No evidence that circulating HIV-specific immune responses contribute to persistent inflammation and immune activation in persons on long-term ART.

OBJECTIVE: People with HIV (PWH) have persistently elevated levels of inflammation and immune activation despite suppressive antiretroviral therapy (ART), with specific biomarkers showing associations with non-AIDS-defining morbidities and mortality. We investigated the potential role of the HIV-specific adaptive immune response, which also persists under ART, in driving levels of these clinically relevant biomarkers. DESIGN: Cohort-based study. METHODS: HIV-specific IFN-γ-producing T-cell responses and antibody concentrations were measured in blood at study entry in the ACTG A5321 cohort, following a median of 7 years of suppressive ART. HIV persistence measures including cell-associated (CA)-DNA, CA-RNA, and plasma HIV RNA (single-copy assay) were also assessed at study entry. Plasma inflammatory biomarkers and T-cell activation and cycling were measured at a pre-ART time point and at study entry. RESULTS: Neither the magnitudes of HIV-specific T-cell responses nor HIV antibody levels were correlated with levels of the inflammatory or immune activation biomarkers, including hs-CRP, IL-6, neopterin, sCD14, sCD163, TNF-α, %CD38 + HLA-DR + CD8 + and CD4 + cells, and %Ki67 + CD8 + and CD4 + cells - including after adjustment for pre-ART biomarker level. Plasma HIV RNA levels were modestly correlated with CD8 + T-cell activation ( r  = 0.25, P  = 0.027), but other HIV persistence parameters were not associated with these biomarkers. In mediation analysis, relationships between HIV persistence parameters and inflammatory biomarkers were not influenced by either HIV-specific T-cell responses or antibody levels. CONCLUSION: Adaptive HIV-specific immune responses do not appear to contribute to the elevated inflammatory and immune activation profile in persons on long-term ART.

Antibody-dependent cellular cytotoxicity responses and susceptibility influence HIV-1 mother-to-child transmission.

HIV-1 vaccine efforts are primarily directed toward eliciting neutralizing antibodies (nAbs). However, vaccine trials and mother-to-child natural history cohort investigations indicate that antibody-dependent cellular cytotoxicity (ADCC), not nAbs, correlate with prevention. The ADCC characteristics associated with lack of HIV-1 acquisition remain unclear. Here, we examine ADCC and nAb properties in pretransmission plasma from HIV-1-exposed infants and from the corresponding transmitting and nontransmitting mothers' breast milk and plasma. Breadth and potency (BP) were assessed against a panel of heterologous, nonmaternal variants. ADCC and neutralization sensitivity were estimated for the strains in the infected mothers. Infants who eventually acquired HIV-1 and those who remained uninfected had similar pretransmission ADCCBP. Viruses circulating in the transmitting and nontransmitting mothers had similar ADCC susceptibility. Infants with higher pretransmission ADCCBP and exposure to more ADCC-susceptible strains were less likely to acquire HIV-1. In contrast, higher preexisting infant neutralization BP and greater maternal virus neutralization sensitivity did not associate with transmission. Infants had higher ADCCBP closer to birth and in the presence of high plasma IgG relative to IgA levels. Mothers with potent humoral responses against their autologous viruses harbored more ADCC-sensitive strains. ADCC sensitivity of the exposure variants and preexisting ADCCBP influenced mother-to-child HIV-1 transmission during breastfeeding. Vaccination strategies that enhance ADCC are likely insufficient to prevent HIV-1 transmission because some strains may have low ADCC susceptibility.

Pre-existing infant antibody-dependent cellular cytotoxicity associates with reduced HIV-1 acquisition and lower morbidity.

In humans, pre-existing anti-HIV-1 neutralizing antibodies (nAbs) have not been associated with decreased HIV-1 acquisition. Here, we evaluate antibody-dependent cellular cytotoxicity (ADCC) present in pre-transmission infant and maternal plasma and breast milk (BM) against the contemporaneous maternal HIV-1 variants. HIV-1-exposed uninfected compared with HIV-1-exposed infected infants have higher ADCC and a combination of ADCC and nAb responses against their corresponding mother's strains. ADCC does not correlate with nAbs, suggesting they are independent activities. The infected infants with high ADCC compared with low ADCC, but not those with higher ADCC plus nAbs, have lower morbidity up to 1 year after birth. A higher IgA to IgG ratio, observed in BM supernatants and in a higher proportion of the infected compared with the uninfected infants, associates with lower ADCC. Against the exposure strains, ADCC, more than nAbs, associates with both lower mother-to-child transmission and decreased post-infection infant morbidity.

HIV-specific T cell responses reflect substantive in vivo interactions with antigen despite long-term therapy.

Antiretroviral therapies (ARTs) abrogate HIV replication; however, infection persists as long-lived reservoirs of infected cells with integrated proviruses, which reseed replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in reducing viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle: by querying the dynamics of HIV-specific T cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. In longitudinal assessments, we show that the rates of change in persisting HIV Nef-specific responses, but not responses to other HIV gene products, were associated with residual frequencies of infected cells. These Nef-specific responses were highly stable over time and disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV antigens. These results indicate substantial visibility of the HIV-infected cells to T cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing infection.

A new cell line for assessing HIV-1 antibody dependent cellular cytotoxicity against a broad range of variants.

Human immunodeficiency virus type 1 (HIV-1) studies suggest that antibody-dependent cellular cytotoxicity (ADCC) influences both virus acquisition and subsequent disease outcome. Technical issues with currently available assays, however, have limited the ability to comprehensively assess the impact of ADCC on transmission and disease progression. Commonly used ADCC assays use a target cell line, CEM.NKr-CCR5-Luc, that often does not support replication of relevant HIV-1 variants. Thus, the extent of ADCC responses against a large panel of HIV-1 strains often cannot be assessed using the currently available methods. We developed two new reporter cell-lines (MT4-CCR5-Luc and PM1-CCR5-Luc) to overcome these issues. MT4-CCR5-Luc cells are resistant, whereas PM1-CCR5-Luc cells are susceptible, to killing by a natural killer cell line, CD16+KHYG-1, in the absence of antibody. Polyclonal HIVIG gave similar ADCC estimates against HIV-1 isolate, NL4-3, regardless of which of the three cell lines were used as the targets. In contrast to CEM.NKr-CCR5-Luc and PM1-CCR5-Luc, however, MT4-CCR5-Luc target cells produce significantly higher luciferase after exposure to various HIV-1 strains, including transmitted founder variants and viruses incorporating specific envelopes of interest. This higher luciferase expression does not yield spurious results because ADCC estimates are similar when killing is assessed by both reporter protein expression and flow cytometry. Furthermore, ADCC estimates derived from MT4-CCR5-Luc cells are not skewed by non-antibody contents present in human plasma. In aggregate, the MT4-CCR5-Luc cell line can be used to estimate monoclonal antibody or plasma-induced ADCC responses against a diverse range of HIV-1 envelopes relevant for transmission and disease progression studies.

T cells with high PD-1 expression are associated with lower HIV-specific immune responses despite long-term antiretroviral therapy.

OBJECTIVE: We evaluated frequencies of T cells with high PD-1 expression (PD-1) before and after long-term effective antiretroviral therapy (ART), and determined if frequencies on-ART correlated positively with measures of HIV persistence and negatively with HIV-specific responses. METHODS: We enrolled individuals who started ART during chronic infection and had durable suppression of viremia for at least 4 years (N = 99). We assessed PD-1 T-cell frequencies at timepoints pre-ART and on-ART using flow cytometry, and evaluated how frequencies on-ART are associated with measures of HIV persistence, HIV-specific immune responses, and immune activation levels. RESULTS: Pre-ART, PD-1 CD4 T cells correlated positively with viremia and negatively with CD4 T-cell count. At year 1 on-ART, %PD-1 CD4 T cells decreased but then remained stable at 4 and 6-15 years on-ART, whereas %PD-1 CD8 T cells on-ART remained similar to pre-ART. PD-1 CD4 T cells correlated positively with HIV DNA pre-ART and on-ART, and with CD4 T-cell activation on-ART. PD-1 CD4 T cells negatively correlated with HIV Gag-specific and Env-specific T-cell responses but not with CMV-specific or EBV-specific responses. PD-1 CD8 T cells trended towards a negative correlation with responses to Gag and Env, but not to CMV and EBV. CONCLUSION: PD-1 T cells persist in blood despite prolonged suppression on ART, correlate with HIV DNA levels, and are associated with lower HIV-specific T-cell responses but not CMV-specific or EBV-specific responses, suggesting that these cells are HIV-specific. The findings support evaluating PD-1 blockade strategies for their effect on HIV persistence and HIV-specific immunity.

Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells.

The presence of persistent, latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models, which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses, which comprise the vast majority of all infected cells, and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here, we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA, but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus, ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus, our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection.

Neutralization and beyond: Antibodies and HIV-1 acquisition.

It is widely accepted that an effective HIV-1 preventative vaccine must elicit antibodies that can block virus acquisition. Although, anti-HIV-1 broadly neutralizing antibodies (bnAbs) have been isolated, unfortunately, no vaccine immunogens have been designed that can elicit these bnAbs in uninfected at-risk individuals. Some studies have suggested that other antibody functionalities, besides neutralization, such as antibody-dependent cellular cytotoxicity (ADCC), may prevent HIV-1 acquisition. In contrast to bnAbs, ADCC-inducing antibodies may be more amenable to elicitation by current vaccine technologies. This review will provide clarity about the role of nAbs and ADCC-inducing antibodies in preventing transmission, highlight mechanisms that potentially explain how ADCC-mediating antibodies may work, and speculate about the generation of these novel protective antibodies. Anti-HIV-1 ADCC-inducing antibodies may provide a new avenue for developing an effective HIV-1 vaccine.

T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy.

HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART), these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18). Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that enhancing the cytolytic activity of Nef-specific T-cells may lead to reductions in infected cell frequencies, even in the absence of therapeutic latency reversal.

Defective HIV-1 Proviruses Are Expressed and Can Be Recognized by Cytotoxic T Lymphocytes, which Shape the Proviral Landscape.

Despite antiretroviral therapy, HIV-1 persists in memory CD4+ T cells, creating a barrier to cure. The majority of HIV-1 proviruses are defective and considered clinically irrelevant. Using cells from HIV-1-infected individuals and reconstructed patient-derived defective proviruses, we show that defective proviruses can be transcribed into RNAs that are spliced and translated. Proviruses with defective major splice donors (MSDs) can activate novel splice sites to produce HIV-1 transcripts, and cells with these proviruses can be recognized by HIV-1-specific cytotoxic T lymphocytes (CTLs). Further, cells with proviruses containing lethal mutations upstream of CTL epitopes can also be recognized by CTLs, potentially through aberrant translation. Thus, CTLs may change the landscape of HIV-1 proviruses by preferentially targeting cells with specific types of defective proviruses. Additionally, the expression of defective proviruses will need to be considered in the measurement of HIV-1 latency reversal.

IFITM1 targets HIV-1 latently infected cells for antibody-dependent cytolysis.

HIV-1 persistence in latent reservoirs during antiretroviral therapy (ART) is the main obstacle to virus eradication. To date, there is no marker that adequately identifies latently infected CD4+ T cells in vivo. Using a well-established ex vivo model, we generated latently infected CD4+ T cells and identified interferon-induced transmembrane protein 1 (IFITM1), a transmembrane antiviral factor, as being overexpressed in latently infected cells. By targeting IFITM1, we showed the efficient and specific killing of a latently infected cell line and CD4+ T cells from ART-suppressed patients through antibody-dependent cytolysis. We hypothesize that IFITM1 could mark natural reservoirs, identifying an immune target for killing of latently infected cells. These novel insights could be explored to develop clinical therapeutic approaches to effectively eradicate HIV-1.

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes.

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.