RESUMO
The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1ß, IL-2, IFN-γ, and TNF-α. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4+ T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Imunidade Celular , Microrganismos Geneticamente Modificados/imunologia , Mycobacterium bovis/imunologia , Neoplasias da Bexiga Urinária/terapia , Adulto , Idoso , Animais , Linfócitos T CD4-Positivos/patologia , Linhagem Celular Tumoral , Citocinas/imunologia , Feminino , Humanos , Masculino , Camundongos , Microrganismos Geneticamente Modificados/genética , Pessoa de Meia-Idade , Mycobacterium bovis/genética , Toxina Pertussis/genética , Toxina Pertussis/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine. Initially, three engineering runs and two cGMP runs were performed in 60-L bioreactors, demonstrating the consistency of the production process, as evaluated by the growth curves, glucose consumption and metabolite formation (lactate and acetate). Cell recovery by tangential filtration was 92 ± 13 %. We optimized the conditions for beta-propiolactone (BPL) inactivation of the bacterial suspensions, establishing a maximum cell density of OD600 between 27 and 30, with a BPL concentration of 1:4000 (v/v) at 150 rpm and 4 °C for 30 h. BPL was hydrolyzed by heating for 2h at 37 °C. The criteria and methods for quality control were defined using the engineering runs and the cGMP Lots passed all specifications. cGMP vaccine Lots displayed high potency, inducing between 80 and 90% survival in immunized mice when challenged with virulent pneumococci. Sera from mice immunized with the cGMP Lots recognized several pneumococcal proteins in the extract of encapsulated strains by Western blot. The cGMP whole cell antigen bulk and whole cell vaccine product lots were shown to be stable for up to 12 and 18 months, respectively, based upon survival assays following i.p. challenge. Our results show the consistency and stability of the cGMP whole cell pneumococcal vaccine lots and demonstrate the feasibility of production in a developing country setting.
Assuntos
Reatores Biológicos , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/biossíntese , Propiolactona/farmacologia , Animais , Anticorpos Antibacterianos/sangue , Feminino , Fermentação , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Vacinas Pneumocócicas/imunologia , Controle de QualidadeRESUMO
Mycobacterium bovis BCG has long been investigated as a candidate for heterologous antigen presentation. We have previously described an rBCG-Pertussis that confers protection against challenge with Bordetella pertussis in neonate and adult mice. In order to obtain stable expression in vivo, we constructed an unmarked BCG lysine auxotrophic and a complementation vector containing the lysine and the genetically detoxified S1 pertussis toxin genes, both under control of the same promoter. Complemented BCG-Delta lysine growth and expression of the pertussis antigen were stable, without the use of an antibiotic marker. Our results show that the complemented rBCG-Delta lysA-S1PT-lysA(+)(kan(-)), which is now suitable to be evaluated in clinical trials, maintains similar characteristics of the original rBCG-pNL71S1PT strain, such as the antigen expression level, cellular immune response and protection against the same model challenge in neonatal-immunized mice.
Assuntos
Antígenos de Bactérias/imunologia , Mycobacterium bovis/imunologia , Toxina Pertussis/imunologia , Vacina contra Coqueluche/imunologia , Coqueluche/prevenção & controle , Animais , Animais Recém-Nascidos , Bordetella pertussis/imunologia , Clonagem Molecular , Vetores Genéticos , Imunidade Celular , Interferon gama/imunologia , Camundongos , Proteínas Recombinantes de Fusão/imunologia , Transdução Genética , Fator de Necrose Tumoral alfa/imunologia , Coqueluche/imunologiaRESUMO
OBJECTIVE: to discuss the current PAHO recommendation that does not support the substitution of traditional cellular DTP vaccine by acellular DTP, and the role of mutations, in humans, as the main cause of rare adverse events, such as epileptic-like convulsions, triggered by pertussis vaccine. DATA REVIEW: the main components related to toxic effects of cellular pertussis vaccines are the lipopolysaccharide of bacterial cell wall and pertussis toxin. The removal of part of lipopolysaccharide layer has allowed the creation of a safer cellular pertussis vaccine, with costs comparable to the traditional cellular vaccine, and which may be a substitute for the acellular vaccine. CONCLUSION: The new methodology introduced by Instituto Butantan allows for the development of a new safer pertussis vaccine with low LPS content (Plow), and the use of the lipopolysaccharide obtained in the process in the production of monophosphoryl lipid A. This component has shown potent adjuvant effect when administered together with influenza inactivated vaccine, making possible to reduce the antigen dose, enhancing the production capacity and lowering costs.
Assuntos
Vacina contra Difteria, Tétano e Coqueluche/efeitos adversos , Vacinas contra Difteria, Tétano e Coqueluche Acelular/efeitos adversos , Lipopolissacarídeos/imunologia , Mutação , Análise Custo-Benefício , Vacina contra Difteria, Tétano e Coqueluche/genética , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Vacinas contra Difteria, Tétano e Coqueluche Acelular/genética , Vacinas contra Difteria, Tétano e Coqueluche Acelular/imunologia , Humanos , Lipopolissacarídeos/efeitos adversos , Organização Mundial da SaúdeRESUMO
Objective: to discuss the current PAHO recommendation that does not support the substitution of traditional cellular DTP vaccine by acellular DTP, and the role of mutations, in humans, as the main cause of rare adverse events, such as epileptic-like convulsions, triggered by pertussis vaccine. Data review: the main components related to toxic effects of cellular pertussis vaccines are the lipopolysaccharide of bacterial cell wall and pertussis toxin. The removal of part of lipopolysaccharide layer has allowed the creation of a safer cellular pertussis vaccine, with costs comparable to the traditional cellular vaccine, and which may be a substitute for the acellular vaccine. Conclusion: The new methodology introduced by Instituto Butantan allows for the development of a new safer pertussis vaccine with low LPS content (Plow), and the use of the lipopolysaccharide obtained in the process in the production of monophosphoryl lipid A. This component has shown potent adjuvant effect when administered together with influenza inactivated vaccine, making possible to reduce the antigen dose, enhancing the production capacity and lowering costs.
Objetivo: Discutir as recomendações da WHO-OPAS que não consideram indicada a substituição da vacina DTP celular clássica pela DTP acelular e o papel de mutações, em humanos, como principal causa dos raros eventos de convulsões epileptiformes desencadeadas pela vacina pertussis. Revisão dos dados: Os principais componentes relacionados aos efeitos tóxicos da vacina pertussis celular são o lipopolissacarídio da parede celular da bactéria e a toxina pertussis. A remoção de parte da camada lipopolissacarídica permitiu a criação de uma vacina pertussis celular, mais segura e de custo comparável ao da vacina celular tradicional, podendo substituir a vacina pertussis acelular. Conclusão: A nova vacina pertussis, com baixo teor de LPS (Plow) desenvolvida pelo Instituto Butantan, além de oferecer uma vacina mais segura, permite o aproveitamento do lipopolissacarídeo para a produção de monofosforil lipídeo A. Esse componente mostrou-se potente como adjuvante e altamente eficiente quando administrado com a vacina de influenza, levando à possibilidade de se reduzir a dose de antígeno, aumentando a capacidade de produção e redução dos custos.
Assuntos
Humanos , Vacina contra Difteria, Tétano e Coqueluche/efeitos adversos , Vacinas contra Difteria, Tétano e Coqueluche Acelular/efeitos adversos , Lipopolissacarídeos/imunologia , Mutação , Análise Custo-Benefício , Vacina contra Difteria, Tétano e Coqueluche/genética , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Vacinas contra Difteria, Tétano e Coqueluche Acelular/genética , Vacinas contra Difteria, Tétano e Coqueluche Acelular/imunologia , Lipopolissacarídeos/efeitos adversos , Organização Mundial da SaúdeRESUMO
The currently used pertussis vaccines are highly efficacious; however, neonates are susceptible to whooping cough up to the sixth month. In agreement, DTP-immunized neonate mice were not protected against intracerebral challenge with Bordetella pertussis. Neonate mice immunized with either DTP or a recombinant-BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin do not show a humoral immune response against PT. On the other hand, rBCG-Pertussis induces higher PT-specific IFN-gamma production and an increase in both IFN-gamma(+) and TNF-alpha(+)-CD4(+)-T cells than the whole cell pertussis vaccine and confers protection against a lethal intracerebral challenge with B. pertussis.
Assuntos
Bordetella pertussis/imunologia , Mycobacterium bovis/genética , Toxina Pertussis/imunologia , Vacina contra Coqueluche/imunologia , Coqueluche/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Bordetella pertussis/genética , Linfócitos T CD4-Positivos/imunologia , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Humanos , Recém-Nascido , Interferon gama/biossíntese , Camundongos , Toxina Pertussis/genética , Vacina contra Coqueluche/genética , Análise de Sobrevida , Fator de Necrose Tumoral alfa/biossíntese , Coqueluche/imunologiaRESUMO
A mycobacterial codon-optimized gene encoding the Sm14 antigen of Schistosoma mansoni was generated using oligonucleotide assembly. This synthetic gene enhanced approximately fourfold the protein expression level in recombinant Mycobacterium bovis Bacille Calmette-Guérin (rBCG) when compared to that obtained using the native gene in the same expression vector. Immunization of mice with rBCG expressing Sm14 via the synthetic gene induced specific cellular Th1-predominant immune responses, as determined by interferon-gamma production of Sm14-stimulated splenocytes, which were comparable to those recorded in animals immunized with an rBCG strain expressing the native gene. Administration of a single dose of the rBCG-Sm14 construct carrying the synthetic gene conferred protection against cercarial challenge in outbred Swiss mice, at a level equivalent to those provided by either a single dose of rBCG expressing the native gene or three doses of Escherichia coli-derived recombinant Sm14. Our data demonstrated that despite improving the level of antigen expression, the codon optimization strategy did not result in enhanced immunity or protection against cercarial S. mansoni challenge.
Assuntos
Vacina BCG/imunologia , Proteínas de Transporte de Ácido Graxo/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/farmacologia , Schistosoma mansoni/química , Esquistossomose mansoni/prevenção & controle , Animais , Vacina BCG/administração & dosagem , Códon/genética , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/imunologia , Proteínas de Transporte de Ácido Graxo/uso terapêutico , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Schistosoma mansoni/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas SintéticasRESUMO
A conjugate meningococcal vaccine against serogroup B/C consisting of capsular PS (polysaccharide) from serogroup C conjugated to OMV (outer membrane vesicle) from serogroup B would be a very useful vaccine in regions where there is a prevalence of both serogroups, for example in Brazil. For this purpose, the conjugation method that uses ADHy (adipic acid dihydrazide) as spacer and a carbodi-imide derivative, EDAC [1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide], as catalyser was optimized looking for synthesis yield and maintenance of the antigenicity of both components. The best synthesis conditions preserving the vaccine immunogenicity resulted in a final yield of approx. 17%. Immunogenicity of the vaccine was highest when 10% of the sialic acid residues of the PS were occupied by the ADHy spacer. Sterilization of the conjugate by filtration through a 0.22-microm-pore-size membrane resulted in a low recovery of protein and PS (approximately 50%), although the vaccine immunogenicity was maintained. Using gamma irradiation on freeze-dried sample, it was possible to maintain the integrity of OMV structure and, consequently, its ability to induce bactericidal antibodies.
Assuntos
Vacinas Meningocócicas/imunologia , Polissacarídeos Bacterianos/imunologia , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia , Adipatos/química , Animais , Cápsulas Bacterianas , Etildimetilaminopropil Carbodi-Imida/química , Feminino , Raios gama , Vacinas Meningocócicas/efeitos da radiação , Camundongos , Teste Bactericida do Soro , Ultrafiltração , Vacinas Conjugadas/efeitos da radiaçãoRESUMO
We have constructed vectors that permit the expression in Escherichia coli of Schistosoma mansoni fatty acid-binding protein 14 (Sm14) in fusion with the nontoxic, but highly immunogenic, tetanus toxin fragment C (TTFC). The recombinant six-His-tagged proteins were purified by nickel affinity chromatography and used in immunization and challenge assays. Animals inoculated with TTFC in fusion with or coadministered with Sm14 showed high levels of tetanus toxin antibodies, while animals inoculated with Sm14 in fusion with or coadministered with TTFC showed high levels of Sm14 antibodies. In both cases, there were no changes in the type of immune response (Th2) obtained with the fusion proteins compared to those obtained with the nonfused proteins. Mice immunized with the recombinant proteins (TTFC in fusion with or coadministered with Sm14) survived the challenge with tetanus toxin and did not show any symptoms of the disease. Control animals inoculated with either phosphate-buffered saline (PBS) or Sm14 died with severe symptoms of tetanus after 24 h. Mice immunized with the recombinant proteins (Sm14 in fusion with or coadministered with TTFC) showed a 50% reduction in worm burden when they were challenged with S. mansoni cercariae, while control animals inoculated with either PBS or TTFC were not protected. The results show that the expression of other antigens in fusion at the carboxy terminus of TTFC is feasible for the development of a multivalent recombinant vaccine.
Assuntos
Proteínas de Transporte/imunologia , Proteínas de Helminto/imunologia , Proteínas de Membrana Transportadoras , Fragmentos de Peptídeos/imunologia , Schistosoma mansoni , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Toxina Tetânica/imunologia , Tétano/imunologia , Tétano/prevenção & controle , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Anti-Helmínticos/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte de Ácido Graxo , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Esquistossomose mansoni/parasitologia , Taxa de Sobrevida , Tétano/induzido quimicamente , Toxina Tetânica/administração & dosagem , Toxina Tetânica/genética , Toxina Tetânica/isolamento & purificação , Toxina Tetânica/toxicidade , Vacinação , Vacinas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Meningococcal outer membrane vesicle (OMV) vaccines are weak antigens in infants. This study aimed at investigating alternative adjuvants for induction of functional antibodies in newborn mice. Serogroup B/C anti-meningococcal vaccines, consisting of capsular polysaccharide from serogroup C (PSC) conjugated to OMV from one serogroup B serosubtype prevalent in Brazil, combined with OMV from another prevalent serosubtype, were tested in newborn and adult mice with the following adjuvants: aluminum hydroxide, MPL (monophosphoryl lipid A), Titermax and MF59. Total IgG, IgG avidity index determination and bactericidal assay were performed with sera from immunized mice. Antibodies induced against PSC in newborn mice showed avidity and bactericidal titers, similar to those obtained in adult mice, independently of the adjuvant. Evidence is presented that the inclusion of MF59 enhanced the immune response against OMV in newborn mice.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Neisseria meningitidis Sorogrupo C/imunologia , Vacinas Conjugadas/imunologia , Animais , Animais Recém-Nascidos , Afinidade de Anticorpos , Proteínas da Membrana Bacteriana Externa/imunologia , Atividade Bactericida do Sangue , Modelos Animais de Doenças , Humanos , Imunoglobulina G/sangue , Infecções Meningocócicas/microbiologia , Vacinas Meningocócicas/administração & dosagem , Camundongos , Polissacarídeos Bacterianos/imunologia , Polissorbatos/administração & dosagem , Esqualeno/administração & dosagem , Vacinas Conjugadas/administração & dosagemRESUMO
The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall. The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability. The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14. Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S. mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli. The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries.
Assuntos
Proteínas de Transporte/imunologia , Proteínas de Helminto/imunologia , Proteínas de Membrana Transportadoras , Mycobacterium bovis/genética , Schistosoma mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Vacinas de DNA/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Bovinos , Células Cultivadas , Proteínas de Transporte de Ácido Graxo , Feminino , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Monócitos , Mycobacterium fortuitum/enzimologia , Mycobacterium fortuitum/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/patogenicidade , Esquistossomose mansoni/parasitologia , Vacinas de DNA/administração & dosagem , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M. fortuitum beta-lactamase promoter, pBlaF*. Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol. Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile. Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response. Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin. Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin. Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antibacterianos/imunologia , Vacina BCG/imunologia , Proteínas de Bactérias/biossíntese , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Toxina Tetânica/imunologia , Animais , Formação de Anticorpos/imunologia , Proteínas de Bactérias/genética , Western Blotting , Chlorocebus aethiops , Toxina Diftérica/antagonistas & inibidores , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/crescimento & desenvolvimento , Testes de Neutralização , Sorotipagem , Toxina Tetânica/antagonistas & inibidores , Toxina Tetânica/biossíntese , Vacinas Combinadas , Vacinas Sintéticas/imunologia , Células VeroRESUMO
Streptococcus pneumoniae is a major public health problem and new strategies for the development of cost-effective alternative vaccines are important. The use of protein antigens such as PspA (pneumococcal surface protein A) is a promising approach to increase coverage at reduced costs. We have previously described the induction of a strong antibody response by a DNA vaccine expressing a C-terminal fragment of PspA. Fusion of this fragment with the cytoplasmic variant of SV40 large T-antigen (CT-Ag) caused reduction in specific interferon-gamma produced by stimulated spleen cells. In this work we show that the DNA vaccine expressing the C-terminal region of PspA elicits significant protection in mice against intraperitoneal challenge with a virulent strain of S. pneumoniae. Furthermore, fusion with CT-Ag completely abrogated the protection elicited by DNA immunization with this fragment. In this case, protection did not correlate with total anti-PspA antibody production nor with total IgG2a levels. The anti-PspA sera obtained from both constructs showed equivalent opsonic activity of pneumococci, indicating that the antibodies produced were functional. We could, though, observe a correlation between a lower IgG1:IgG2a ratio, which is indicative of a stronger bias towards Th1 responses, and protection. We also show that a vector expressing the most variable N-terminal alpha-helical region induces higher antibody formation, with increased protection of mice against intraperitoneal challenge with a more virulent strain of S. pneumoniae. As a whole, these results indicate that antibodies elicited against PspA would not be solely responsible for the protection induced by DNA vaccination and that cell-mediated immune responses could also be involved in protection against pneumococcal sepsis.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Células Th1/imunologia , Animais , Proteínas de Bactérias/genética , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Opsonizantes , Fagocitose , Vacinas Pneumocócicas/administração & dosagem , Streptococcus pneumoniae/patogenicidade , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
Streptococcus pneumoniae is a major cause of disease, especially in developing countries, and cost-effective alternatives to the currently licensed vaccines are needed. We constructed DNA vaccines based on pneumococcal surface protein A (PspA), an antigen shown to induce protection against pneumococcal bacteremia. PspA fragments can be divided into three families, which can be subdivided into six clades, on the basis of PspA amino acid sequence divergence (S. K. Hollingshead, R. Becker, and D. E. Briles, Infect. Immun. 68:5889-5900, 2000). Since most clinical isolates belong to family 1 or family 2, PspA fragments from members of both of these families were analyzed. Vectors encoding the complete N-terminal regions of PspAs elicited significant humoral responses, and cross-reactivity was mainly restricted to the same family. DNA vaccines encoding fusions between PspA fragments from family 1 and family 2 were also constructed and were able to broaden the cross-reactivity, with induction of antibodies that showed reactions with members of both families. At least for the pneumococcal strains tested, the cross-reactivity of antibodies was not reflected in cross-protection. Animals immunized with DNA vaccines expressing the complete N-terminal regions of PspA fragments were protected only against intraperitoneal challenge with a strain expressing PspA from the same clade.