANLEY,D., 2005) e de apresentarem pouco ou nenhum efeito colateral (KOIZUMI,N.;WATANABE,H., 2005b). Para a produção destas subunidades recombinantes tem-se utilizado sistemas de expressão baseados em procariotos e em eucariotos.
Certos procariotos não têm a capacidade de auxiliar no folding da proteína e nem realizar modificações pós-traducionais, as proteínas produzidas neste modelo são expressas na maioria das vezes na forma insolúvel, originando corpúsculos de inclusão, o que leva ao emprego de etapas adicionais de solubilização e re-folding destas proteínas (JENKINS,N. et al., 1996;MELDGAARD,M.;SVENDSEN,I., 1994). A alternativa para a ampla gama de proteínas que não podem ser expressas com
sucesso em Escherichia coli, é produzi-las na levedura metilotrófica Pichia pastoris. Este eucarioto emergiu como um poderoso sistema de expressão heteróloga de proteínas recombinantes (CEREGHINO,J.L.;CREGG,J.M., 2000). A utilização desta plataforma oferece vantagens sobre os sistemas de expressão em procariotos, destacando o alto crescimento em meios de cultura relativamente simples, possibilidade de expandir a produção para escalas industriais, bem como, a presença neste sistema de um forte promotor induzível com metanol (DALY,R.;HEARN,M.T., 2006;MACAULEY-PATRICK,S. et al., 2005). O uso da levedura P. pastoris permite a produção de proteínas com modificações pós-traducionais, como glicosilação e adição de pontes dissulfeto, além disso, há a possibilidade de secreção de proteínas heterólogas de forma solúvel no meio, o que
simplifica etapas de purificação (CEREGHINO,G.P. et al.,
2002;CEREGHINO,J.L.;CREGG,J.M., 2000;GELLISSEN,G., 2000). Até o presente momento, não existem relatos na literatura da avaliação do potencial imunoprotetor de proteínas recombinantes de Leptospira produzidas na levedura P. pastoris.
Este trabalho foi delineado visando produzir proteínas recombinantes de L. interrogans em um sistema eucarioto baseado na levedura metilotrófica P. pastoris. As hipóteses deste estudo foram que as proteínas expressas neste sistema fossem solúveis e apresentassem um rendimento superior ao sistema de expressão baseado em E. coli. Além disso, a secreção destas proteínas permitiria sua glicosilação, característica esta que poderia interferir em sua antigenicidade, imunogenicidade e potencial imunoprotetor. Desta forma, tínhamos como objetivo geral produzir duas proteínas de L. interrogans, LigANI e LipL32, utilizando P. pastoris como sistema de expressão e avaliar seu potencial imunoprotetor em hamsters. Para isso, traçamos os seguintes objetivos específicos: (i) clonar os genes
ligANI e lipL32 no plasmídeo pPICZαB de expressão em P. pastoris, (ii) expressar e
purificar as proteínas LigANI e LipL32 e (iii) avaliar o potencial antigênico, imunogênico e imunoprotetor das proteínas produzidas neste sistema eucarioto.
Os dados gerados nesta tese estão apresentados na forma de artigos científicos. Esta forma de apresentação, comparada ao modelo de tese tradicional, visa propiciar uma divulgação objetiva e rápida dos resultados obtidos. Neste contexto, o artigo 1 trata de uma revisão sobre vacinas e imunidade contra a leptospirose. Neste artigo abordamos os avanços no estudo da imunidade contra Leptospira e também o potencial imunoprotetor em modelos animais de antígenos
avaliados entre leptospiras patogênicas. Esse trabalho está formatado segundo as normas do periódico Expert Review of Vaccines.
Em seguida, o artigo 2 descreve a utilização da levedura P. pastoris na expressão das proteínas LigANI e LipL32 de L. interrogans. Este trabalho relata a expressão secretória destas proteínas em sua forma glicosilada, com rendimento significativamente maior que o obtido quando produzidas em E. coli. Este trabalho está aceito para publicação no periódico Microbial Cell Factories.
Como prosseguimento deste estudo, avaliamos o potencial imunogênico e imunoprotetor das proteínas LigANI e LipL32 produzidas em P. pastoris. Neste estudo, utilizamos o modelo animal hamster em ensaio desafio com cepa virulenta de L. interrogans. Este trabalho originou o artigo 3 desta tese, que está formatado segundo as normas do periódico Clinical and Vaccine Immunology.
2. ARTIGO 1
Leptospirosis: recent advances in vaccines and immune profile
(Revisão formatada segundo as normas do periódico Expert Review of Vaccines)
Leptospirosis: recent advances in vaccines and immune profile
Daiane Drawanz Hartwig1; Fabiana Kömmling Seixas1; Odir Antônio Dellagostin1*
1
Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil
§
Corresponding author: Odir A. Dellagostin, Centro de Biotecnologia, Universidade
Federal de Pelotas, Campus Universitário, Caixa Postal 354, CEP 96010-900, Pelotas, RS, Brazil. Tel. +55 53 3275 7587; Fax +55 53 3275 7551
Summary
The immune response induced by vaccines against leptospirosis composed by whole-cell preparations prevents the disease. However, it has several drawbacks including incomplete, short-term, serovar-specific effects and poor immunological memory. These limitations of the killed whole-cell vaccines highlight the need for obtaining an effective multivalent vaccine preparation and the development of improved immunization protocols. Several leptospiral recombinant proteins have been evaluated regarding their potential for use as vaccine candidates. In this paper, we summarized the current findings on immunity against Leptospira and on leptospiral antigens that have been evaluated as immunogens and that induce protective immunity in animal models.
Keywords: Leptospira; leptospirosis; immunity; vaccines.
Introduction
Leptospirosis, one of the most widespread zoonotic diseases in the world is caused by spirochete Leptospira (1,2,3). It has a higher incidence in tropical and subtropical regions (4). Leptospirosis is an occupational disease which affects humans and animals that come into frequently contact with rodents or polluted water and soil
(4,5)
. Leptospira infection occurs after penetration of the bacterium through mucosa or skin lesion, and is usually an acute disease, however organisms sometimes escape immune defenses and may induced a chronic disease (6). Symptoms range from a mild influenza-like illness, often confused with other febrile diseases, to a severe infection with renal and hepatic failure (Weil’s disease), or severe pulmonary haemorrhage syndrome (SPHS) with a case-fatality rate of 50% or more (7,8).
The immunity against Leptospira is reported traditionally as humoral. It involves the stimulation and maturation of B cells producer of immunoglobulins (Ig) with specificities primarily directed at the polysaccharide components of the leptospiral lipopolysaccharides (LPS) (3). Recently, the role of the cell-mediated immunity in protection against leptospirosis, characterized by CD4 and gammadelta (γδ) T cells, was examined (9,10,11,12). Moreover, it was demonstrated that pathogenic leptospires can stimulate production of type 1 cell-mediated immune (Th1) cytokines (13). The establishment of correlation between the Th1 and Th2 anti-Leptospira immunity is of major importance to understanding the pathogenesis of induced or natural infection as well as to obtain a successful vaccine against leptospirosis.
There are more than 270 pathogenic serovars of Leptospira and this antigenic diversity has been attributed to distribution and composition of the LPS (14). This serological diversity precludes the obtaining of an effective multivalent vaccine and the development of immunization protocols based on whole-cell or membrane preparations. Scientists who work on vaccine development have focused on bacterial mobility, LPS, outer membrane proteins (OMPs) and virulence factors, revised by Wang et. al (15). Recently, many antigens have been evaluated regarding antigenicity and immunogenicity properties. Based on antibody production, lymphocyte proliferation and determination of cytokine profile, studies have shown that constructs tested as vaccine modulated both Th1 and Th2 immune response (16,17,18,19,20,21).
In this review we present the recent advances in the field of immunity and vaccines against leptospirosis. The immunity induced by Leptospira, novel vaccination strategies, vaccine candidates (subunit, vectored, DNA and DNA prime/protein boost vaccines), new forms of antigen presentation and the immunity induced by them are discussed.
Immunity against Leptospira
The first step in the activation of the immune system by Leptospira is the antibody production, but the events involved remain undefined. During the initial stages of infection leptospires evade the host innate immune system and some reports indicate that they acquire complement factor H and fluid-phase regulators (22) using the leptospiral endostatinlike (Len) proteins as ligands (23,24). Spirochete invasion and toxicity of outer membrane components cause robust inflammatory host responses (25). The high production of the pro-inflammatory cytokines causes deleterious effects in the host. The up-regulated gene expression of both pro- and anti-inflammatory immune effectors together with a higher Leptospira burden, suggest that these gene expression levels could be predictors of adverse outcome in leptospirosis (26).
An important finding regarding the innate immune response against leptospiral was that the macrophages activation by leptospiral LPS occurred through CD14 and the Toll-like receptor 2 (TLR2) (27). L. interrogans produces an atypical LPS that differs in several biochemical, physical and biological properties, as degree of acylation, phosphorylation, or the length of acyl chains (28), and this can be responsible for modified pro-inflammatory properties of LPS. Indeed, the TLR2 is the predominant receptor for Gram-positive bacteria and for other bacterial products that are distinct from Gram-negative LPS (29,30,31). Other microorganisms that have an atypical LPS have been reported to signal through TLR2 pathway, like Porphyromonas gingivalis,
Rhizobium, Legionella pneumophila and Helicobacter pylori (32,33,34). L. pneumophila and H. pylori present an atypical lipid A that shows some similarities with the lipid A from Leptospira. This characteristic of the lipid A in Leptospira may be responsible for its ability to adapt and colonize different hosts. However, the role of TLR4 in immunity
against leptospirosis is not ruled out, mediated by a leptospiral ligand(s) other than LPS
(35)
. Nahori et al. demonstrated the existence of an important difference between human and mouse specificity in TLR recognition (36). This may have important consequences for leptospiral LPS sensing and subsequent susceptibility toleptospirosis.
After the entry of the spirochete in the host, T and B cells are stimulated. The initial removal is done by phagocytes, the majority of leptospires is digested in the vacuoles of macrophages and neutrophils, where the phagocytic activity is initiated by opsonizant antibodies (Sambasiva et al., 2003). The antibody response against leptospirosis is classic, starting with a peak of IgM, which is quickly followed by increased IgG levels and this persist for a longer period.
The paradigm in the study of immunity induced by Leptospira is that the protective immunity is not exclusively humoral (3) and the mechanism by which leptospires activate the immune system and the role of cell-mediated immunity in host defense to Leptospira remains poorly understood. Indeed, there were evidences that anti-LPS antibodies are not the only mechanism that play a role in naturally acquired protective immunity (37). This fact was reexamined by other authors and in these works it was showed that the immunity in vaccinated cattle with a protective monovalent serovar Hardjo vaccine is associated with induction of a Th1 response, because the animals produced gamma interferon (IFN-γ) by gammadelta (γδ) T cells, with the remaining cells being CD4 T cells (11,12,38,9). It is speculated that this might be due to the fact that γδ T cell are the first to be stimulated in an infection or inflammatory reaction and the CD4 T cells may be more efficient once they are engaged and expanded.
Direct injury by microbial factors and cytokines produced in response to infection has been proposed to be involved in pathogenesis of leptospirosis. The evaluation of cytokine production against virulent leptospires has been performed in a
lethal hamster model of leptospirosis. The expression levels of cytokine mRNA in the peripheral blood mononuclear cells was evaluated in a kinetic study, and a pronounced expression of Th1 cytokine mRNA, such as the tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-12 (IL-12) was observed (13). In another study the Leptospira infection resulted also in the production of anti-inflammatory cytokines, including transforming growth factor beta (TGF-β) and IL-10 (39). In humans the TNF-α have been reported to be involved in leptospirosis cases and it was demonstrated a significant increase in patients with this disease (40). The expression of this factor in plasma represents a host global response and it was associated with severity of disease and mortality (41). Recently, it has been demonstrated that the human leptospirosis does not seem to generate memory T cells specific for Leptospira or its protein antigens (42). In addition, the first report on global responses of pathogenic Leptospira to innate immunity was published (43). In this work it was revealed that as an immune evasion strategy of L. interrogans it down-regulates the major outer membrane proteins (OMP) and a putative transcription factors may be involved in governing these down-regulations. Concluding, the interaction of
Leptospira with the host immune system components requires further studies for
providing qualified information for selection of vaccine candidates.
Novel vaccination strategies
The drawbacks presented by vaccines prepared from killed whole leptospiral cells highlight the need of new vaccine strategies for the prevention of the leptospirosis. The identification of proteins that elicit protective immunity has become a major focus of current leptospirosis vaccine research. Additionally, the way these antigens are administrated is important. Several leptospiral recombinant vaccines have been
constructed using advanced methods and evaluated in animal models. These include subunit vaccines, DNA vaccines and vectored vaccines.
Subunit vaccines
Research on interaction of spirochetes with the host's immune system has a strong emphasis on OMPs. In fact, these structures have been convincingly shown to activate immune cells via CD14 and TLR2, and recent data also indicate an interaction with LPS binding protein (LBP) (44). Immunization with a combination of the LipL41, a surface-exposed lipoprotein and OmpL1, a transmembrane porin, provided synergistic protection in hamsters (71% survival), higher than protection obtained with these proteins alone (45). This synergism in immune protection may be due to the combination of two membrane proteins classes in the immune system stimulation. The LipL41-attached lipid being required for immunogenicity and/or the membrane conformation of the OmpL1 porin being required to conserve conformational epitopes (46). Other lipoproteins, including rLIC12730 (44%), rLIC10494 (40%) and rLIC12922 (30%) were also evaluated in the same animal model challenged with a lethal dose of a virulent strain of Leptospira (47).
The recombinant Lig proteins (LigA and LigB) induced complete protection in CH3/HeJ mice (48), however the mouse model is not the ideal for leptospirosis studies, because large infective doses are required for disease development. The classic model for leptospirosis is the hamster, due to its susceptibility to infection and reproducibility of the results (49). Using the hamster model, recombinant LigA was evaluated as vaccine candidate against infection by L. interrogans serovar Pomona (50). LigA was truncated into conserved (rLigAcon) and variable (rLigAvar) regions and expressed in
Escherichia coli as a fusion protein with glutathione-S-transferase (GST). The
significant using aluminum hydroxide as adjuvant, and the vaccine conferred sterilizing immunity. One year later the proteins LigA and LigB from L. interrogans serovar Copenhageni were used in the immunization of hamsters using Freund's adjuvant (51). A single fragment, named LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immune protection against mortality (67-100%) in homologue challenge, but this fragment did not confer sterilizing immunity. LigB did not present significant immune protection in this study, but in another (52) this protein was truncated into conserved (LigBcon) and variable (varB1, varB2) fragments and expressed as GST/His-tag fusion proteins. The challenge experiment was performed in hamster model with a virulent L. interrogans serovar Pomona. rLigBcon was able to aford protection (71%), followed by rVarB1 (54%) and rVarB2 (33%). The administration of all three fragments enhanced the protective efficacy of the vaccine (83%).
The efficacy of the subunit vaccine is usually variable and it is attributed to incorrect folding of the recombinant protein (51), or due to low expression, when the recombinant protein is toxic for the cells (53,50). Considering the importance of the protein structural integrity to confer immune protection, new strategies have been developed for recombinant proteins refolding. The recombinant OmpA was produced in
E. coli as an insoluble form and high hydrostatic pressure (HHP) in association with
redox-shuffling reagents (oxidized and reduced glutathione) and guanidine hydrochloride or l-arginine were used to refold aggregated as inclusion bodies (54). About 40% of the protein was refolded and the circular dichroism revealed the presence of secondary structure, and high antibody titers were seen after immunization with this protein, and sera from infected hamsters reacted with soluble OmpA70 (54).
OmpA-like proteins were also evaluated and may serve as novel vaccine candidates for leptospirosis (55). Of the proteins studied, Lp4337 was able to impart maximum protection (75%), followed by Lp3685 (58%) and Lp0222 (42%), against lethal infection of Leptospira in the immunized animals. In a synergist study 12 OMPs were evaluated and three proteins, rLp1454, rLp1118 and rMceII were found to be protective in a hamster model of leptospirosis (71%, 75% and 100%, respectively) and synergistically (87%) against serovar Pomona infection, which may help us to develop a multicomponent vaccine for leptospirosis (56).
Vectored vaccines
A vaccine vectored by adenovirus was tested with the Hap1 (Hemolysis-Associated Protein 1), also known as LipL32 (57) in a gerbil model (58). The adenovirus vector containing this antigen stimulated significant protection against a heterologous
Leptospira challenge, while the recombinant protein did not confer protection (59). Substantial evidences suggest that the immune system immunomodulation and induction of the protective immunity is dependent on cellular mechanism.
The bacillus Calmette-Guerin (BCG), a live attenuated Mycobacterium bovis is used to protect against tuberculosis (60), and is considered a promising candidate as a vector system for delivery of foreign antigens to the immune system. The gene coding for LipL32 was cloned into several mycobacterial vectors for expression in BCG (61). Hamsters immunized with recombinant BCG (rBCG) expressing LipL32 were protected against mortality upon challenge with a lethal inoculum of L. interrogans serovar Copenhageni. Autopsy examination did not reveal macroscopic or histological evidence of disease in rBCG immunized hamsters that survived the lethal challenge. The efficiency of these vectored vaccines may be in its capacity of induced a strong cellular
and humoral immune response against foreign antigens, suggesting that the way the immune system is induced is important for protection against leptospirosis.
DNA vaccines and DNA prime/protein boost
In leptospirosis vaccine development there are reports of DNA immunization and a variation of this technique, called DNA prime, a combination of the DNA and protein immunization. DNA vaccines take advantage of the fact that plasmid DNA can directly transfect animal cells, provide prolonged antigen expression in vivo leading to amplification of the immune response (62). These vaccines appear to offer several advantages, such as easy construction, temperature stability, low cost of mass production and capacity to induce both humoral and cellular immunity (63,64,65).
The first report of Leptospira DNA vaccine evaluation, that presented survival rate, was the immunization of guinea pigs with DNA recombinant plasmid rpDJt expressing protein P68 derived from a genomic library of serovar lai strain 017 (66). The survival percentage of P68 immunized group was 100% and the group rpDJt was 77%, a high percentage for a negative control group. The same animal model was used by outer authors for evaluation of the immune protection induced by the plasmid VR1012 encoding the 33 kDa endoflagellin of L. interrogans serovar lai (67). In this study it was reported 90% of survival compared to control group. Five years later the use of DNA constructs encoding leptospiral protein Hap1 was tested (59). The immune protection was demonstrated using a hamster model with a survival rate of the 60% against a serovar canicola challenge.
The protein OmpL1 of serovar Copenhageni was cloned in a mammalian expression vector pcDNA3.1(+) and the survival evaluated in hamsters challenged with the heterologous serovar Pomona (68). The authors reported that the animals immunized
with pcDNA3.1(+)/ompL1 plasmid DNA presented a survival rate of 33%. This vector was used for expressing the OMP LipL21 of serovar Lai, but in this study guinea pigs were used as model. All animal survived the lethal challenge, and the titer of specific antibodies and stimulation index of splenocytes increased (69). Furthermore, no obvious pathologic changes were observed in the pcDNA3.1(+)/lipL21 immunized guinea pigs. Still on the use of OMPs in the DNA vaccines evaluation, three antigens were cloned into a pVAX1 plasmid using a linking prime PCR method to construct a
lipL32-lipL41-ompL1 fusion gene (70). BALB/c mice were immunized using DNA-DNA, DNA-protein (DNA prime) and protein-protein strategies. The groups receiving the recombined LipL32-LipL41-OmpL1 vaccine had anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategy stimulated more antibodies than DNA-DNA and yielded greater cytokine and splenocyte proliferation than protein-protein. In this study the authors did not evaluate the immune protective potential.
As mentioned before, the recombinant protein LigA induced significant protection against serovar Pomona challenge in hamsters. In another study it was demonstrated the protective efficacy of a LigA DNA vaccine (21). The LigA DNA vaccine was constructed in two truncated forms: a conserved portion (LigAcon) and a variable portion (LigAvar) and challenge with a virulent serovar Pomona. In this study all groups immunized with LigA constructs presented 100% of survival, however the control groups also had high level (62%).
New forms of antigen delivery
The development of the novel ways of antigen presentation and availability of improved adjuvants suitable for clinical use is highly desirable and necessary. Adjuvants play a pivotal role in vaccination, principally when the vaccine antigen itself
has only weak immunogenicity. Actually, aluminum hydroxide is the adjuvant licenced for use in vaccine formulations for human use, however if it is used for many times, it can cause severe toxics reactions such as erythema, subcutaneous nodules and contact hypersensivity. Additionally, it is unable to activate the cell mediated immunity (71,72,73).
Therefore, delivery vehicles that act as adjuvants have been evaluated against various infectious diseases, such as leptospirosis. Liposomes from total polar lipids of non-pathogenic L. biflexa serovar Patoc were evaluated as deliveries of Lp0607, Lp1118 and Lp1454 of L. interrogans serovar Pomona in a hamster model (74). The protective efficacy of the leptosomes (so called by the authors) based vaccines was 75%. These leptossomes are phospholipids vesicles that elicit humoral and cell mediated immunity (75,76). These authors that tested leptosomes in preliminary studies, evaluated smegmossomes (vesicles originated of the polar lipids from Mycobacterium
smegmatis), testing the same antigens (77). The vaccine constructions evaluated by them demonstrate that 75% of the animals survival the challenge, compared to only 37% survival rate in the aluminum hydroxide group.
PLGA microspheres were used for LigA delivery (78). Microspheres are composed of poly-lactide co-glycolides, that are biodegradable and biocompatible components (79). LigA protein presented by this vehicle to the immune system demonstrated that 75% of the hamsters were protected, but aluminum hydroxide alone protects 50% of them. The use of particulate adjuvants in subunit vaccines present success because prevent antigen degradation, enhancing its presentation to professional APCs including macrophages and dendritic cells, immunostimulating components such as TLR ligants, toxins and cytokines, thus inducing humoral and cell mediated immune responses.
Immunity stimulated by new vaccines
Currently, a considerable number of antigens used in vaccine formulations have been evaluated regarding the immune response profile induced based on antibody production, lymphocyte proliferation and determination of cytokine profile. Most recombinant vaccines induced strong humoral responses with high levels of IgG, Th2 citokynes (IL-4, IL-10) and cell mediated immunity marked by T cell proliferation and Th1 citokynes (IFN-γ) production (77,74,78,16,21). The cytokines are responsible for activation, differentiation and cell proliferation, acting on its target cells through specific receptors and may provide a useful method for the accurate study of mechanisms of anti-Leptospira immunity, indications of prognostic factors and evaluation of the effectiveness of the vaccine against leptospirosis (26). IL-4 is secreted by Th2 cells, which are the major modulating cells of humoral immunity. IL-4 can promote proliferation of B cells and it can also regulate the Th1/Th2 cytokine balance
(80)
. IL-10 is classically described as an anti-inflammatory cytokine with effects in immune regulation and inflammation by down-regulating the expression of Th1 cytokines (81). IFN-γ is a potent pro-inflammatory cytokine (82). Its production was shown as dependent on IL12p40 in human blood stimulated by L. interrogans notably inhibiting Th2 cell activity (83).
Conclusions
The findings reviewed in this work represent recent progress made in the
Leptospira immnunity and recombinant vaccine development against leptospirosis.
Many antigens have been expressed in different heterologous systems and some have shown to provide protection. A number of different factors have been evaluated and identified as important in the induction of immune response. With these important
findings, the search for an efficient and broad serovar-range vaccine against leptospirosis rapidly progressing.
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3. ARTIGO 2
High yield expression of leptospirosis vaccine candidates LigA and
LipL32 in the methylotrophic yeast Pichia pastoris
High yield expression of leptospirosis vaccine candidates LigA and
LipL32 in the methylotrophic yeast Pichia pastoris
Daiane D. Hartwig1, Thaís L. Oliveira1, Fabiana K. Seixas1, Karine M. Forster1, Caroline Rizzi1,Cláudia P. Hartleben1, Alan J. A. McBride2, Odir A. Dellagostin1§
1
Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil
2
Laboratório de Patologia e Biologia Molecular, Instituto Gonçalo Moniz, Fiocruz-BA, Salvador, BA, Brazil
§
Corresponding author: Alan J. A. McBride; Odir A. Dellagostin, Centro de Biotecnologia,
Universidade Federal de Pelotas, Campus Universitário, Caixa Postal 354, CEP 96010-900, Pelotas, RS, Brazil. Tel. +55 53 3275 7587; Fax +55 53 3275 7551
Email addresses:
DDH: daianehartwig@gmail.com CR: ccrizzi@yahoo.com.br
TLO: thais.larreoliveira@gmail.com CPH: claudia.fernandes@ufpel.tche.br FKS: seixas.fk@gmail.com AJAM: alanm@bahia.fiocruz.br KMF: kmacielforster@yahoo.com.br OAD: odir@ufpel.edu.br
Abstract
BackgroundLeptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.
Results
The vaccine candidates LigANI and LipL32 were cloned and expressed in P. pastoris as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis.
Conclusions
The expression of LigANI and LipL32 in P. pastoris resulted in a significant increase in yield compared to expression in E. coli. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.
