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HIV-infected patients of all ages frequently underperform in response to seasonal influenza vaccination, despite virologic control of HIV. The molecular mechanisms governing this impairment, as well as predictive biomarkers for responsiveness, remain unknown. This study was performed in samples obtained prevaccination (T0) from HIV-infected children who received the 2012–2013 seasonal influenza vaccine. Response status was determined based on established criterion for hemagglutination inhibition titer; participants with a hemagglutination titer ≥1:40 plus a ≥4-fold increase over T0 at 3 wk postvaccination were designated as responders. All children had a history of prior influenza vaccinations. At T0, the frequencies of CD4 T cell subsets, including peripheral T follicular helper (pTfh) cells, which provide help to B cells for developing into Ab-secreting cells, were similar between responders and nonresponders. However, in response to in vitro stimulation with influenza A/California/7/2009 (H1N1) Ag, differential gene expression related to pTfh cell function was observed by Fluidigm high-density RT-PCR between responders and nonresponders. In responders, H1N1 stimulation at T0 also resulted in CXCR5 induction (mRNA and protein) in CD4 T cells and IL21 gene induction in pTfh cells that were strongly associated with H1N1-specific B cell responses postvaccination. In contrast, CD4 T cells of nonresponders exhibited increased expression of IL2 and STAT5 genes, which are known to antagonize peripheral Tfh cell function. These results suggest that the quality of pTfh cells at the time of immunization is important for influenza vaccine responses and provide a rationale for targeted, ex vivo Ag-driven molecular profiling of purified immune cells to detect predictive biomarkers of the vaccine response.

Vaccine-induced protection against infection by HIV or highly pathogenic and virulent SIV strains has been limited. 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 SIVmac251. We vaccinated three cohorts of 12 macaques each with live, irradiated vaccine cells secreting the modified endoplasmic reticulum chaperone gp96-Ig. Cohort 1 was vaccinated with cells secreting gp96SIVIg carrying SIV peptides. In addition, Cohort 2 received recombinant envelope protein SIV-gp120. Cohort 3 was injected with cells secreting gp96-Ig (no SIV Ags) vaccines. Cohort 2 was protected from infection. After seven rectal challenges with highly pathogenic SIVmac251, the hazard ratio was 0.27, corresponding to a highly significant, 73% reduced risk for viral acquisition. The apparent success of the novel vaccine modality recommends further study.

TNFR superfamily (TNFRSF)4 (OX40, CD134) and TNFRSF25 are costimulatory receptors that influence CD4+ and CD8+ T cell responses to cognate Ag. Independently, these receptors have been described to stimulate overlapping functions, including enhanced proliferation and activation for both regulatory T cells (CD4+Foxp3+; Tregs) and conventional T cells (CD4+Foxp3− or CD8+Foxp3−; Tconvs). To determine the relative functionality of TNFRSF4 and TNFRSF25 in T cell immunity, the activity of TNFRSF4 and TNFRS25 agonistic Abs was compared in the context of both traditional protein/adjuvant (OVA/aluminum hydroxide) and CD8+-specific heat shock protein-based (gp96-Ig) vaccine approaches. These studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine-induced CD8+ T cell proliferation following both primary and secondary Ag challenge. In contrast, the activities of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4+ T cell immunity. TNFRSF4 agonists were potent costimulators of OVA/aluminum hydroxide-induced CD4+ Tconv proliferation, but they only weakly costimulated Treg proliferation and IgG2a production, whereas TNFRSF25 agonists were strong costimulators of Treg proliferation, producers of IgG1, IgG2a, and IgG2b, and weak costimulators of CD4+ Tconv proliferation. Interestingly, Ag-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically affected by the presence of TNFRSF4 or TNFRSF25 costimulation. These studies highlight the overlapping but nonredundant activities of TNFRSF4 and TNFRSF25 in T cell immunity, which may guide the application of receptor agonistic agents as vaccine adjuvants for infectious disease and tumor immunity.

Perturbations in B cells occur in both, chronological aging and in HIV infection. We investigated B cells in young (Y

The secreted gp96 vaccine approach is based on the use of cellular vaccines (293 cells) that continuously secrete gp96-Ig associated with antigenic peptides that are cross presented to CD8 T cells in vivo. Cellular gp96 vaccines are highly active in generation CTL for cancer therapy and SIV prophylaxis. Here, we investigate the use of gp96-Ig cDNA as vaccine platform. An optimized electroporation approach was used to deliver the B45 plasmid encoding secreted gp96-Ig and HIV-gag-gfp or ovalbumin genes. The B45 plasmid is a bovine papilloma virus derived vector from which the potentially transforming genes E5, E6, and E7 have been removed as well as the L1 and L2 viral capsid genes. The plasmid (50ug) was injected into the muscle (musculus tibialis anterior) of C57/Bl6 mice followed by electric field pulse that induced temporary and reversible pores in the muscle cell membrane. 48h after i.m. injection and EP-delivery, we found gfp (HIV-gag-gfp) expression in the muscle cells. To determine gp96-Ig secretion and cross presentation to CD8 cells we used cDNA for ovalbumin as antigen in conjunction with gp96-Ig and expansion of OT-I TCR-transgenic cells as readout. Vaccination by electroporation increased the frequency of OT-I in the spleen and in the mucosal compartment (lamina propria, small intestine).This simple, efficient and reproducible method of delivery may have great potential for further development of DNA gp96-Ig vaccination.

NOD.SCID il-2r γc-/- (NSG) KO mice have no functional adaptive immune system and readily accept human xenografts. We have generated NSG mice with human adaptive immunity (NSG-hu) by transplanting CD34+ hematopoietic stem cells isolated from fetal hepatic tissue. These early progenitors give rise to all members of the human adaptive and innate lineages and can be found localized to immunologically relevant compartments (spleen, thymus, mLN, IEL and LPL). Human engraftment appears stable over time and reaches a high proportion of human CD45+ cells when compared to murine CD45+ cells in peripheral blood (54%±24%). Higher proportions of human CD45+ cells were detected in specific tissues (spleen, PEC, IEL and mLN) (82%±29%). We have also found that by using ACK2, an antagonistic anti-mouse stem cell antibody, to further ablate murine hematopoietic stem cells, human engraftment can be boosted (81%±6%). Previously, our lab has developed a cell secreted heat shock protein gp96 based vaccine expressing HIV gag (293gp96HIVgag). NSG-hu vaccinated with 293gp96HIVgag i.p. generates HIV gag-pentamer positive CD8 T cells in the gut. In addition, an interferon gamma ELIPOST response is also observed in the mucosa when stimulated with relevant peptide but not in spleen. We conclude that NSG-hu mice are able to establish and maintain stable hematopoiesis from CD34+ precursor cells. Furthermore, NSG-hu mice are capable of mounting a human HIV-specific immune response after gp96 vaccination.

NOD.SCID common gamma chain (NSG) knockout mice have no functional adaptive immune system of their own; they readily accept human CD34+ hematopoietic stem cell xenografts (NSG-hu). Therefore, NSG-hu mice present a model system in which to study human immunology. Previously, we developed a cell based vaccine combining secreted heat shock protein gp96 and HIV gag protein (293-gp96Ig-HIVgag). Here we use NSG-hu mice vaccinated with 293-gp96Ig-HIVgag to study human immunological responses. Flow cytometry analysis of peripheral blood in pre-vaccination (8-9 week old) animals revealed human engraftment (mCD45 vs. hCD45) ranging from 25%-50%. In addition, human CD45+ cells were detected in specific tissues (spleen, PEC, IEL and MLN); CD4+, CD8+ T cells and NK cells were found within secondary murine lymphoid organs. Post vaccination (12-13 week old) human engraftment of peripheral blood was observed ranging from 15%-79%. In an ELISPOT for IFN-γ, PECs from NSG-hu vaccinated with 293-gp96Ig-HIVgag i.p. generated an HIV specific immune response when stimulated with SLYNTVATL peptide, producing an average of 778 spots per 2x105 cells plated whereas mice vaccinated with 293 cells alone generated an average of 88.5 spots per 2x105 cells plated (p

TNF receptor super family member 25 (TNFR25, DR3) is constitutively and highly expressed by CD4+FoxP3+ regulatory T cells (Treg). Here we have utilized a TNFR25 agonistic monoclonal antibody, 4C12, to study the effects of TNFR25 signaling on Treg in vivo. We have previously demonstrated that signaling through TNFR25, in the absence of TCR signals, induces rapid and selective expansion of natural Treg in vivo to 30-35% of all CD4+ T cells within 4 days. TNFR25-expanded Treg remained highly suppressive ex vivo. Here we demonstrate that although certain IL-2 antibody complexes expand Treg to a similar degree, only in vivo TNFR25-expanded Treg are protective against allergic lung inflammation, a model for asthma, by reversing the T-effector to T-regulatory ratio in the lung, suppressing IL-13 and Th2 cytokine production and blocking eosinophil exudation into bronchio-alveolar fluid. Unlike TNFR25 responses by T effector cells, which require TCR engagement, expansion of Treg by TNFR25 signaling is not dependent upon stimulation by exogenous antigens. Our studies define a novel mechanism for Treg control and important functions for TNFR25/TL1A in regulating auto aggression that balance its known role in enhancing auto immunity.

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.

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.