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Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum , the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti- P. vivax vaccine.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Millennium Institute for Gene Therapy (Brazil) Recently, we described a heterologous prime-boost strategy using plasmid DNA followed by replication-defective human recombinant adenovirus type 5 as a powerful strategy to elicit long-lived CD8(+) T-cell-mediated protective immunity against experimental systemic infection of mice with a human intracellular protozoan parasite, Trypanosoma cruzi. in the present study, we further characterized the protective long-lived CD8(+) T cells. We compared several functional and phenotypic aspects of specific CD8(+) T cells present 14 or 98 days after the last immunizing dose and found the following: (i) the numbers of specific cells were similar, as determined by multimer staining or by determining the number of gamma interferon (IFN-gamma)-secreting cells by enzyme-linked immunospot (ELISPOT) assay; (ii) these cells were equally cytotoxic in vivo; (iii) following in vitro stimulation, a slight decline in the frequency of multifunctional cells (CD107a(+) IFN-gamma(+) or CD107a(+) IFN-gamma(+) tumor necrosis factor alpha positive [TNF-alpha(+)]) was paralleled by a significant increase of CD107a singly positive cells after 98 days; (iv) the expression of several surface markers was identical, except for the reexpression of CD127 after 98 days; (v) the use of genetically deficient mice revealed a role for interleukin-12 (IL-12)/IL-23, but not IFN-gamma, in the maintenance of these memory cells; and (vi) subsequent immunizations with an unrelated virus or a plasmid vaccine or the depletion of CD4(+) T cells did not significantly erode the number or function of these CD8(+) T cells during the 15-week period. From these results, we concluded that heterologous plasmid DNA prime-adenovirus boost vaccination generated a stable pool of functional protective long-lived CD8(+) T cells with an effector memory phenotype. Universidade Federal de São Paulo, Escola Paulista Med, UNIFESP, CTCMol, BR-04044010 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04044010 São Paulo, Brazil Fiocruz MS, Ctr Pesquisas Rene Rachou, BR-30190002 Belo Horizonte, MG, Brazil Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, BR-31270901 Belo Horizonte, MG, Brazil Univ Massachusetts, Sch Med, Dept Med, Div Infect Dis & Immunol, Worcester, MA 01655 USA Univ Fed Minas Gerais, Dept Microbiol, Inst Ciencias Biol, BR-31270901 Belo Horizonte, MG, Brazil Universidade Federal de São Paulo, Escola Paulista Med, UNIFESP, CTCMol, BR-04044010 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04044010 São Paulo, Brazil FAPESP: 2006/1983-4 FAPESP: 2009/06820-4 CNPq: 420067/2005-1 Web of Science

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Millennium Institute for Vaccine Development and Technology Millennium Institute for Gene Therapy Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4(+) and CD8(+) T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4(+) and CD8(+) T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-gamma) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. in spite of a normal numeric expansion, specific CD8(+) T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-gamma or IFN-gamma/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-gamma in the presence of highly cytotoxic T cells. Vaccinated IFN-gamma-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-gamma in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy. Universidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, BR-04044010 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04044010 São Paulo, Brazil Univ Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Ctr Ciencias Saude, BR-21941 Rio de Janeiro, Brazil Fiocruz MS, Inst Oswaldo Cruz, Lab Biol Celular & Biol Interacoes, BR-21045900 Rio de Janeiro, Brazil Univ Fed Minas Gerais, Dept Bioquim & Imunol, Inst Ciencias Biol, BR-31270901 Belo Horizonte, MG, Brazil Fiocruz MS, Ctr Pesquisas Rene Rachou, BR-30190002 Belo Horizonte, MG, Brazil Univ Massachusetts, Sch Med, Dept Med, Div Infect Dis & Immunol, Worcester, MA 01605 USA Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Microbiol, BR-31270901 Belo Horizonte, MG, Brazil Universidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, BR-04044010 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04044010 São Paulo, Brazil FAPESP: 2006/1983-4 Millennium Institute for Vaccine Development and Technology: CNPq-420067/2005-1 FAPEMIG: EDT 24.000 Web of Science

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) duringBrucella abortusinfection. CHO reporter cell lines transfected with CD14 and TLRs showed thatB. abortustriggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived fromBrucellarough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-α) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A fromRhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers ofBrucellawere monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks followingB. abortusinfection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance toB. abortusinfection and that S lipid A has potent adjuvant activity.