Enhanced Immunogenicity and Protective Effects against SARS-CoV-2 Following Immunization with a Recombinant RBD-IgG Chimeric Protein.

The unprecedented global impact caused by SARS-CoV-2 imposed huge health and economic challenges, highlighting the urgent need for safe and effective vaccines. The receptor-binding domain (RBD) of SARS-CoV-2 is the major target for neutralizing antibodies and for vaccine formulations. Nonetheless, the low immunogenicity of the RBD requires the use of alternative strategies to enhance its immunological properties. Here, we evaluated the use of a subunit vaccine antigen generated after the genetic fusing of the RBD with a mouse IgG antibody. Subcutaneous administration of RBD-IgG led to the extended presence of the protein in the blood of immunized animals and enhanced RBD-specific IgG titers. Furthermore, RBD-IgG immunized mice elicited increased virus neutralizing antibody titers, measured both with pseudoviruses and with live original (Wuhan) SARS-CoV-2. Immunized K18-hACE2 mice were fully resistant to the lethal challenge of the Wuhan SARS-CoV-2, demonstrated by the control of body-weight loss and virus loads in their lungs and brains. Thus, we conclude that the genetic fusion of the RBD with an IgG molecule enhanced the immunogenicity of the antigen and the generation of virus-neutralizing antibodies, supporting the use of IgG chimeric antigens as an approach to improve the performance of SARS-CoV-2 subunit vaccines.

Diagnostic and vaccine potential of Zika virus envelope protein (E) derivates produced in bacterial and insect cells.

Introduction: In the present study we evaluated the features of different recombinant forms of Zika virus (ZIKV) proteins produced in either bacterial (Eschericha coli) or insect cells (Drosophila melanogaster). The ZIKV-envelope glycoprotein (EZIKV) is responsible for virus entry into host cells, is the main target of neutralizing antibodies and has been used as a target antigen either for serological tests or for the development of subunit vaccines. The EZIKV is composed of three structural and functional domains (EDI, EDII, and EDIII), which share extensive sequence conservation with the corresponding counterparts expressed by other flaviviruses, particularly the different dengue virus (DENV) subtypes. Methods: In this study, we carried out a systematic comparison of the antigenicity and immunogenicity of recombinant EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells. For the antigenicity analysis we collected 88 serum samples from ZIKV-infected participants and 57 serum samples from DENV-infected. For immunogenicity, C57BL/6 mice were immunized with two doses of EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells to evaluate humoral and cellular immune response. In addition, AG129 mice were immunized with EZIKV and then challenge with ZIKV. Results: Testing of samples collected from ZIKV-infected and DENV-infected participants demonstrated that the EZIKV and EDIIIZIKV produced in BL21 cells presented better sensitivity and specificity compared to proteins produced in S2 cells. In vivo analyses were carried out with C57BL/6 mice and the results indicated that, despite similar immunogenicity, antigens produced in S2 cells, particularly EZIKV and EDIIIZIKV, induced higher ZIKV-neutralizing antibody levels in vaccinated mice. In addition, immunization with EZIKV expressed in S2 cells delayed the onset of symptoms and increased survival rates in immunocompromised mice. All recombinant antigens, either produced in bacteria or insect cells, induced antigen-specific CD4+ and CD8+ T cell responses. Conclusion: In conclusion, the present study highlights the differences in antigenicity and immunogenicity of recombinant ZIKV antigens produced in two heterologous protein expression systems.

Conventional type 1 dendritic cells induce TH 1, TH 1-like follicular helper T cells and regulatory T cells after antigen boost via DEC205 receptor.

Conventional dendritic cells (cDCs) are specialized in antigen presentation. In the mouse spleen, cDCs are classified in cDC1s and cDC2s, and express DEC205 and DCIR2 endocytic receptors, respectively. Monoclonal antibodies (mAbs) αDEC205 (αDEC) and αDCIR2 have been fused to different antigens to deliver them to cDC1s or cDC2s. We immunized mice with αDEC and αDCIR2 fused to an antigen using Poly(I:C) as adjuvant. The initial immune response was analyzed from days 3 to 6 after the immunization. We also studied the influence of a booster dose. Our results showed that antigen targeting to cDC1s promoted a pro-inflammatory TH 1 cell response. Antigen targeting to cDC2s induced TFH cells, GCs, and plasma cell differentiation. After boost, antigen targeting to cDC1s improved the TH 1 cell response and induced TH 1-like TFH cells that led to an increase in specific antibody titers and IgG class switch. Additionally, a population of regulatory T cells was also observed. Antigen targeting to cDC2s did not improve the specific antibody response after boost. Our results add new information on the immune response induced after the administration of a booster dose with αDEC and αDCIR2 fusion mAbs. These results may be useful for vaccine design using recombinant mAbs.

Dendritic Cell Targeting Using a DNA Vaccine Induces Specific Antibodies and CD4+ T Cells to the Dengue Virus Envelope Protein Domain III.

Dengue fever has become a global threat, causing millions of infections every year. An effective vaccine against all four serotypes of dengue virus (DENV) has not been developed yet. Among the different vaccination strategies available today, DNA vaccines are safe and practical, but currently induce relatively weak immune responses in humans. In order to improve immunogenicity, antigens may be targeted to dendritic cells (DCs), the main antigen presenting cells and orchestrators of the adaptive immune response, inducing T and B cell activation. It was previously shown that a DNA vaccine encoding a fusion protein comprised of an antigen and a single-chain Fv antibody (scFv) specific for the DC endocytic receptor DEC205 induced strong immune responses to the targeted antigen. In this work, we evaluate this strategy to improve the immunogenicity of dengue virus (DENV) proteins. Plasmids encoding the scFv αDEC205, or an isotype control (scFv ISO), fused to the DENV2 envelope protein domain III (EDIII) were generated, and EDIII specific immune responses were evaluated in immunized mice. BALB/c mice were intramuscularly (i.m.) immunized three times with plasmid DNAs encoding either scDEC-EDIII or scISO-EDIII followed by electroporation. Analyses of the antibody responses indicated that EDIII fusion with scFv targeting the DEC205 receptor significantly enhanced serum anti-EDIII IgG titers that inhibited DENV2 infection. Similarly, mice immunized with the scDEC-EDIII plasmid developed a robust CD4+ T cell response to the targeted antigen, allowing the identification of two linear epitopes recognized by the BALB/c haplotype. Taken together, these results indicate that targeting DENV2 EDIII protein to DCs using a DNA vaccine encoding the scFv αDEC205 improves both antibody and CD4+ T cell responses. This strategy opens perspectives for the use of DNA vaccines that encode antigens targeted to DCs as a strategy to increase immunogenicity.

CpG Oligodeoxinucleotides and Flagellin Modulate the Immune Response to Antigens Targeted to CD8α+ and CD8α- Conventional Dendritic Cell Subsets.

Dendritic cells (DCs) are antigen-presenting cells essential for the induction of adaptive immune responses. Their unprecedented ability to present antigens to T cells has made them excellent targets for vaccine development. In the last years, a new technology based on antigen delivery directly to different DC subsets through the use of hybrid monoclonal antibodies (mAbs) to DC surface receptors fused to antigens of interest opened new perspectives for the induction of robust immune responses. Normally, the hybrid mAbs are administered with adjuvants that induce DC maturation. In this work, we targeted an antigen to the CD8α+ or the CD8α- DC subsets in the presence of CpG oligodeoxinucleotides (ODN) or bacterial flagellin, using hybrid αDEC205 or αDCIR2 mAbs, respectively. We also accessed the role of toll-like receptors (TLRs) 5 and 9 signaling in the induction of specific humoral and cellular immune responses. Wild-type and TLR5 or TLR9 knockout mice were immunized with two doses of the hybrid αDEC205 or αDCIR2 mAbs, as well as with an isotype control, together with CpG ODN 1826 or flagellin. A chimeric antigen containing the Plasmodium vivax 19 kDa portion of the merozoite surface protein (MSP119) linked to the Pan-allelic DR epitope was fused to each mAb. Specific CD4+ T cell proliferation, cytokine, and antibody production were analyzed. We found that CpG ODN 1826 or flagellin were able to induce CD4+ T cell proliferation, CD4+ T cells producing pro-inflammatory cytokines, and specific antibodies when the antigen was targeted to the CD8α+ DC subset. On the other hand, antigen targeting to CD8α- DC subset promoted specific antibody responses and proliferation, but no detectable pro-inflammatory CD4+ T cell responses. Also, specific antibody responses after antigen targeting to CD8α+ or CD8α- DCs were reduced in the absence of TLR9 or TLR5 signaling, while CD4+ T cell proliferation was mainly affected after antigen targeting to CD8α+ DCs and in the absence of TLR9 signaling. These results extend our understanding of the modulation of specific immune responses induced by antigen targeting to DCs in the presence of different adjuvants. Such knowledge may be useful for the optimization of DC-based vaccines.