Vaccinia Virus Vectors Targeting Peptides for MHC Class II Presentation to CD4+ T Cells

Abstract

CD4+ helper T cells play important roles in providing help to B cells, macrophages, and cytotoxic CD8+ T cells, but also exhibit direct effector functions against a variety of different pathogens. In contrast to CD8+ T cells, CD4+ T cells typically exhibit broader specificities and undergo less clonal expansion during many types of viral infections, which often makes the identification of virus-specific CD4+ T cells technically challenging. In this study, we have generated recombinant vaccinia virus (VacV) vectors that target I-Ab–restricted peptides for MHC class II (MHC-II) presentation to activate CD4+ T cells in mice. Conjugating the lymphocytic choriomeningitis virus immunodominant epitope GP61–80 to either LAMP1 to facilitate lysosomal targeting or to the MHC-II invariant chain (Ii) significantly increased the activation of Ag-specific CD4+ T cells in vivo. Immunization with VacV-Ii-GP61–80 activated endogenous Ag-specific CD4+ T cells that formed memory and rapidly re-expanded following heterologous challenge. Notably, immunization of mice with VacV expressing an MHC-II–restricted peptide from Leishmania species (PEPCK335–351) conjugated to either LAMP1 or Ii also generated Ag-specific memory CD4+ T cells that underwent robust secondary expansion following a visceral leishmaniasis infection, suggesting this approach could be used to generate Ag-specific memory CD4+ T cells against a variety of different pathogens. Overall, our data show that VacV vectors targeting peptides for MHC-II presentation is an effective strategy to activate Ag-specific CD4+ T cells in vivo and could be used to study Ag-specific effector and memory CD4+ T cell responses against a variety of viral, bacterial, or parasitic infections.

Cellular and Humoral Immune Responses in Mice Immunized with Vaccinia Virus Expressing the SARS-CoV-2 Spike Protein

“…6C). In contrast, Ii conjugated to the gp 6180 I-A b restricted peptide from the LCMV glycoprotein was expressed in cells infected with VacV-Ii-gp 6180 , which confirms our previous finding demonstrating the utility of using VacV-expressing Ii conjugated to peptide Ags (32). Thus, these data show that a C-terminal conjugation of Ii to full-length SARS-CoV-2 spike fails to produce a stable chimeric Ag in vitro.…”

“…After three rounds of plaque purification in the presence of BrdU, individual viral plaques were screened by DNA sequencing, and successfully recombined viruses were expanded and quantified using BSC-40 cells and standard procedures. VacV-Ii-gp 6180 has been previously described (32).…”

“…We have previously reported that conjugating the MHC-II Ii to immunogenic peptides expressed by VacV resulted in stronger activation of Ag-specific CD4 1 T cells in vivo (32). To determine if this also held true using a full-length, >1200 aa transmembrane viral Ag rather than a defined MHC-II restricted peptide, we generated VacV that expressed the full-length spike protein that contained the MHC-II Ii conjugated to its C terminus (VacV-S-Ii).…”

Design and Immunological Validation of Macaca fascicularis Papillomavirus Type 3 Based Vaccine Candidates in Outbred Mice: Basis for Future Testing of a Therapeutic Papillomavirus Vaccine in NHPs

“…Coupling of antigens to certain immune regulatory molecules and T cell adjuvants could be beneficial in terms of breaking the immunosuppressive environment of tumors (53,58). All immunogens expressed via rAds contain the T cell adjuvant Ii (except for E1E2E6E7 which serves as reference), which is known to enhance CD4 + and CD8 + T cell responses when delivered by viral vectors (25,27,43,59,60). A trend towards higher magnitudes of responses against E1 and E2 could be observed in outbred mice when vaccinated with rAd Ii-E1E2E6E7 as compared to E1E2E6E7 (Figure 5) and, importantly, a significantly higher level of specific killing in animals vaccinated with Ii-adjuvanted vaccines has been shown (Figure 6).…”