The immunodominant antibody response to Zika virus NS1 protein is characterized by cross-reactivity to self.

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations.

A recombinant multi-antigen vaccine formulation containing Babesia bovis merozoite surface antigens MSA-2a1, MSA-2b and MSA-2c elicits invasion-inhibitory antibodies and IFN-γ producing cells.

BACKGROUND: Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides. RESULTS: The antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4+ T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c. CONCLUSIONS: These data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.

A Human Trypanosome Suppresses CD8+ T Cell Priming by Dendritic Cells through the Induction of Immune Regulatory CD4+ Foxp3+ T Cells.

Although CD4+ Foxp3+ T cells are largely described in the regulation of CD4+ T cell responses, their role in the suppression of CD8+ T cell priming is much less clear. Because the induction of CD8+ T cells during experimental infection with Trypanosoma cruzi is remarkably delayed and suboptimal, we raised the hypothesis that this protozoan parasite actively induces the regulation of CD8+ T cell priming. Using an in vivo assay that eliminated multiple variables associated with antigen processing and dendritic cell activation, we found that injection of bone marrow-derived dendritic cells exposed to T. cruzi induced regulatory CD4+ Foxp3+ T cells that suppressed the priming of transgenic CD8+ T cells by peptide-loaded BMDC. This newly described suppressive effect on CD8+ T cell priming was independent of IL-10, but partially dependent on CTLA-4 and TGF-ß. Accordingly, depletion of Foxp3+ cells in mice infected with T. cruzi enhanced the response of epitope-specific CD8+ T cells. Altogether, our data uncover a mechanism by which T. cruzi suppresses CD8+ T cell responses, an event related to the establishment of chronic infections.

The Combined Deficiency of Immunoproteasome Subunits Affects Both the Magnitude and Quality of Pathogen- and Genetic Vaccination-Induced CD8+ T Cell Responses to the Human Protozoan Parasite Trypanosoma cruzi.

The ß1i, ß2i and ß5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-γ (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected ß1i, ß2i and ß5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-γ+/TNF+) or single-positive (IFN-γ+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses.

Adenovirus vector-induced CD8⁺ T effector memory cell differentiation and recirculation, but not proliferation, are important for protective immunity against experimental Trypanosoma cruzi Infection.

Heterologous prime-boost vaccination using plasmid DNA followed by replication-defective adenovirus vector generates a large number of specific CD8⁺ T effector memory (TEM) cells that provide long-term immunity against a variety of pathogens. In the present study, we initially characterized the frequency, phenotype, and function of these T cells in vaccinated mice that were subjected to infectious challenge with the human protozoan parasite Trypanosoma cruzi. We observed that the frequency of the specific CD8⁺ T cells in the spleens of the vaccinated mice increased after challenge. Specific TEM cells differentiated into cells with a KLRG1(High) CD27(Low) CD43(Low) CD183(Low)T-bet(High) Eomes(Low) phenotype and capable to produce simultaneously the antiparasitic mediators IFNγ and TNF. Using the gzmBCreERT2/ROSA26EYFP transgenic mouse line, in which the cells that express Granzyme B after immunization, are indelibly labeled with enhanced yellow fluorescent protein, we confirmed that CD8⁺ T cells present after challenge were indeed TEM cells that had been induced by vaccination. Subsequently, we observed that the in vivo increase in the frequency of the specific CD8⁺ T cells was not because of an anamnestic immune response. Most importantly, after challenge, the increase in the frequency of specific cells and the protective immunity they mediate were insensitive to treatment with the cytostatic toxic agent hydroxyurea. We have previously described that the administration of the drug FTY720, which reduces lymphocyte recirculation, severely impairs protective immunity, and our evidence supports the model that when large amounts of antigen-experienced CD8⁺ TEM cells are present after heterologous prime-boost vaccination, differentiation, and recirculation, rather than proliferation, are key for the resultant protective immunity.

Immunogenicity of a prime-boost vaccine containing the circumsporozoite proteins of Plasmodium vivax in rodents.

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.

Relevance of long-lived CD8(+) T effector memory cells for protective immunity elicited by heterologous prime-boost vaccination.

Owing to the importance of major histocompatibility complex class Ia-restricted CD8(+) T cells for host survival following viral, bacterial, fungal, or parasitic infection, it has become largely accepted that these cells should be considered in the design of a new generation of vaccines. For the past 20 years, solid evidence has been provided that the heterologous prime-boost regimen achieves the best results in terms of induction of long-lived protective CD8(+) T cells against a variety of experimental infections. Although this regimen has often been used experimentally, as is the case for many vaccines, the mechanism behind the efficacy of this vaccination regimen is still largely unknown. The main purpose of this review is to examine the characteristics of the protective CD8(+) T cells generated by this vaccination regimen. Part of its efficacy certainly relies on the generation and maintenance of large numbers of specific lymphocytes. Other specific characteristics may also be important, and studies on this direction have only recently been initiated. So far, the characterization of these protective, long-lived T cell populations suggests that there is a high frequency of polyfunctional T cells; these cells cover a large breadth and display a T effector memory (TEM) phenotype. These TEM cells are capable of proliferating after an infectious challenge and are highly refractory to apoptosis due to a control of the expression of pro-apoptotic receptors such as CD95. Also, they do not undergo significant long-term immunological erosion. Understanding the mechanisms that control the generation and maintenance of the protective activity of these long-lived TEM cells will certainly provide important insights into the physiology of CD8(+) T cells and pave the way for the design of new or improved vaccines.

Pathogen-induced proapoptotic phenotype and high CD95 (Fas) expression accompany a suboptimal CD8+ T-cell response: reversal by adenoviral vaccine.

MHC class Ia-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Re-circulation of lymphocytes mediated by sphingosine-1-phosphate receptor-1 contributes to resistance against experimental infection with the protozoan parasite Trypanosoma cruzi.

T-cell mediated immune responses are critical for acquired immunity against infection by the intracellular protozoan parasite Trypanosoma cruzi. Despite its importance, it is currently unknown where protective T cells are primed and whether they need to re-circulate in order to exert their anti-parasitic effector functions. Here, we show that after subcutaneous challenge, CD11c(+)-dependent specific CD8(+) T-cell immune response to immunodominant parasite epitopes arises almost simultaneously in the draining lymph node (LN) and the spleen. However, until day 10 after infection, we observed a clear upregulation of activation markers only on the surface of CD11C(+)PDCA1(+) cells present in the LN and not in the spleen. Therefore, we hypothesized that CD8(+) T cells re-circulated rapidly from the LN to the spleen. We investigated this phenomenon by administering FTY720 to T. cruzi-infected mice to prevent egress of T cells from the LN by interfering specifically with signalling through sphingosine-1-phosphate receptor-1. In T. cruzi-infected mice receiving FTY720, CD8 T-cell immune responses were higher in the draining LN and significantly reduced in their spleen. Most importantly, FTY720 increased susceptibility to infection, as indicated by elevated parasitemia and accelerated mortality. Similarly, administration of FTY720 to mice genetically vaccinated with an immunodominant parasite antigen significantly reduced their protective immunity, as observed by the parasitemia and survival of vaccinated mice. We concluded that re-circulation of lymphocytes mediated by sphingosine-1-phosphate receptor-1 greatly contributes to acquired and vaccine-induced protective immunity against experimental infection with a human protozoan parasite.

Neutralizing antibodies to human and simian adenoviruses in humans and New-World monkeys.

Vaccines based on adenovirus (Ad) vectors are currently in development against several pathogens. However, neutralizing antibodies (NAb) to human adenovirus type 5 (AdHu5), the best-studied vector, are highly prevalent in humans worldwide. Less-prevalent adenoviruses, including human and simian serotypes, provide alternative vaccine platforms. In this study, sera from 200 Brazilian human subjects and New-World monkeys were tested for NAb titers to human serotypes AdHu5 and AdHu26 and chimpanzee-origin Ad viruses of serotype 6 (AdC6) and serotype 68 (AdC68). Seroprevalence rates of NAb in humans were 69.5% for AdHu5, 44% for AdHu26, 21% for AdC6 and 23.5% for AdC68. In addition, NAb titers to human Ad were consistently higher than those found to simian serotypes. Surprisingly, sera from some New-World monkey species were able to neutralize AdC6 and/or AdC68. A possible explanation for these findings and the implications for the development of Ad-vector vaccines are discussed in detail.

HIV-1 vaccine clinical trials: the Brazilian experience.

Although the development of an effective HIV-1 vaccine has proved very challenging for more than two decades, it remains the best hope to control the HIV pandemic. Since Brazil has particular epidemiological features, as well as adequate policies and infrastructure, the country has been an interesting site for HIV vaccine trials. Since 1995, eight trials were performed in Brazil enrolling over 2000 subjects. Peptide vaccine candidates were initially designed to elicit neutralising antibodies as an attempt to provide sterilising immunity against HIV-1. This strategy, however, has proved extremely difficult, and candidates were poorly immunogenic. Therefore, the next vaccine candidates focused mainly on the induction of cell mediated immune responses that would limit AIDS progression and transmission by suppressing viremia. Such candidates were naked DNA or viral vectors in either prophylactic or therapeutic approaches. Even though several candidates were immunogenic, protective immune responses against HIV-1 remain to be achieved. However, several studies with non-human primates and human elite controllers demonstrate that effective immune responses against HIV-1 may be elicited, supporting the belief that an HIV-1 vaccine is possible. Much has been learned, and now the development of an effective HIV-1 vaccine requires resetting priorities with focus on basic research, considering the merits of neutralising antibodies and CMI, as well as the role of innate immunity on HIV-1 protection. In this new perspective, large-scale trials should be replaced by smaller preliminary efficacy studies.