Heat shock protein Gp96-Ig-peptide complexes based vaccine induce predominant immune responses at mucosal sites (46.13)

Abstract We have developed a vaccine design that uses the unique ability of the endoplasmic reticulum (ER) chaperone, heat shock protein gp96, to bind antigenic peptides and deliver them to antigen-presenting cells. Secreted form of gp96, gp96-Ig, was generated by replacing the ER retention sequence KDEL with the IgG1-Fc domain. To generate a cellular SIV vaccine, HEK-293 cells were transfected with gp96-Ig and the cDNAs encoding the SIV antigens gag, env and retanef. Irradiated, transfected 293 cells that secrete 1, 5 or 50 micrograms gp96-Ig-SIV complexes in 24h, were injected intraperitoneally (IP) in Mamu-A*01+ Rhesus Macaques at 0, 4 and 25 weeks. To determine whether gp96-Ig vaccination induces mucosal immunity, we used the 3T3-OVA-gp96-Ig/OT-1 model. Our data indicate that the best route of vaccination for induction of mucosal CD8-CTL immunity is the IP route. Gp96-Ig immunization increases frequency of CD11chighMHC classIIhighCD103+ cells in peritoneal cavity and efficiently induces CCR9 on responding T cells. In nonhuman primate model, the frequency of SIV-specific cells in the rectal mucosa reached 30-50% after the third immunization. We conclude that gp96-Ig vaccination startegy induces antigen-specific effector memory CD8T cells migrating to the intestinal mucosa and thus could be extremely useful for improving protection against a range of mucosal pathogens

Powerful mucosal immune response in macaques (Macaca mulatta) in response to SIV gp96-Ig immunization (39.35)

Abstract Induction of mucosal immunity is critical for protection from enteric pathogens. We have developed a vaccine design that utilizes the unique property of the endoplasmic reticulum chaperon, heat shock protein (HSP) gp96, to bind antigenic peptides and deliver them to APCs. The aim of our study was to evaluate safety and systemic and mucosal SIV-immunity with secreted gp96-Ig vaccines in non-human primates. We have generated a secreted form gp96 by replacing the ER retention sequence KDEL with the IgG1-Fc tag. HEK-293 cells were transfected with gp96-Ig and with the cDNAs encoding the SIV antigens gag, pol, env and retanef (reassorted rev, tat, nef). Irradiated, transfected 293 cells that secrete 1, 5 or 50 micrograms gp96-Ig-SIV-peptide complexes in 24h, were injected intraperitoneally in Mamu-A*01+ macaques at 0, 4 and 24 weeks. The frequency of SIVGag- and SIVTat- tetramer+ cells in the rectal mucosa reached 30-50% in some macaques after the third immunization. Tetramer+ cells expressed appropriate functional (granzymeB) and migration markers (alpha4beta7, CD103). The cells secrete IL-2 and IFN-gamma in response to different peptide (gag, tat, env) stimulation. We conclude that the cell secreted SIV-gp96-Ig vaccine is safe and can induce strong poly-specific, multifunctional CD8 responses in mucosal compartments that are thought to be critical for protection from SIV/HIV infection

Multiple Low-Dose Challenges in a Rhesus Macaque AIDS Vaccine Trial Result in an Evolving Host Response That Affects Protective Outcome

Using whole-blood transcriptional profiling, we investigated differences in the host response to vaccination and challenge in a rhesus macaque AIDS vaccine trial. Samples were collected from animals prior to and after vaccination with live, irradiated vaccine cells secreting the modified endoplasmic reticulum chaperone gp96-Ig loaded with simian immunodeficiency virus (SIV) peptides, either alone or in combination with a SIV-gp120 protein boost. Additional samples were collected following multiple low-dose rectal challenges with SIV(mac251). Animals in the boosted group had a 73% reduced risk of infection. Surprisingly, few changes in gene expression were observed during the vaccination phase. Focusing on postchallenge comparisons, in particular for protected animals, we identified a host response signature of protection comprised of strong interferon signaling after the first challenge, which then largely abated after further challenges. We also identified a host response signature, comprised of early macrophage-mediated inflammatory responses, in animals with undetectable viral loads 5 days after the first challenge but with unusually high viral titers after subsequent challenges. Statistical analysis showed that prime-boost vaccination significantly lowered the probability of infection in a time-consistent manner throughout several challenges. Given that humoral responses in the prime-boost group were highly significant prechallenge correlates of protection, the strong innate signaling after the first challenge suggests that interferon signaling may enhance vaccine-induced antibody responses and is an important contributor to protection from infection during repeated low-dose exposure to SIV

Cutting Edge: Novel Vaccination Modality Provides Significant Protection against Mucosal Infection by Highly Pathogenic Simian Immunodeficiency Virus

Vaccine induced protection against infection by HIV or highly pathogenic and virulent SIV-strains has been limited. Here, in a proof of concept study, we show that a novel vaccine approach significantly protects Rhesus macaques from mucosal infection by the highly pathogenic strain SIV(mac251). We vaccinated 3 cohorts of 12 macaques each with live, irradiated vaccine cells secreting the modified ER chaperone gp96(−)Ig. Cohort 1 was vaccinated with cells secreting gp96(SIV)Ig carrying SIV peptides. Cohort 2 in addition received recombinant envelope protein SIV-gp120. Cohort 3 was injected with cells secreting gp96-Ig (no SIV antigens) vaccines. Cohort 2 was protected from infection. After seven rectal challenges with highly pathogenic SIV(mac251) the hazard ratio was 0.27 corresponding to a highly significant, 73% reduced risk of viral acquisition. The apparent success of the novel vaccine modality recommends further study

Gp96SIVIg immunization induces potent polyepitope specific, multifunctional memory responses in rectal and vaginal mucosa

The ER-resident chaperone gp96, when released by cell lysis, induces an immunogenic chemokine signature and causes innate immune activation of DC and NK cells. Here we show that intraperitoneal immunization with a genetically engineered, secreted form of gp96, gp96-Ig chaperoning SIV antigens, induces high levels of antigen specific CD8 CTL in the rectal and vaginal mucosa of Rhesus macaques. The frequency of SIV Gag- and SIV Tat-tetramer positive CD8 CTL in the intestinal mucosa reached 30-50% after the third immunization. Tetramer positive CD8 CTL expressed appropriate functional (granzyme B) and migration markers (CD103). The polyepitope specificity of the mucosal CD8 and CD4 response is evident from a strong, multifunctional cytokine response upon stimulation with peptides covering the gag, tat and env proteins. Induction of powerful mucosal effector CD8 CTL responses by cell-based gp96(SIV)-Ig immunization may provide a pathway to the development of safe and effective SIV/HIV vaccines

Essential Role of Human T Cell Leukemia Virus Type 1 orf-I in Lethal Proliferation of CD4+ Cells in Humanized Mice

Human T cell leukemia virus type 1 (HTLV-1) is the ethological agent of adult T cell leukemia/lymphoma (ATLL) and a number of lymphocyte-mediated inflammatory conditions, including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 orf-I encodes two proteins, p8 and p12, whose functions in humans are to counteract innate and adaptive responses and to support viral transmission. However, the in vivo requirements for orf-I expression vary in different animal models. In macaques, the ablation of orf-I expression by mutation of its ATG initiation codon abolishes the infectivity of the molecular clone HTLV-1p12KO. In rabbits, HTLV-1p12KO is infective and persists efficiently. We used humanized mouse models to assess the infectivity of both wild-type HTLV-1 (HTLV-1WT) and HTLV-1p12KO. We found that NOD/SCID/γC-/- c-kit+ mice engrafted with human tissues 1 day after birth (designated NSG-1d mice) were highly susceptible to infection by HTLV-1WT, with a syndrome characterized by the rapid polyclonal proliferation and infiltration of CD4+ CD25+ T cells into vital organs, weight loss, and death. HTLV-1 clonality studies revealed the presence of multiple clones of low abundance, confirming the polyclonal expansion of HTLV-1-infected cells in vivo. HTLV-1p12KO infection in a bone marrow-liver-thymus (BLT) mouse model prone to graft-versus-host disease occurred only following reversion of the orf-I initiation codon mutation within weeks after exposure and was associated with high levels of HTLV-1 DNA in blood and the expansion of CD4+ CD25+ T cells. Thus, the incomplete reconstitution of the human immune system in BLT mice may provide a window of opportunity for HTLV-1 replication and the selection of viral variants with greater fitness.IMPORTANCE Humanized mice constitute a useful model for studying the HTLV-1-associated polyclonal proliferation of CD4+ T cells and viral integration sites in the human genome. The rapid death of infected animals, however, appears to preclude the clonal selection typically observed in human ATLL, which normally develops in 2 to 5 of individuals infected with HTLV-1. Nevertheless, the expansion of multiple clones of low abundance in these humanized mice mirrors the early phase of HTLV-1 infection in humans, providing a useful model to investigate approaches to inhibit virus-induced CD4+ T cell proliferation

Interleukin-21 administration to rhesus macaques chronically infected with simian immunodeficiency virus increases cytotoxic effector molecules in T cells and NK cells and enhances B cell function without increasing immune activation or viral replication

We have previously shown that Interleukin-21, a pleiotropic C γ-chain signaling cytokine, induces the expression of the cytotoxic molecules granzyme B (GrB) and perforin in vitro in CD8 T cells and NK cells of chronically HIV infected individuals. In this pilot study, four chronically SIV infected Rhesus macaques (RM) in late- stage disease were given two doses of recombinant MamuIL-21, 50μg/kg, intravenously 7 days apart, followed by one subcutaneous dose, 100μg/kg, 23 days after the second dose. Three animals served as controls. After each dose of IL-21, increases were noted in frequency and mean fluorescence intensity of GrB and perforin expression in memory and effector subsets of CD8 T cells in peripheral blood (PB), in peripheral and mesenteric lymph node (LN) cells, in PB memory and effector CD4 T cells and in NK cells. Frequencies of SIV-gag specific CD107a(+)IFNγ(+) CD8 increased 3.8 fold in PB and 1.8 fold in LN. In addition, PB CD27(+) memory B cells were 2 fold higher and serum SIV antibodies increased significantly after IL-21 administration. No changes were observed in markers of T cell activation, T cell proliferation or plasma virus load. Thus, administration of IL-21 to chronically SIV infected viremic animals was safe, well tolerated and could augment the cytotoxic potential of T cells and NK cells, promote B cell differentiation with increases in SIV antibody titers without discernable increase in cellular activation. Further studies are warranted to elucidate the effects and potential benefit of IL-21 administration in the context of SIV/HIV infection and in HIV/SIV vaccine design

Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition

A recombinant vaccine containing Aventis Pasteur's canarypox vector (ALVAC)-HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC-simian immunodeficiency virus (SIV) and gp120 alum (ALVAC-SIV + gp120) equivalent vaccine, but not an ALVAC-SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies