Vaccine-Mediated Inhibition of the Transporter Associated with Antigen Processing Is Insufficient To Induce Major Histocompatibility Complex E-Restricted CD8+ T Cells in Nonhuman Primates.

Major histocompatibility complex E (MHC-E) is a highly conserved nonclassical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs coexpressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus 2 ICP47, bovine herpes virus 1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia but not MHC-E. These data demonstrate that, in contrast to results in mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.IMPORTANCE Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.

The Role of MHC-E in T Cell Immunity Is Conserved among Humans, Rhesus Macaques, and Cynomolgus Macaques.

MHC-E is a highly conserved nonclassical MHC class Ib molecule that predominantly binds and presents MHC class Ia leader sequence-derived peptides for NK cell regulation. However, MHC-E also binds pathogen-derived peptide Ags for presentation to CD8+ T cells. Given this role in adaptive immunity and its highly monomorphic nature in the human population, HLA-E is an attractive target for novel vaccine and immunotherapeutic modalities. Development of HLA-E-targeted therapies will require a physiologically relevant animal model that recapitulates HLA-E-restricted T cell biology. In this study, we investigated MHC-E immunobiology in two common nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM). Compared to humans and MCM, RM expressed a greater number of MHC-E alleles at both the population and individual level. Despite this difference, human, RM, and MCM MHC-E molecules were expressed at similar levels across immune cell subsets, equivalently upregulated by viral pathogens, and bound and presented identical peptides to CD8+ T cells. Indeed, SIV-specific, Mamu-E-restricted CD8+ T cells from RM recognized antigenic peptides presented by all MHC-E molecules tested, including cross-species recognition of human and MCM SIV-infected CD4+ T cells. Thus, MHC-E is functionally conserved among humans, RM, and MCM, and both RM and MCM represent physiologically relevant animal models of HLA-E-restricted T cell immunobiology.

Repeated semen exposure decreases cervicovaginal SIVmac251 infection in rhesus macaques.

Semen is the vehicle for virion dissemination in the female reproductive tract (FRT) in male-to-female HIV transmission. Recent data suggests that higher frequency semen exposure is associated with activation of anti-HIV mechanisms in HIV negative sex workers. Here, we use a non-human primate (NHP) model to show that repeated vaginal exposure to semen significantly reduces subsequent infection by repeated low-dose vaginal SIVmac251 challenge. Repeated semen exposures result in lower CCR5 expression in circulating CD4+ T-cells, as well as higher expression of Mx1 (in correlation with IFNε expression) and FoxP3 in the cervicovaginal mucosa, and increased infiltration of CD4+ T-cells. Establishing in vivo evidence of competing effects of semen on transmission impacts our basic understanding of what factors may determine HIV infectivity in humans. Our results clearly indicate that repeated semen exposure can profoundly modulate the FRT microenvironment, paradoxically promoting host resistance against HIV acquisition.

DNA vaccines: developing new strategies to enhance immune responses.

We have focused our research on understanding the basic biology of and developing novel therapeutic and prophylactic DNA vaccines. We have among others three distinct primary areas of interest which include: 1. Enhancing in vivo delivery and transfection of DNA vaccine vectors 2. Improving DNA vaccine construct immunogenicity 3. Using molecular adjuvants to modulate and skew immune responses. Key to the immunogenicity of DNA vaccines is the presentation of expressed antigen to antigen-presenting cells. To improve expression and presentation of antigen, we have investigated various immunization methods with current focus on a combination of intramuscular injection and electroporation. To improve our vaccine constructs, we also employed methods such as RNA/codon optimization and antigen consensus to enhance expression and cellular/humoral cross-reactivity, respectively. Our lab also researches the potential of various molecular adjuvants to skew Th1/Th2 responses, enhance cellular/humoral responses, and improve protection in various animal models. Through improving our understanding of basic immunology as it is related to DNA vaccine technology, our goal is to develop the technology to the point of utility for human and animal health.

Serial cervicovaginal exposures with replication-deficient SIVsm induce higher dendritic cell (pDC) and CD4+ T-cell infiltrates not associated with prevention but a more severe SIVmac251 infection of rhesus macaques.

OBJECTIVE: Intravaginal exposure to simian immunodeficiency virus (SIV) acutely recruits interferon-alpha (IFN-α) producing plasmacytoid dendritic cells (pDC) and CD4 T-lymphocyte targets to the endocervix of nonhuman primates. We tested the impact of repeated cervicovaginal exposures to noninfectious, defective SIV particles over 72 hours on a subsequent cervicovaginal challenge with replication competent SIV. METHODS: Thirty-four female Indian Rhesus macaques were given a 3-day twice-daily vaginal exposures to either SIVsmB7, a replication-deficient derivative of SIVsmH3 produced by a T lymphoblast CEMx174 cell clone (n = 16), or to CEM supernatant controls (n = 18). On the fourth day, animals were either euthanized to assess cervicovaginal immune cell infiltration or intravaginally challenged with SIVmac251. Challenged animals were tracked for plasma viral load and CD4 counts and euthanized at 42 days after infection. RESULTS: At the time of challenge, macaques exposed to SIVsmB7, had higher levels of cervical CD123 pDCs (P = 0.032) and CD4 T cells (P = 0.036) than those exposed to CEM control. Vaginal tissues showed a significant increase in CD4 T-cell infiltrates (P = 0.048) and a trend toward increased CD68 cellular infiltrates. After challenge, 12 SIVsmB7-treated macaques showed 2.5-fold greater daily rate of CD4 decline (P = 0.0408), and viral load rise (P = 0.0036) as compared with 12 control animals. CONCLUSIONS: Repeated nonproductive exposure to viral particles within a short daily time frame did not protect against infection despite pDC recruitment, resulting instead in an accelerated CD4 T-cell loss with an increased rate of viral replication.