Efficient ex vivo expansion of conserved element vaccine-specific CD8+ T-cells from SHIV-infected, ART-suppressed nonhuman primates

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells’ recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo-expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells’ predominant CD8+ effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans

DNA immunization in combination with effective antiretroviral drug therapy controls viral rebound and prevents simian AIDS after treatment is discontinued

AbstractDNA immunization in conjunction with antiretroviral therapy was evaluated in SIV-infected rhesus macaques treated with [R]-9-[2-phosphonylmethoxypropyl]adenine (PMPA). Macaques were immunized monthly with DNA vaccines expressing either SIV gag/tat or SIV gag/tat and 19 CD8+ T cell epitopes during 7 months of therapy. Half the animals from each group were additionally immunized before infection. Only 60% of the animals (4 controls, 20 vaccinated) responded to PMPA (ART responders). All 4 ART responder controls demonstrated viral rebound or CD4 decline after PMPA was withdrawn. In contrast, 17 of 20 vaccinated ART responders contained viral rebound for over 7 months after PMPA was withdrawn. Viral control correlated with stable CD4 counts, higher lymphoproliferation and an increase in the magnitude and breadth of the CD8+ T cell response. Immunizing before infection or with multi-epitopes enhanced these effects. These results demonstrate that DNA immunization during antiretroviral therapy may be an effective strategy to treat HIV infection

Therapeutic conserved elements (CE) DNA vaccine induces strong T-cell responses against highly conserved viral sequences during simian-human immunodeficiency virus infection

HIV-specific T-cell responses play a key role in controlling HIV infection, and therapeutic vaccines for HIV that aim to improve viral control will likely need to improve on the T-cell responses induced by infection. However, in the setting of chronic infection, an effective therapeutic vaccine must overcome the enormous viral genetic diversity and the presence of pre-existing T-cell responses that are biased toward immunodominant T-cell epitopes that can readily mutate to evade host immunity and thus potentially provide inferior protection. To address these issues, we investigated a novel, epidermally administered DNA vaccine expressing SIV capsid (p27Gag) homologues of highly conserved elements (CE) of the HIV proteome in macaques experiencing chronic but controlled SHIV infection. We assessed the ability to boost or induce de novo T-cell responses against the conserved but immunologically subdominant CE epitopes. Two groups of animals were immunized with either the CE DNA vaccine or a full-length SIV p57gag DNA vaccine. Prior to vaccination, CE responses were similar in both groups. The full-length p57gag DNA vaccine, which contains the CE, increased overall Gag-specific responses but did not increase CE responses in any animals (0/4). In contrast, the CE DNA vaccine increased CE responses in all (4/4) vaccinated macaques. In SIV infected but unvaccinated macaques, those that developed stronger CE-specific responses during acute infection exhibited lower viral loads. We conclude that CE DNA vaccination can re-direct the immunodominance hierarchy towards CE in the setting of attenuated chronic infection and that induction of these responses by therapeutic vaccination may improve immune control of HIV

Immunogenicity of hybrid DNA vaccines expressing hepatitis B core particles carrying human and simian immunodeficiency virus epitopes in mice and rhesus macaques

An effective HIV vaccine will likely need to induce broad and potent CTL responses. Epitope-based vaccines offer significant potential for inducing multi-specific CTL, but often require conjugation to T helper epitopes or carrier moieties to induce significant responses. We tested hybrid DNA vaccines encoding one or more HIV or SIV CTL epitopes fused to a hepatitis B core antigen ( HBcAg) carrier gene as a means to improve the immunogenicity of epitope-based DNA vaccines. Immunization of mice with a HBcAg-HIV epitope DNA vaccine induced CD8 + T cell responses that significantly exceeded levels induced with DNA encoding either the whole HIV antigen or the epitope alone. In rhesus macaques, a multi-epitope hybrid HBcAg-SIV DNA vaccine induced CTL responses to 13 different epitopes, including 3 epitopes that were previously not detected in SIV-infected macaques. These data demonstrate that immunization with hybrid HBcAg-epitope DNA vaccines is an effective strategy to increase the magnitude and breadth of HIV-specific CTL responses

Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation

<div><p>A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.</p></div

Laparoscopic Technique for Serial Collection of Para-Colonic, Left Colic, and Inferior Mesenteric Lymph Nodes in Macaques.

Unlike peripheral lymph nodes (PLN), the mesenteric lymph nodes (MLN) draining the gastrointestinal (GI) tract are exposed to microbes and microbial products from the intestines and as such, are immunologically distinct. GI draining (MLN) have also been shown to be sites of early viral replication and likely impact early events that determine the course of HIV infection. They also are important reservoir sites that harbor latently-infected cells and from which the virus can emerge even after prolonged combination antiretroviral therapy (cART). Changes in the microbial flora and increased permeability of the GI epithelium associated with lentiviral infection can impact the gut associated lymphoid tissue (GALT) and induce changes to secondary lymphoid organs limiting immune reconstitution with cART. Nonhuman primate models for AIDS closely model HIV infection in humans and serial sampling of the GALT and associated secondary lymphoid organs in this model is crucial to gain a better understanding of the critical early events in infection, pathogenesis, and the role of immune responses or drugs in controlling virus at these sites. However, current techniques to sample GI draining (MLN) involve major surgery and/or necropsy, which have, to date, limited the ability to investigate mechanisms mediating the initiation, persistence and control of infection in this compartment. Here, we describe a minimally invasive laparoscopic technique for serial sampling of these sites that can be used with increased sampling frequency, yields greater cell numbers and immune cell subsets than current non-invasive techniques of the GALT and reduces the potential for surgical complications that could complicate interpretation of the results. This procedure has potential to facilitate studies of pathogenesis and evaluation of preventive and treatment interventions, reducing sampling variables that can influence experimental results, and improving animal welfare

Correction: Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation.

[This corrects the article DOI: 10.1371/journal.pone.0198154.]

Early cellular innate immune responses drive Zika viral persistence and tissue tropism in pigtail macaques

The immune response to Zika virus is required to curtail the infection and avoid immunopathology, but may be involved in the associated pathophysiology. Here the authors show that viral persistence and tissue tropism is shaped by an early innate immune response in a pigtail macaque model of infection