Small molecule inhibitors of transcriptional cyclin-dependent kinases impose HIV-1 latency, presenting "block and lock" treatment strategies.

Current antiretroviral therapy for HIV-1 infection does not represent a cure for infection as viral rebound inevitably occurs following discontinuation of treatment. The "block and lock" therapeutic strategy is intended to enforce proviral latency and durably suppress viremic reemergence in the absence of other intervention. The transcription-associated cyclin-dependent protein kinases (tCDKs) are required for expression from the 5´ HIV-1 long-terminal repeat, but the therapeutic potential of inhibiting these kinases for enforcing HIV-1 latency has not been characterized. Here, we expanded previous observations to directly compare the effect of highly selective small molecule inhibitors of CDK7 (YKL-5-124), CDK9 (LDC000067), and CDK8/19 (Senexin A), and found each of these prevented HIV-1 provirus expression at concentrations that did not cause cell toxicity. Inhibition of CDK7 caused cell cycle arrest, whereas CDK9 and CDK8/19 inhibitors did not, and could be continuously administered to establish proviral latency. Upon discontinuation of drug administration, HIV immediately rebounded in cells that had been treated with the CDK9 inhibitor, while proviral latency persisted for several days in cells that had been treated with CDK8/19 inhibitors. These results identify the mediator kinases CDK8/CDK19 as potential "block and lock" targets for therapeutic suppression of HIV-1 provirus expression.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and this diversity persists within infected cell reservoirs during antiretroviral therapy (ART). Achieving a better understanding of on-ART proviral evolutionary dynamics, and a better appreciation of how the overall persisting pool of (largely genetically defective) proviruses differs from the much smaller replication-competent HIV reservoir, is critical to HIV cure efforts. We reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study who experienced HIV seroconversion, and used these data to characterize the diversity, lineage origins, and ages of proviral env-gp120 sequences sampled longitudinally up to 12 years on ART. We also studied HIV sequences emerging from the reservoir in two participants. We observed that proviral clonality generally increased over time on ART, with clones frequently persisting long term. While on-ART proviral integration dates generally spanned the duration of untreated infection, HIV emerging in plasma was exclusively younger (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained stable during ART in all but one participant, in whom there was evidence that younger proviruses had been preferentially eliminated after 12 years on ART. Analysis of the gag region in three participants corroborated our env-gp120-based observations, indicating that our observations are not influenced by the HIV region studied. Our results underscore the remarkable genetic stability of the distinct proviral sequences that persist in blood during ART. Our results also suggest that the replication-competent HIV reservoir is a genetically restricted, younger subset of this overall proviral pool.IMPORTANCECharacterizing the genetically diverse HIV sequences that persist in the reservoir despite antiretroviral therapy (ART) is critical to cure efforts. Our observations confirm that proviruses persisting in blood on ART, which are largely genetically defective, broadly reflect the extent of within-host HIV evolution pre-ART. Moreover, on-ART clonal expansion is not appreciably accompanied by the loss of distinct proviral lineages. In fact, on-ART proviral genetic composition remained stable in all but one participant, in whom, after 12 years on ART, proviruses dating to around near ART initiation had been preferentially eliminated. We also identified recombinant proviruses between parental sequence fragments of different ages. Though rare, such sequences suggest that reservoir cells can be superinfected with HIV from another infection era. Overall, our finding that the replication-competent reservoir in blood is a genetically restricted, younger subset of all persisting proviruses suggests that HIV cure strategies will need to eliminate a reservoir that differs in key respects from the overall proviral pool.

Effects of COVID-19 mRNA vaccination on HIV viremia and reservoir size.

OBJECTIVE: The immunogenic nature of COVID-19 mRNA vaccines led to some initial concern that these could stimulate the HIV reservoir. We analyzed changes in plasma HIV loads (pVL) and reservoir size following COVID-19 mRNA vaccination in 62 people with HIV (PWH) receiving antiretroviral therapy (ART), and analyzed province-wide trends in pVL before and after the mass vaccination campaign. DESIGN: Longitudinal observational cohort and province-wide analysis. METHODS: 62 participants were sampled pre-vaccination, and one month after their first and second COVID-19 immunizations. Vaccine-induced anti-SARS-CoV-2-Spike antibodies in serum were measured using the Roche Elecsys Anti-S assay. HIV reservoirs were quantified using the Intact Proviral DNA Assay; pVL were measured using the cobas 6800 (LLOQ:20 copies/mL). The province-wide analysis included all 290,401 pVL performed in British Columbia, Canada between 2012-2022. RESULTS: Pre-vaccination, the median intact reservoir size was 77 (IQR:20-204) HIV copies/million CD4+ T-cells, compared to 74 (IQR:27-212) and 65 (IQR:22-174) post-first and -second dose, respectively (all comparisons p>0.07). Pre-vaccination, 82% of participants had pVL<20 copies/mL (max:110 copies/mL), compared to 79% post-first dose (max:183 copies/mL) and 85% post-second dose (max:79 copies/mL) (p > 0.4). There was no evidence that the magnitude of the vaccine-elicited anti-SARS-CoV-2-Spike immune response influenced pVL nor changes in reservoir size (p > 0.6). We found no evidence linking the COVID-19 mass vaccination campaign to population-level increases in detectable pVL frequency among all PWH in the province, nor among those who maintained pVL suppression on ART. CONCLUSION: We found no evidence that COVID-19 mRNA vaccines induced changes in HIV reservoir size nor plasma viremia.

Dynamics of T-cell Responses Following COVID-19 mRNA Vaccination and Breakthrough Infection in Older Adults.

Introduction: While older adults generally mount weaker antibody responses to a primary COVID-19 vaccine series, T-cell responses remain less well characterized in this population. We compared SARS-CoV-2 spike-specific T-cell responses after 2- and 3-dose COVID-19 mRNA vaccination and subsequent breakthrough infection in older and younger adults. Methods: We quantified CD4+ and CD8+ T-cells reactive to overlapping peptides spanning the ancestral SARS-CoV-2 spike protein in 40 older adults (median age 79) and 50 younger health care workers (median age 39), all COVID-19 naive, using an activation-induced marker assay. T-cell responses were further assessed in 24 participants, including 8 older adults, who subsequently experienced their first SARS-CoV-2 breakthrough infection. Results: A third COVID-19 mRNA vaccine dose significantly boosted spike-specific CD4+ and CD8+ T-cell frequencies to above 2-dose levels in older and younger adults. T-cell frequencies did not significantly differ between older and younger adults after either dose. Multivariable analyses adjusting for sociodemographic, health, and vaccine-related variables confirmed that older age was not associated with impaired cellular responses. Instead, the strongest predictors of CD4+ and CD8+ T-cell frequencies post-third-dose were their corresponding post-second-dose frequencies. Breakthrough infection significantly increased both CD4+ and CD8+ T-cell frequencies, to comparable levels in older and younger adults. Exploratory analyses revealed an association between HLA-A*02:03 and higher post-vaccination CD8+ T-cell frequencies, which may be attributable to numerous strong-binding HLA-A*02:03-specific CD8+ T-cell epitopes in the spike protein. Conclusion: Older adults mount robust T-cell responses to 2- and 3-dose COVID-19 mRNA vaccination, which are further boosted following breakthrough infection.

Viral and host mediators of non-suppressible HIV-1 viremia.

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as 'producer proviruses', and those that did not as 'non-producer proviruses'. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.

HIV reservoirs are dominated by genetically younger and clonally enriched proviruses.

IMPORTANCE: Characterizing the human immunodeficiency virus (HIV) reservoir that endures despite antiretroviral therapy (ART) is critical to cure efforts. We observed that the oldest proviruses persisting during ART were exclusively defective, while intact proviruses (and rebound HIV) dated to nearer ART initiation. This helps explain why studies that sampled sub-genomic proviruses on-ART (which are largely defective) routinely found sequences dating to early infection, whereas those that sampled replication-competent HIV found almost none. Together with our findings that intact proviruses were more likely to be clonal, and that on-ART low-level/isolated viremia originated from proviruses of varying ages (including possibly defective ones), our observations indicate that (i) on-ART and rebound viremia can have distinct within-host origins, (ii) intact proviruses have shorter lifespans than grossly defective ones and thus depend more heavily on clonal expansion for persistence, and (iii) an HIV reservoir predominantly "dating" to near ART initiation will be substantially adapted to within-host pressures, complicating immune-based cure strategies.

Effects of COVID-19 mRNA vaccination on HIV viremia and reservoir size.

Objective: The immunogenic nature of COVID-19 mRNA vaccines led to some initial concern that these could stimulate the HIV reservoir. We analyzed changes in plasma HIV loads (pVL) and reservoir size following COVID-19 mRNA vaccination in 62 people with HIV (PWH) receiving antiretroviral therapy (ART), and analyzed province-wide trends in pVL before and after the mass vaccination campaign. Design: Longitudinal observational cohort and province-wide analysis. Methods: 62 participants were sampled pre-vaccination, and one month after their first and second COVID-19 immunizations. Vaccine-induced anti-SARS-CoV-2-Spike antibodies in serum were measured using the Roche Elecsys Anti-S assay. HIV reservoirs were quantified using the Intact Proviral DNA Assay; pVL were measured using the cobas 6800 (LLOQ:20 copies/mL). The province-wide analysis included all 290,401 pVL performed in British Columbia, Canada between 2012-2022. Results: Pre-vaccination, the median intact reservoir size was 77 (IQR:20-204) HIV copies/million CD4+ T-cells, compared to 74 (IQR:27-212) and 65 (IQR:22-174) post-first and -second dose, respectively (all comparisons p>0.07). Pre-vaccination, 82% of participants had pVL<20 copies/mL (max:110 copies/mL), compared to 79% post-first dose (max:183 copies/mL) and 85% post-second dose (max:79 copies/mL) (p>0.4). The magnitude of the vaccine-elicited anti-SARS-CoV-2-Spike antibody response did not correlate with changes in reservoir size nor detectable pVL frequency (p>0.6). We found no evidence linking the COVID-19 mass vaccination campaign to population-level increases in detectable pVL frequency among all PWH in the province, nor among those who maintained pVL suppression on ART. Conclusion: We found no evidence that COVID-19 mRNA vaccines induced changes in HIV reservoir size nor plasma viremia.

Near full-length HIV sequencing in multiple tissues collected postmortem reveals shared clonal expansions across distinct reservoirs during ART.

HIV persists in tissues during antiretroviral therapy (ART), but the relative contribution of different anatomical compartments to the viral reservoir in humans remains unknown. We performed an extensive characterization of HIV reservoirs in two men who donated their bodies to HIV cure research and who had been on suppressive ART for years. HIV DNA is detected in all tissues, with large variations across anatomical compartments and between participants. Intact HIV genomes represent 2% and 25% of all proviruses in the two participants and are mainly detected in secondary lymphoid organs, with the spleen and mediastinal lymph nodes harboring intact viral genomes in both individuals. Multiple copies of identical HIV genomes are found in all tissues, indicating that clonal expansions are common in anatomical sites. The majority (>85%) of these expanded clones are shared across multiple tissues. These findings suggest that infected cells expand, migrate, and possibly circulate between anatomical sites.

CDK8 inhibitors antagonize HIV-1 reactivation and promote provirus latency in T cells.

Latent HIV-1 provirus represents the barrier toward a cure for infection and is dependent upon the host RNA Polymerase (Pol) II machinery for reemergence. Here, we find that inhibitors of the RNA Pol II mediator kinases CDK8/19, Senexin A and BRD6989, inhibit induction of HIV-1 expression in response to latency-reversing agents and T cell signaling agonists. These inhibitors were found to impair recruitment of RNA Pol II to the HIV-1 LTR. Furthermore, HIV-1 expression in response to several latency reversal agents was impaired upon disruption of CDK8 by shRNA or gene knockout. However, the effects of CDK8 depletion did not entirely mimic CDK8/19 kinase inhibition suggesting that the mediator kinases are not functionally redundant. Additionally, treatment of CD4+ peripheral blood mononuclear cells isolated from people living with HIV-1 and who are receiving antiretroviral therapy with Senexin A inhibited induction of viral replication in response to T cell stimulation by PMA and ionomycin. These observations indicate that the mediator kinases, CDK8 and CDK19, play a significant role for regulation of HIV-1 transcription and that small molecule inhibitors of these enzymes may contribute to therapies designed to promote deep latency involving the durable suppression of provirus expression. IMPORTANCE A cure for HIV-1 infection will require novel therapies that can force elimination of cells that contain copies of the virus genome inserted into the cell chromosome, but which is shut off, or silenced. These are known as latently-infected cells, which represent the main reason why current treatment for HIV/AIDS cannot cure the infection because the virus in these cells is unaffected by current drugs. Our results indicate that chemical inhibitors of Cdk8 also inhibit the expression of latent HIV provirus. Cdk8 is an important enzyme that regulates the expression of genes in response to signals to which cells need to respond and which is produced by a gene that is frequently mutated in cancers. Our observations indicate that Cdk8 inhibitors may be employed in novel therapies to prevent expression from latent provirus, which might eventually enable infected individuals to cease treatment with antiretroviral drugs.

Antibody neutralization capacity after coronavirus disease 2019 vaccination in people with HIV in Canada.

OBJECTIVES: Many vaccines require higher/additional doses or adjuvants to provide adequate protection for people with HIV (PWH). Here, we compare coronavirus disease 2019 (COVID-19) vaccine-induced antibody neutralization capacity in PWH vs. HIV-negative individuals following two vaccine doses. DESIGN: In Canadian prospective observational cohorts, including a multicentre study of PWH receiving at least two COVID-19 vaccinations (mRNA or ChAdOx1-S), and a parallel study of HIV-negative controls (Stop the Spread Ottawa Cohort), we measured vaccine-induced neutralization capacity 3 months post dose 2 (±1 month). METHODS: COVID-19 neutralization efficiency was measured by calculating the half maximal inhibitory dilution (ID50) using a high-throughput protein-based neutralization assay for Ancestral (Wuhan), Delta and Omicron (BA.1) spike variants. Univariable and multivariable quantile regression were used to compare COVID-19-specific antibody neutralization capacity by HIV status. RESULTS: Neutralization assays were performed on 256 PWH and 256 controls based on specimen availability at the timepoint of interest, having received two vaccines and known date of vaccination. There was a significant interaction between HIV status and previous COVID-19 infection status in median ID50. There were no differences in median ID50 for HIV+ vs. HIV-negative persons without past COVID-19 infection. For participants with past COVID-19 infection, median ICD50 was significantly higher in controls than in PWH for ancestral SARS-CoV-2 and Omicron variants, with a trend for the Delta variant in the same direction. CONCLUSION: Vaccine-induced SARS-CoV-2 neutralization capacity was similar between PWH vs. HIV-negative persons without past COVID-19 infection, demonstrating favourable humoral-mediated immunogenicity. Both HIV+ and HIV-negative persons demonstrated hybrid immunity. TRIAL REGISTRATION: clinicaltrials.gov NCT04894448.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and members of these diverse forms persist within infected cell reservoirs, even during antiretroviral therapy (ART). Characterizing the diverse viral sequences that persist during ART is critical to HIV cure efforts, but our knowledge of on-ART proviral evolutionary dynamics remains incomplete, as does our understanding of the differences between the overall pool of persisting proviral DNA (which is largely genetically defective) and the subset of intact HIV sequences capable of reactivating. Here, we reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study (WIHS) who experienced HIV seroconversion. We measured diversity, lineage origins and ages of proviral sequences (env-gp120) sampled up to four times, up to 12 years on ART. We used the same techniques to study HIV sequences emerging from the reservoir in two participants. Proviral clonality generally increased over time on ART, with clones frequently persisting across multiple time points. The integration dates of proviruses persisting on ART generally spanned the duration of untreated infection (though were often skewed towards years immediately pre-ART), while in contrast, reservoir-origin viremia emerging in plasma was exclusively "younger" (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained highly stable during ART in all but one participant in whom, after 12 years, there was evidence that "younger" proviruses had been preferentially eliminated. Analysis of within-host recombinant proviral sequences also suggested that HIV reservoirs can be superinfected with virus reactivated from an older era, yielding infectious viral progeny with mosaic genomes of sequences with different ages. Overall, results underscore the remarkable genetic stability of distinct proviral sequences that persist on ART, yet suggest that replication-competent HIV reservoir represents a genetically-restricted and overall "younger" subset of the overall persisting proviral pool in blood.

Current HIV/SIV Reservoir Assays for Preclinical and Clinical Applications: Recommendations from the Experts 2022 NIAID Workshop Summary.

Since the first HIV-cured person was reported in 2009, a strong interest in developing highly sensitive HIV and SIV reservoir assays has emerged. In particular, the question arose about the comparative value of state-of-the-art assays to measure and characterize the HIV reservoir, and how these assays can be applied to accurately detect changes in the reservoir during efforts to develop a cure for HIV infection. Second, it is important to consider the impact on the outcome of clinical trials if these relatively new HIV reservoir assays are incorporated into clinical trial endpoints and/or used for clinical decision-making. To understand the advantages and limitations and the regulatory implications of HIV reservoir assays, the National Institute of Allergy and Infectious Diseases (NIAID) sponsored and convened a meeting on September 16, 2022, to discuss the state of knowledge concerning these questions and best practices for selecting HIV reservoir assays for a particular research question or clinical trial protocol.

HIV reservoirs are dominated by genetically younger and clonally enriched proviruses.

In order to cure HIV, we need to better understand the within-host evolutionary origins of the small reservoir of genome-intact proviruses that persists within infected cells during antiretroviral therapy (ART). Most prior studies on reservoir evolutionary dynamics however did not discriminate genome-intact proviruses from the vast background of defective ones. We reconstructed within-host pre-ART HIV evolutionary histories in six individuals and leveraged this information to infer the ages of intact and defective proviruses sampled after an average >9 years on ART, along with the ages of rebound and low-level/isolated viremia occurring during this time. We observed that the longest-lived proviruses persisting on ART were exclusively defective, usually due to large deletions. In contrast, intact proviruses and rebound HIV exclusively dated to the years immediately preceding ART. These observations are consistent with genome-intact proviruses having shorter lifespans, likely due to the cumulative risk of elimination following viral reactivation and protein production. Consistent with this, intact proviruses (and those with packaging signal defects) were three times more likely to be genetically identical compared to other proviral types, highlighting clonal expansion as particularly important in ensuring their survival. By contrast, low-level/isolated viremia sequences were genetically heterogeneous and sometimes ancestral, where viremia may have originated from defective proviruses. Results reveal that the HIV reservoir is dominated by clonally-enriched and genetically younger sequences that date to the untreated infection period when viral populations had been under within-host selection pressures for the longest duration. Knowledge of these qualities may help focus strategies for reservoir elimination.

Viral and Host Mediators of Non-Suppressible HIV-1 Viremia.

Non-suppressible HIV-1 viremia (NSV) can occur in persons with HIV despite adherence to combination antiretroviral therapy (ART) and in the absence of significant drug resistance. Here, we show that plasma NSV sequences are comprised primarily of large clones without evidence of viral evolution over time. We defined proviruses that contribute to plasma viremia as "producer", and those that did not as "non-producer". Compared to ART-suppressed individuals, NSV participants had a significantly larger producer reservoir. Producer proviruses were enriched in chromosome 19 and in proximity to the activating H3K36me3 epigenetic mark. CD4+ cells from NSV participants demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, NSV participants showed no elevation in HIV-specific CD8+ cell responses and producer proviruses were enriched for HLA escape mutations. We identified critical host and viral mediators of NSV that represent potential targets to disrupt HIV persistence and promote viral silencing.

HIV-1 subtype C Nef-mediated SERINC5 down-regulation significantly contributes to overall Nef activity.

BACKGROUND: Nef performs multiple cellular activities that enhance HIV-1 pathogenesis. The role of Nef-mediated down-regulation of the host restriction factor SERINC5 in HIV-1 pathogenesis is not well-defined. We aimed to investigate if SERINC5 down-regulation activity contributes to HIV-1 subtype C disease progression, to assess the relative contribution of this activity to overall Nef function, and to identify amino acids required for optimal activity. We measured the SERINC5 down-regulation activity of 106 subtype C Nef clones, isolated from individuals in early infection, for which the Nef activities of CD4 and HLA-I down-regulation as well as alteration of TCR signalling were previously measured. The relationship between SERINC5 down-regulation and markers of disease progression, and the relative contribution of SERINC5 down-regulation to a Nef fitness model-derived E value (a proxy for overall Nef fitness in vivo), were assessed. RESULTS: No overall relationship was found between SERINC5 down-regulation and viral load set point (p = 0.28) or rate of CD4+ T cell decline (p = 0.45). CD4 down-regulation (p = 0.02) and SERINC5 down-regulation (p = 0.003) were significant determinants of E values in univariate analyses, with the greatest relative contribution for SERINC5 down-regulation, and only SERINC5 down-regulation remained significant in the multivariate analysis (p = 0.003). Using a codon-by-codon analysis, several amino acids were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. Site-directed mutagenesis experiments of selected mutants confirmed a substantial reduction in SERINC5 down-regulation activity associated with the mutation 173T, while mutations 10K, 135F, and 176T were associated with more modest reductions in activity that were not statistically significant. CONCLUSIONS: These results suggest that SERINC5 down-regulation is a significant contributor to overall Nef function and identify potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.

Impact of Age and Severe Acute Respiratory Syndrome Coronavirus 2 Breakthrough Infection on Humoral Immune Responses After Three Doses of Coronavirus Disease 2019 mRNA Vaccine.

Background: Longer-term immune response data after 3 doses of coronavirus disease 2019 (COVID-19) mRNA vaccine remain limited, particularly among older adults and after Omicron breakthrough infection. Methods: We quantified wild-type- and Omicron-specific serum immunoglobulin (Ig)G levels, angiotensin-converting enzyme 2 displacement activities, and live virus neutralization up to 6 months after third dose in 116 adults aged 24-98 years who remained COVID-19 naive or experienced their first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during this time. Results: Among the 78 participants who remained COVID-19 naive throughout follow up, wild-type- and Omicron-BA.1-specific IgG concentrations were comparable between younger and older adults, although BA.1-specific responses were consistently significantly lower than wild-type-specific responses in both groups. Wild-type- and BA.1-specific IgG concentrations declined at similar rates in COVID-19-naive younger and older adults, with median half-lives ranging from 69 to 78 days. Antiviral antibody functions declined substantially over time in COVID-19-naive individuals, particularly in older adults: by 6 months, BA.1-specific neutralization was undetectable in 96% of older adults, versus 56% of younger adults. Severe acute respiratory syndrome coronavirus 2 infection, experienced by 38 participants, boosted IgG levels and neutralization above those induced by vaccination alone. Nevertheless, BA.1-specific neutralization remained significantly lower than wild-type, with BA.5-specific neutralization lower still. Higher Omicron BA.1-specific neutralization 1 month after third dose was an independent correlate of lower SARS-CoV-2 infection risk. Conclusions: Results underscore the immune benefits of the third COVID-19 mRNA vaccine dose in adults of all ages and identify vaccine-induced Omicron-specific neutralization as a correlate of protective immunity. Systemic antibody responses and functions however, particularly Omicron-specific neutralization, decline rapidly in COVID-19-naive individuals, particularly in older adults, supporting the need for additional booster doses.

SARS-CoV-2 Vaccine-Induced T-Cell Response after Three Doses in People Living with HIV on Antiretroviral Therapy Compared to Seronegative Controls (CTN 328 COVAXHIV Study).

People living with HIV (PLWH) may be at risk for poor immunogenicity to certain vaccines, including the ability to develop immunological memory. Here, we assessed T-cell immunogenicity following three SARS-CoV-2 vaccine doses in PLWH versus uninfected controls. Blood was collected from 38 PLWH on antiretroviral therapy and 24 age-matched HIV-negative controls, pre-vaccination and after 1st/2nd/3rd dose of SARS-CoV-2 vaccines, without prior SARS-CoV-2 infection. Flow cytometry was used to assess ex vivo T-cell immunophenotypes and intracellular Tumor necrosis factor (TNF)-α/interferon(IFN)-γ/interleukin(IL)-2 following SARS-CoV-2-Spike-peptide stimulation. Comparisons were made using Wilcoxon signed-rank test for paired variables and Mann-Whitney for unpaired. In PLWH, Spike-specific CD4 T-cell frequencies plateaued post-2nd dose, with no significant differences in polyfunctional SARS-CoV-2-specific T-cell proportions between PLWH and uninfected controls post-3rd dose. PLWH had higher frequencies of TNFα+CD4 T-cells and lower frequencies of IFNγ+CD8 T-cells than seronegative participants post-3rd dose. Regardless of HIV status, an increase in naive, regulatory, and PD1+ T-cell frequencies was observed post-3rd dose. In summary, two doses of SARS-CoV-2 vaccine induced a robust T-cell immune response in PLWH, which was maintained after the 3rd dose, with no significant differences in polyfunctional SARS-CoV-2-specific T-cell proportions between PLWH and uninfected controls post-3rd dose.

SARS-CoV-2 live virus neutralization after four COVID-19 vaccine doses in people with HIV receiving suppressive antiretroviral therapy.

OBJECTIVE: Limited data exist regarding the immune benefits of fourth COVID-19 vaccine doses in people with HIV (PWH) receiving antiretroviral therapy (ART), particularly now that most have experienced a SARS-CoV-2 infection. We quantified wild-type, Omicron-BA.5 and Omicron-BQ.1-specific neutralization up to 1 month post-fourth COVID-19 vaccine dose in 63 (19 SARS-CoV-2-naive and 44 SARS-CoV-2-experienced) PWH. DESIGN: A longitudinal observational cohort. METHODS: Quantification of wild-type-, Omicron-BA.5, and Omicron-BQ.1-specific neutralization using live virus assays. RESULTS: Participants received monovalent (44%) and bivalent (56%) mRNA fourth doses. In COVID-19-naive PWH, fourth doses enhanced wild-type and Omicron-BA.5-specific neutralization modestly above three-dose levels ( P  = 0.1). In COVID-19-experienced PWH, fourth doses enhanced wild-type specific neutralization modestly ( P  = 0.1) and BA.5-specific neutralization substantially ( P  = 0.002). Consistent with humoral benefits of 'hybrid' immunity, COVID-19-experienced PWH exhibited the highest neutralization post-fourth dose, wherein those with Omicron-era infections displayed higher wild-type specific ( P  = 0.04) but similar BA.5 and BQ.1-specific neutralization than those with pre-Omicron-era infections. Nevertheless, BA.5-specific neutralization was significantly below wild-type in everyone regardless of COVID-19 experience, with BQ.1-specific neutralization lower still (both P  < 0.0001). In multivariable analyses, fourth dose valency did not affect neutralization magnitude. Rather, an mRNA-1273 fourth dose (versus a BNT162b2 one) was the strongest correlate of wild-type specific neutralization, while prior COVID-19, regardless of pandemic era, was the strongest correlate of BA.5 and BQ.1-specific neutralization post-fourth dose. CONCLUSION: Fourth COVID-19 vaccine doses, irrespective of valency, benefit PWH regardless of prior SARS-CoV-2 infection. Results support recommendations that all adults receive a fourth COVID-19 vaccine dose within 6 months of their third dose (or their most recent SARS-CoV-2 infection).

Inhibition of the TRIM24 bromodomain reactivates latent HIV-1.

Expression of the HIV-1 genome by RNA Polymerase II is regulated at multiple steps, as are most cellular genes, including recruitment of general transcription factors and control of transcriptional elongation from the core promoter. We recently discovered that tripartite motif protein TRIM24 is recruited to the HIV-1 Long Terminal Repeat (LTR) by interaction with TFII-I and causes transcriptional elongation by stimulating association of PTEF-b/ CDK9. Because TRIM24 is required for stimulation of transcription from the HIV-1 LTR, we were surprised to find that IACS-9571, a specific inhibitor of the TRIM24 C-terminal bromodomain, induces HIV-1 provirus expression in otherwise untreated cells. IACS-9571 reactivates HIV-1 in T cell lines bearing multiple different provirus models of HIV-1 latency. Additionally, treatment with this TRIM24 bromodomain inhibitor encourages productive HIV-1 expression in newly infected cells and inhibits formation of immediate latent transcriptionally repressed provirus. IACS-9571 synergizes with PMA, ionomycin, TNF-α and PEP005 to activate HIV-1 expression. Furthermore, co-treatment of CD4 + T cells from individuals with HIV-1 on antiretroviral therapy (ART) with PEP005 and IACS-9571 caused robust provirus expression. Notably, IACS-9571 did not cause global activation of T cells; rather, it inhibited induction of IL2 and CD69 expression in human PBMCs and Jurkat T cells treated with PEP005 or PMA. These observations indicate the TRIM24 bromodomain inhibitor IACS-9571 represents a novel HIV-1 latency reversing agent (LRA), and unlike other compounds with this activity, causes partial suppression of T cell activation while inducing expression of latent provirus.

COVID-19 vaccine immunogenicity in people with HIV.

OBJECTIVES: Many vaccines require higher/additional doses or adjuvants to provide adequate protection for people with HIV (PWH). Our objective was to compare COVID-19 vaccine immunogenicity in PWH to HIV-negative individuals. DESIGN: In a Canadian multi-center prospective, observational cohort of PWH receiving at least two COVID-19 vaccinations, we measured vaccine-induced immunity at 3 and 6 months post 2nd and 1-month post 3rd doses. METHODS: The primary outcome was the percentage of PWH mounting vaccine-induced immunity [co-positivity for anti-IgG against SARS-CoV2 Spike(S) and receptor-binding domain proteins] 6 months post 2nd dose. Univariable and multivariable logistic regressions were used to compare COVID-19-specific immune responses between groups and within subgroups. RESULTS: Data from 294 PWH and 267 controls were analyzed. Immunogenicity was achieved in over 90% at each time point in both groups. The proportions of participants achieving comparable anti-receptor-binding domain levels were similar between the group at each time point. Anti-S IgG levels were similar by group at month 3 post 2nd dose and 1-month post 3rd dose. A lower proportion of PWH vs. controls maintained vaccine-induced anti-S IgG immunity 6 months post 2nd dose [92% vs. 99%; odds ratio: 0.14 (95% confidence interval: 0.03, 0.80; P = 0.027)]. In multivariable analyses, neither age, immune non-response, multimorbidity, sex, vaccine type, or timing between doses were associated with reduced IgG response. CONCLUSION: Vaccine-induced IgG was elicited in the vast majority of PWH and was overall similar between groups. A slightly lower proportion of PWH vs. controls maintained vaccine-induced anti-S IgG immunity 6 months post 2nd dose demonstrating the importance of timely boosting in this population.