Objective: In HIV-infected individuals on antiretroviral therapy (ART), latent HIV is enriched in CD4⁺ T cells expressing immune checkpoint molecules, in particular programmed cell death-1 (PD-1). We therefore assessed the effect of blocking PD-1 on latency, both in vitro and in vivo. Methods: HIV latency was established in vitro following coculture of resting CD4⁺ T cells with myeloid dendritic cells. Expression of PD-1 was quantified by flow cytometry, and latency assessed in sorted PD-1^high and PD-1^low/− nonproliferating CD4⁺ memory T cells. The role of PD-1 in the establishment of latency was determined by adding anti-PD-1 (pembrolizumab) to cocultures before and after infection. In addition, a single infusion of anti-PD-1 (nivolumab) was administered to an HIV-infected individual on ART with metastatic melanoma, and cell-associated HIV DNA and RNA, and plasma HIV RNA were quantified. Results: HIV latency was significantly enriched in PD-1^high compared with PD-1^low/- nonproliferating, CD4⁺ memory T cells. Sorting for an additional immune checkpoint molecule, T-cell immunoglobulin domain and mucin domain-3, in combination with PD-1, further enriched for latency. Blocking PD-1 prior to HIV infection, in vitro, resulted in a modest but significant decrease in latently infected cells in all donors (n = 6). The administration of anti-PD-1 to an HIV-infected individual on ART resulted in a significant increase in cell-associated HIV RNA in CD4⁺ T cells, without significant changes in HIV DNA or plasma HIV RNA, consistent with reversal of HIV latency. Conclusion: PD-1 contributes to the establishment and maintenance of HIV latency and should be explored as a target, in combination with other immune checkpoint molecules, to reverse latency.

The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease.

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Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-κB element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon.

In people living with HIV on antiretroviral therapy, HIV latency is the major barrier to a cure. HIV persists preferentially in CD4⁺ T cells expressing multiple immune checkpoint (IC) molecules, including programmed death (PD)-1, T cell Ig and mucin domain-containing protein 3 (TIM-3), lymphocyte associated gene 3 (LAG-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). We aimed to determine whether these and other IC molecules have a functional role in maintaining HIV latency and whether blocking IC molecules with Abs reverses HIV latency. Using an in vitro model that establishes latency in both nonproliferating and proliferating human CD4⁺ T cells, we show that proliferating cells express multiple IC molecules at high levels. Latent infection was enriched in proliferating cells expressing PD-1. In contrast, nonproliferating cells expressed IC molecules at significantly lower levels, but latent infection was enriched in cells expressing PD-1, TIM-3, CTL-associated protein 4 (CTLA-4), or B and T lymphocyte attenuator (BTLA). In the presence of an additional T cell–activating stimulus, staphylococcal enterotoxin B, Abs to CTLA-4 and PD-1 reversed HIV latency in proliferating and nonproliferating CD4⁺ T cells, respectively. In the absence of staphylococcal enterotoxin B, only the combination of Abs to PD-1, CTLA-4, TIM-3, and TIGIT reversed latency. The potency of latency reversal was significantly higher following combination IC blockade compared with other latency-reversing agents, including vorinostat and bryostatin. Combination IC blockade should be further explored as a strategy to reverse HIV latency. Key Points Blocking CTLA-4 or PD-1 combined with T cell activation reverses HIV latency. Without T cell activation, blocking multiple ICs reverses latency.

HIV latency is the major barrier to a cure for people living with HIV (PLWH) on antiretroviral therapy (ART) because the virus persists in long-lived non-proliferating and proliferating latently infected CD4⁺ T cells. Latently infected CD4⁺ T cells do not express viral proteins and are therefore not visible to immune mediated clearance. Therefore, identifying interventions that can reverse latency and also enhance immune mediated clearance is of high interest. Interferons (IFNs) have multiple immune enhancing effects and can inhibit HIV replication in activated CD4⁺ T cells. However, the effects of IFNs on the establishment and reversal of HIV latency is not understood. Using an in vitro model of latency, we demonstrated that plasmacytoid dendritic cells (pDC) inhibit the establishment of HIV latency through secretion of type I IFNα, IFNβ and IFNω but not IFNε or type III IFNλ1 and IFNλ3. However, once latency was established, IFNα but no other IFNs were able to efficiently reverse latency in both an in vitro model of latency and CD4⁺ T cells collected from PLWH on suppressive ART. Binding of IFNα to its receptor expressed on primary CD4⁺ T cells did not induce activation of the canonical or non-canonical NFκB pathway but did induce phosphorylation of STAT1, 3 and 5 proteins. STAT5 has been previously demonstrated to bind to the HIV long terminal repeat and activate HIV transcription. We demonstrate diverse effects of interferons on HIV latency with type I IFNα; inhibiting the establishment of latency but also reversing HIV latency once latency is established. Antiretroviral therapy (ART) cannot cure HIV or eliminate infection from long-lived and proliferating latently infected CD4⁺ T cells. Plasmacytoid dendritic cells (pDC) are major producers of interferons (IFNs), which have multiple effects on viral replication and immunity including suppression of viral expression that could favor HIV latency. Van Der Sluis et al. show that type I IFNs inhibit the establishment of HIV latency, however, once established, latency can be reversed by IFNα but not by other type I or type III IFNs. These observations demonstrate that pDC through type I IFNs are important in HIV latency and can potentially be manipulated to eliminate latent infection.