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ERASE HIV, Research Focus 2:

In the setting of HIV infection, CD8+ T cells can mediate two distinct activities to suppress viral replication: the major histocompatibility complex (MHC)-restricted, antigen (Ag)-specific cytolytic response, which directly eliminates virus-producing cells, and the non-MHC-restricted, non-cytolytic response, which silences HIV transcription and favors the establishment of latency. By promoting viral latency, the non-cytolytic CD8+ T cell response enables infected cells to avoid clearance by cytotoxic T lymphocytes (CTLs) due to insufficient antigen expression; thereby paradoxically enabling viral persistence. Furthermore, cytotoxic HIV-specific CD8+ T cells are incapable of effectively killing CD4+ T cells in which the reactivation of virus production leads to systemic viral rebound due to their exhausted effector potential from chronic antigen stimulation; a skewed memory profile that limits survival and proliferative capacity; and their exclusion from lymphoid tissue. We hypothesize that any therapeutic strategy aimed at achieving viral remission requires rejuvenated cytotoxic CD8+ T cells localized to lymphoid sites of viral persistence.

ERASE HIV, Research Focus 3:

The “shock and kill” approach for HIV eradication aims to reactivate virus in latently infected cells coupled with their immune-mediated clearance; however, clinical trials have yet to demonstrate the efficacy of this concept. Notably, CD8+ T cells exert a pro-latency effect during ART and contribute to the inefficiency of latency reversing agents (LRAs) in inducing virus reactivation in vivo. Therefore, approaches are needed to simultaneously suppress the pro-latency effects of CD8+ T cells while promoting the immune-mediated clearance of CD4+ T cells harboring reactivated virus. We hypothesize that inhibiting pro-survival pathway will reduce the reservoir size by promoting apoptosis in cells with reactivated virus production (“shock and suicide”).

Complementing broadly neutralizing antibodies to restrict virus escape:

The HIV-1 envelope (Env) trimer is a challenging target for humoral immunity, as it is highly glycosylated, masks its conserved regions, and is tolerant of variation in exposed regions. During infection, neutralizing antibody responses drive virus selection, but lag rapidly adapting virus populations. In rare individuals, these successive waves of virus evolution and antibody responses lead to the development of unique broadly neutralizing antibodies (bNAbs) that successfully bind conserved regions in Env; however, virus resistance remains the central vulnerability of effective bNAb use. Effective strategies to combat HIV-1 via bNAbs require a better understanding of the selective pressure imposed by bNAbs and novel approaches to limit virus escape. Notably, bNAbs can exhibit mutually exclusive resistance pathways when targeting the same epitope, albeit with fine specificity differences, or targeting distinct, but conformationally related, epitopes. By mapping escape variants, we seek to identify putative complementary bNAbs, which exhibit inverse resistance pathways (i.e. antagonistic mutations), such that escape from one enhances sensitivity to the other.

The role of platelets in promoting HIV reservoir seeding in the CNS-resident myeloid cells:

Seeding of the HIV-1 reservoir in long-lived perivascular macrophages and microglial cells within the central nervous system (CNS) occurs almost immediately following onset of infection. The viral seeding of the CNS likely occurs via the transmigration of infected monocytes across the blood-brain barrier (BBB) as suggested by studies blocking CNS integrins. Paradoxically, monocytes are generally resistant to infection and levels of chemoattractant cytokines remain low in the cerebrospinal fluid (CSF) following infection; therefore, it is challenging to explain how monocytes seed the CNS viral reservoir. Our preliminary data suggests that platelets, anuclear cells involved in blood clotting, may promote HIV spread during the acute phase of infection by harboring and transmitting infectious virus to the susceptible monocytes through formation of platelet-monocyte complexes (PMCs). Therefore, we hypothesize that the disruption of PMC formation during the acute phase of infection will limit the seeding and maintenance of the CNS viral reservoir.

Determining the contribution of CD4+ T cells and macrophages to HIV persistence:

The long-term maintenance of the HIV-1 viral reservoir during antiretroviral therapy (ART) and the kinetics of viral rebound after ART interruption (ATI) are determined by the type, quantity, and anatomic distribution of infected cells prior to ART initiation. The viral reservoir is largely composed of latently infected, resting memory CD4+ T cells; yet tissue-resident macrophages may plausibly contribute to viral persistence given their expression of HIV cell entry receptors; their susceptibility to infection in vitro; and their harboring cell-associated HIV-DNA in multiple tissues in vivo. Relatedly, the depletion of CD4+ T cells prior to infection results in rapid AIDS progression characterized by the accumulation of viral DNA and RNA in macrophages and CNS-resident microglial cells. Using this model of “macrophage-dominated” viral reservoir establishment, we will test the hypothesis that CD4+ T cells and macrophages have fundamental differences in their propensity to be productively infected and persist amid long-term ART.



Th17 homeostasis and the role of IL-21 in SIV:

CD4+ Th17 cells, which express IL-17 and play a key role maintaining gut barrier integrity, are preferentially depleted from the gastrointestinal (GI) tract during pathogenic SIV/HIV infection; thereby driving microbial translation and inducing chronic immune activation, key determinants of disease progression and viral persistence (Ryan et al., PLoS Pathog, 2016). The disparity in Th17 depletion between pathogenic and nonpathogenic models of infection correlates with the loss of IL-21 producing CD4+ T-cells in GI tract, suggesting the IL-21 signaling is a regulator of Th17 homeostasis (Micci et al., Blood, 2012). IL-21, when administered during acute SIV infection, preserves intestinal Th17 cells and limits microbial translation (Pallikkuth et al., PLoS Pathog, 2013). When administered in combination with ART, IL-21 treatment reduced inflammation and the content of replication competent virus (Micci et al., J Clin Invest, 2015); however, the latter was not associated with an enhancement of SIV-specific T cell responses. Rather, IL-21 therapy results in the formation of terminally differentiated Natural Killer cells (NKTDs), which exhibit potent MHC-E-restricted anti-SIV activity and correlate with the reduction in replication competent virus (Harper et al., Nat Commun, 2021). The formation of NKTDs are blocked in early acute infection in rhesus macaques (RMs), but not in natural hosts, such as African green monkeys (AGMs; Huot et al., Nat Commun, 2021). These studies provided, for the first time, direct in vivo evidence of the benefit of IL-21 in reducing inflammation during ART and limiting viral persistence through innate-like immune responses, which underscores the clinical potential of this therapy for reducing morbidity in HIV-infected individuals. 

T cell exhaustion and survival in SIV:

As a consequence of chronic antigen stimulation, CD4+ and CD8+ T cells upregulate inhibitory immune checkpoint receptors (ICRs; e.g. PD-1, CTLA-4, TIM-3, LAG3, TIGIT). These ICRs antagonize T cell priming by antigen presenting cells resulting in an “exhausted” hypo-responsive state characterized by impaired cytokine production, diminished proliferative capacity, and aberrant effector differentiation. During long-term ART, the latent viral reservoir during is enriched for CD4+ T cells expressing ICRs, including PD-1+ T follicular helper (TFH) cells in the lymphoid B cell follicle (BCF), an immune-privileged sanctuary for viral persistence. Furthermore, CTLA-4+ CD4+ T cells in the lymphoid T cell zone (TCZ) harbor high levels of replication competent SIV-DNA, which increasingly contributes to composition of the viral reservoir with ongoing ART given their enhanced Bcl-2/STAT5-mediated survival capacity (McGary et al., Immunity, 2017). Combination immune checkpoint blockade (ICB; e.g. anti-PD-1 and/or anti-CTLA-4) synergize to induce T cell proliferation resulting in clonally diverse viral reactivation in plasma, which contributes to a reduction in the viral reservoir size in lymph node (LN) via activation-induced death in a subset of responding cells; however, ICB is insufficient to impact the activity of SIV-specific T cell responses (Harper et al., Nat Med, 2020). Based on the reinvigoration of SIV-specific T cells following ART interruption (ATI), these results demonstrate that the reversal of T cell exhaustion alone, in the absence of viral antigen, is insufficient to stimulate adaptive responses. In parallel, we have shown that levels of IL-10, an immunosuppressive cytokine that regulates TFH differentiation and BCF maintenance, and/or its transcriptomic signatures are induced by SIV infection and not fully normalized with ART; correlate with the content of SIV-DNA in tissue during chronic infection and ART; and co-localize with cells harboring SIV-DNA in the lymphoid BCF. Furthermore, in vivo neutralization of soluble IL-10 during short-term ART lowers the frequency of T cells expressing ICRs, such as PD-1 and CTLA-4, while enhancing immune activation (Harper et al., J Clin Invest, 2022). Preliminary data suggests that PD-1 blockade in combination with IL-10 neutralization synergize to potently control viremia following ART interruption via an unknown mechanism that is currently under investigation (Ribeiro et al., manuscript in preparation).

Inflammation and vaccination responses in SARS-CoV-2:

Using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected rhesus macaques (RMs) as a model of COVID-19 immunopathology, we have shown that Baricitinib, a JAK1/2 inhibitor, attenuates viral-induced lung inflammation by suppressing macrophage chemokine production, which in turn diminishes the infiltration of pro-inflammatory cells (i.e. macrophages and neutrophils) and reduces NETosis activity (Hoang et al., Cell, 2020). Baricitinib’s therapeutic benefit was confirmed in subsequent human clinical trials (Kalil et al., N Engl J Med, 2021; Ely et al., Lancet Respir Med, 2022) leading to emergency use authorization by the FDA. Additional single-cell RNA sequencing analyses revealed populations of lung interstitial (e.g. CD206+MRC1-) and infiltrating (e.g. TREM2+) macrophages as the principal orchestrators of SARS-CoV-2 induced inflammation (Upadhyay et al., manuscript submitted). Furthermore, we evaluated the immune responses and protection of a modified vaccinia Ankara (MVA)-based vaccine expressing the receptor-binding domain (RBD) of the viral spike in combination with a TLR-7/8 agonist adsorbed to alum, 3M-052, following SARS-CoV-2 challenge (Pino et al., Sci Immunol, 2021).

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