Heterologous DNA Prime- Subunit Protein Boost with Chikungunya Virus E2 Induces Neutralizing Antibodies and Cellular-Mediated Immunity.

Chikungunya virus (CHIKV) has become a significant public health concern due to the increasing number of outbreaks worldwide and the associated comorbidities. Despite substantial efforts, there is no specific treatment or licensed vaccine against CHIKV to date. The E2 glycoprotein of CHIKV is a promising vaccine candidate as it is a major target of neutralizing antibodies during infection. In this study, we evaluated the immunogenicity of two DNA vaccines (a non-targeted and a dendritic cell-targeted vaccine) encoding a consensus sequence of E2CHIKV and a recombinant protein (E2*CHIKV). Mice were immunized with different homologous and heterologous DNAprime-E2* protein boost strategies, and the specific humoral and cellular immune responses were accessed. We found that mice immunized with heterologous non-targeted DNA prime- E2*CHIKV protein boost developed high levels of neutralizing antibodies, as well as specific IFN-γ producing cells and polyfunctional CD4+ and CD8+ T cells. We also identified 14 potential epitopes along the E2CHIKV protein. Furthermore, immunization with recombinant E2*CHIKV combined with the adjuvant AS03 presented the highest humoral response with neutralizing capacity. Finally, we show that the heterologous prime-boost strategy with the non-targeted pVAX-E2 DNA vaccine as the prime followed by E2* protein + AS03 boost is a promising combination to elicit a broad humoral and cellular immune response. Together, our data highlights the importance of E2CHIKV for the development of a CHIKV vaccine.

ZIKV-envelope proteins induce specific humoral and cellular immunity in distinct mice strains.

Recent outbreaks of Zika virus (ZIKV) infection have highlighted the need for a better understanding of ZIKV-specific immune responses. The ZIKV envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface and it is the main target of the protective immune response. EZIKV protein contains the central domain (EDI), a dimerization domain containing the fusion peptide (EDII), and a domain that binds to the cell surface receptor (EDIII). In this study, we performed a systematic comparison of the specific immune response induced by different EZIKV recombinant proteins (EZIKV, EDI/IIZIKV or EDIIIZIKV) in two mice strains. Immunization induced high titers of E-specific antibodies which recognized ZIKV-infected cells and neutralized the virus. Furthermore, immunization with EZIKV, EDI/IIZIKV and EDIIIZIKV proteins induced specific IFNγ-producing cells and polyfunctional CD4+ and CD8+ T cells. Finally, we identified 4 peptides present in the envelope protein (E1-20, E51-70, E351-370 and E361-380), capable of inducing a cellular immune response to the H-2Kd and H-2Kb haplotypes. In summary, our work provides a detailed assessment of the immune responses induced after immunization with different regions of the ZIKV envelope protein.

ZIKV-envelope proteins induce specific humoral and cellular immunity in distinct mice strains

Recent outbreaks of Zika virus (ZIKV) infection have highlighted the need for a better understanding of ZIKV-specific immune responses. The ZIKV envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface and it is the main target of the protective immune response. EZIKV protein contains the central domain (EDI), a dimerization domain containing the fusion peptide (EDII), and a domain that binds to the cell surface receptor (EDIII). In this study, we performed a systematic comparison of the specific immune response induced by different EZIKV recombinant proteins (EZIKV, EDI/IIZIKV or EDIIIZIKV) in two mice strains. Immunization induced high titers of E-specific antibodies which recognized ZIKV-infected cells and neutralized the virus. Furthermore, immunization with EZIKV, EDI/IIZIKV and EDIIIZIKV proteins induced specific IFNγ-producing cells and polyfunctional CD4+ and CD8+ T cells. Finally, we identified 4 peptides present in the envelope protein (E1–20, E51–70, E351–370 and E361–380), capable of inducing a cellular immune response to the H-2Kd and H-2Kb haplotypes. In summary, our work provides a detailed assessment of the immune responses induced after immunization with different regions of the ZIKV envelope protein.

Prime-boost with Chikungunya virus E2 envelope protein combined with Poly (I:C) induces specific humoral and cellular immune responses.

Chikungunya virus (CHIKV) is an arbovirus transmitted to humans mainly by the bite of infected Aedes aegypti and Aedes albopictus mosquitoes. CHIKV illness is characterized by fever and long-lasting arthritic symptoms, and in some cases it is a deadly disease. The CHIKV envelope E2 (E2CHIKV) glycoprotein is crucial for virus attachment to the cell. Furthermore, E2CHIKV is the immunodominant protein and the main target of neutralizing antibodies. To date, there is no available prophylactic vaccine or specific treatment against CHIKV infection. Here, we designed and produced a DNA vaccine and a recombinant protein containing a consensus sequence of E2CHIKV. C57BL/6 mice immunized twice with the E2CHIKV recombinant protein in the presence of the adjuvant Poly (I:C) induced the highest E2CHIKV-specific humoral and cellular immune responses, while the immunization with the homologous DNA vaccine pVAX-E2CHIKV was able to induce specific IFN-γ producing cells. The heterologous prime-boost strategy was also able to induce specific cellular and humoral immune responses that were, in general, lower than the responses induced by the homologous E2CHIKV recombinant protein immunization. Furthermore, recombinant E2CHIKV induced the highest titers of neutralizing antibodies. Collectively, we believe this is the first report to analyze E2CHIKV-specific humoral and cellular immune responses after immunization with E2CHIKV recombinant protein and DNA pVAX-E2CHIKV vaccine platforms.

Homologous prime-boost with Zika virus envelope protein and poly (I:C) induces robust specific humoral and cellular immune responses.

The recent outbreaks of Zika virus (ZIKV) infection and the potential association with Guillain-Barré syndrome in adults and with congenital abnormalities have highlighted the urgency for an effective vaccine. The ZIKV Envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface, and has been evaluated together with the pre-membrane protein (prM) of the viral coat as a vaccine candidate in clinical trials. In this study, we performed a head-to-head comparison of the immune response induced by different EZIKV-based vaccine candidates in mice. We compared different platforms (DNA, recombinant protein), adjuvants (poly (I:C), CpG ODN 1826) and immunization strategies (homologous, heterologous). The hierarchy of adjuvant potency showed that poly (I:C) was a superior adjuvant than CpG ODN. While poly (I:C) assisted immunization reached a plateau in antibody titers after two doses, the CpG ODN group required an extra immunization dose. Besides, the administration of poly (I:C) induced higher EZIKV-specific cellular immune responses than CpG ODN. We also show that immunization with homologous prime-boost EZIKV protein + poly (I:C) regimen induced a more robust humoral response than homologous DNA (pVAX-EZIKV) or heterologous regimens (DNA/protein or protein/DNA). A detailed analysis of cellular immune responses revealed that homologous (EZIKV + poly (I:C)) and heterologous (pVAX-EZIKV/EZIKV + poly (I:C)) prime-boost regimens induced the highest magnitude of IFN-γ secreting cells and cytokine-producing CD4+ T cells. Overall, our data demonstrate that homologous EZIKV + poly (I:C) prime-boost immunization is sufficient to induce more robust specific-EZIKV humoral and cellular immune responses than the other strategies that contemplate homologous DNA (pVAX-EZIKV) or heterologous (pVAX-EZIKV/EZIKV + poly (I:C), and vice-versa) immunizations.

Homologous prime-boost with Zika virus envelope protein and poly (I:C) induces robust specific humoral and cellular immune responses

The recent outbreaks of Zika virus (ZIKV) infection and the potential association with Guillain-Barré syndrome in adults and with congenital abnormalities have highlighted the urgency for an effective vaccine. The ZIKV Envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface, and has been evaluated together with the pre-membrane protein (prM) of the viral coat as a vaccine candidate in clinical trials. In this study, we performed a head-to-head comparison of the immune response induced by different EZIKV-based vaccine candidates in mice. We compared different platforms (DNA, recombinant protein), adjuvants (poly (I:C), CpG ODN 1826) and immunization strategies (homologous, heterologous). The hierarchy of adjuvant potency showed that poly (I:C) was a superior adjuvant than CpG ODN. While poly (I:C) assisted immunization reached a plateau in antibody titers after two doses, the CpG ODN group required an extra immunization dose. Besides, the administration of poly (I:C) induced higher EZIKV-specific cellular immune responses than CpG ODN. We also show that immunization with homologous prime-boost EZIKV protein + poly (I:C) regimen induced a more robust humoral response than homologous DNA (pVAX-EZIKV) or heterologous regimens (DNA/protein or protein/DNA). A detailed analysis of cellular immune responses revealed that homologous (EZIKV + poly (I:C)) and heterologous (pVAX-EZIKV/EZIKV + poly (I:C)) prime-boost regimens induced the highest magnitude of IFN-? secreting cells and cytokine-producing CD4+ T cells. Overall, our data demonstrate that homologous EZIKV + poly (I:C) prime-boost immunization is sufficient to induce more robust specific-EZIKV humoral and cellular immune responses than the other strategies that contemplate homologous DNA (pVAX-EZIKV) or heterologous (pVAX-EZIKV/EZIKV + poly (I:C), and vice-versa) immunizations

Homologous prime-boost with Zika virus envelope protein and poly (I:C) induces robust specific humoral and cellular immune responses

The recent outbreaks of Zika virus (ZIKV) infection and the potential association with Guillain-Barré syndrome in adults and with congenital abnormalities have highlighted the urgency for an effective vaccine. The ZIKV Envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface, and has been evaluated together with the pre-membrane protein (prM) of the viral coat as a vaccine candidate in clinical trials. In this study, we performed a head-to-head comparison of the immune response induced by different EZIKV-based vaccine candidates in mice. We compared different platforms (DNA, recombinant protein), adjuvants (poly (I:C), CpG ODN 1826) and immunization strategies (homologous, heterologous). The hierarchy of adjuvant potency showed that poly (I:C) was a superior adjuvant than CpG ODN. While poly (I:C) assisted immunization reached a plateau in antibody titers after two doses, the CpG ODN group required an extra immunization dose. Besides, the administration of poly (I:C) induced higher EZIKV-specific cellular immune responses than CpG ODN. We also show that immunization with homologous prime-boost EZIKV protein + poly (I:C) regimen induced a more robust humoral response than homologous DNA (pVAX-EZIKV) or heterologous regimens (DNA/protein or protein/DNA). A detailed analysis of cellular immune responses revealed that homologous (EZIKV + poly (I:C)) and heterologous (pVAX-EZIKV/EZIKV + poly (I:C)) prime-boost regimens induced the highest magnitude of IFN-? secreting cells and cytokine-producing CD4+ T cells. Overall, our data demonstrate that homologous EZIKV + poly (I:C) prime-boost immunization is sufficient to induce more robust specific-EZIKV humoral and cellular immune responses than the other strategies that contemplate homologous DNA (pVAX-EZIKV) or heterologous (pVAX-EZIKV/EZIKV + poly (I:C), and vice-versa) immunizations

Adjuvants: Classification, Modus Operandi, and Licensing.

Vaccination is one of the most efficient strategies for the prevention of infectious diseases. Although safer, subunit vaccines are poorly immunogenic and for this reason the use of adjuvants is strongly recommended. Since their discovery in the beginning of the 20th century, adjuvants have been used to improve immune responses that ultimately lead to protection against disease. The choice of the adjuvant is of utmost importance as it can stimulate protective immunity. Their mechanisms of action have now been revealed. Our increasing understanding of the immune system, and of correlates of protection, is helping in the development of new vaccine formulations for global infections. Nevertheless, few adjuvants are licensed for human vaccines and several formulations are now being evaluated in clinical trials. In this review, we briefly describe the most well known adjuvants used in experimental and clinical settings based on their main mechanisms of action and also highlight the requirements for licensing new vaccine formulations.

Modulating APOBEC expression enhances DNA vaccine immunogenicity.

DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double-stranded DNA (dsDNA) sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC (apolipoprotein B (APOB) mRNA-editing, catalytic polypeptide)-mediated plasmid degradation. We showed that plasmid sensing via STING (stimulator of interferon (IFN) genes) and TBK-1 (TANK-binding kinase 1) leads to IFN-ß induction, which results in APOBEC3A mRNA upregulation through a mechanism involving protein kinase C signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to a 10-fold reduction in intracellular plasmid levels and plasmid-encoded mRNA, and a 2.6-fold reduction in GFP-expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2-specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN-γ-secreting T cells. Our study brings new insights into the intracellular machinery involved in dsDNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.