Development of a Vaccine for HIV-AIDS: Cellular Immunity

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We are pursuing an HIV vaccine approach based on replication-competent Adenovirus (Ad)-recombinants. The rationale for this strategy is based on the fact that live attenuated vaccines historically have been the most protective, eliciting essentially life-long immunity. Examples include vaccines for smallpox, polio, measles, and yellow fever. We conduct pre-clinical vaccine studies in rhesus macaques and challenge with SIV or SHIV (a chimeric SIV virus containing an HIV envelope), viruses that model HIV-infection of humans. We use a prime-boost strategy, first immunizing mucosally with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a boosting intramuscularly with HIV/SIV envelope protein. Ad replicates in epithelial cells that line mucosal inductive sites, and therefore elicits strong, persistent cellular immunity at mucosal effector sites as well as in the blood. In studying vaccine-induced cellular immunity, we focused on several specific cell types. Natural killer (NK) cells have a rapid response potential, can kill target cells directly, and mediate antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC) which has been shown to contribute to protective efficacy. They can also persist for long periods of time in vivo and have the capacity to establish immunologic memory. We investigated NK cell and innate lymphoid cell (ILC) dynamics and function in rhesus macaque rectal tissue and blood following our vaccination strategy and subsequent repeated low-dose intravaginal SIV exposures. Mucosal memory-like NK and ILC subsets in rectal and vaginal tissues of chronically infected macaques were also evaluated. Mucosal IL-17+NKp44+ ILCs were previously associated with a decreased risk of SIV acquisition and IFN-g+ double-negative (DN) ILCs with an increased risk of acquisition. We confirmed and extended these observations, showing that not only did NKp44+ and DN cells exhibit similar associations, but the respective effects continued past SIV infection, contributing to regulation of viral loads. The data suggested that mucosal NKp44+ and DN ILC subsets play protective and pathogenic roles contributing to viremia control and disease progression, respectively. We also identified gamma-chain deficient, Syk-deficient memory-like delta-gamma NK cells in rectal, endocervical, ectocervical, and vaginal tissues obtained at necropsy from the rhesus macaques. The overall frequency of the mucosal delta-gamma cell populations was higher in the mucosal tissues of macaques with low VLs compared to macaques with high VLs suggesting they might contribute to viremia control. Overall, our results suggested that vaccines that favor expansion of NKp44+ delta-gamma cells, while avoiding DN delta-gamma cells, might be beneficial for control of SIV/HIV infection. Dendritic cells (DCs) orchestrate both innate and adaptive effector immune cell responses. We investigated their role in vaccine-induced immunity in rhesus macaques. Following mucosal immunizations with replicating Ad type 5 host range mutant (Ad5hr)-SIV recombinants, DC subsets and their activation were examined in rectal tissue, blood, and lymph nodes (LN) at 3 timepoints after each immunization. Plasmacytoid DCs, myeloid DCs, and Langerhans cells were significantly increased in the rectal mucosa, but only myeloid DCs were significantly increased in blood post-immunizations. All rectal DC subsets showed increased frequencies of cells expressing activation markers and cytokines post-immunization, blood DCs showed mixed results, and LN DCs showed few changes. Rectal DCs responded strongly to the vector rather than expressed SIV antigens, but rectal DC frequencies positively correlated with induced rectal antigen-specific memory T and B cells. In vitro co-cultures confirmed that rectal Ad-SIV DCs induced proliferation and antigen-specific cytokine production by autologous naive T cells. These results highlighted the rapid response of DCs to Ad immunization and their role in mucosal immune activation and identified initial cellular mechanisms of the replicating Ad-SIV vaccine in the rhesus macaque model. Alveolar macrophages (AMs) play a critical role in lung innate immunity. Using the SIV rhesus macaque model, we investigated the effect of SIV infection on the phenotypic and functional properties of AMs. AM expression of proinflammatory cytokines TNF-a, IL-6, IL-1B, and chemokine RANTES drastically increased 2 weeks post-infection (wpi) compared to AMs of uninfected macaques but dropped significantly with progression to chronic infection. Phagocytic activity of AMs 2-and 4-wpi was elevated compared to AMs of uninfected animals but again significantly decreased by 12-wpi. By 20-wpi the ability of AMs from chronically infected animals to perform SIV-specific antibody-dependent phagocytosis (ADP) was also diminished. PD-1 was expressed on AMs and showed a strong trend toward correlation with plasma viral load, indicating that similar to over-expression on T-cells, PD-1 expression on AMs may be associated with disease progression. Importantly, blockade of PD-1 improved phagocytic function. These findings provide new insight into the dynamics of SIV infection leading to AM dysfunction and alteration of pulmonary innate immunity and suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in HIV-infected individuals. We furthered these studies by evaluating cervicovaginal macrophages (CVM) in rhesus macaques over the course of an SIV vaccine regimen. As one of the first cells exposed to SIV, their interaction with the virus might lead to activation and recruitment of additional susceptible target cells but might also provide important early protection. Knowledge of their response to mucosal vaccination is critical for design and development of improved vaccine strategies. As our mucosal priming immunizations were administered to the upper respiratory tract, we also investigated alveolar macrophages (AM) over the course of immunization. We found that while vaccination induced chemokine responses by CVM, recruitment of susceptible cells did not result. Further, induction of FcgammaRIII expression on CVM was associated with delayed SIV acquisition. At the same time vaccination induced AM activation and phagocytic activity along with expression of factors associated with B cell help. Overall, these findings suggest that vaccine enhancement of macrophage responses may lead to greater efficacy. In view of the association of PD-1-expressing cells with increased viral loads we investigated other cell populations expressing this molecule. We identified a dysfunctional CD8+ T cell population co-expressing CD6 and PD-1 in lymphoid tissues and bronchoalveolar lavage of rhesus macaques chronically infected with SIV. This cell population was expanded compared to uninfected cells and displayed impaired proliferation, cytokine secretion, and cytotoxicity. The frequency of the cells positively correlated with viremia. The cells expressed elevated levels of the inhibitory receptor LAG-3 and SHP-2 phosphatase compared to CD6-PD-1+ CD8+ T cells suggesting a mechanism by which CD6 might induce T cell dysfunction. Combined targeting of CD6 and PD-1 in vitro revived CD8+ T cell effector function of splenocytes of infected macaques suggesting a potential therapeutic strategy.