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.

People With Human Immunodeficiency Virus Receiving Suppressive Antiretroviral Therapy Show Typical Antibody Durability After Dual Coronavirus Disease 2019 Vaccination and Strong Third Dose Responses.

BACKGROUND: Longer-term humoral responses to 2-dose coronavirus disease 2019 (COVID-19) vaccines remain incompletely characterized in people living with human immunodeficiency virus (HIV) (PLWH), as do initial responses to a third dose. METHODS: We measured antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, angiotensin-converting enzyme 2 (ACE2) displacement, and viral neutralization against wild-type and Omicron strains up to 6 months after 2-dose vaccination, and 1 month after the third dose, in 99 PLWH receiving suppressive antiretroviral therapy and 152 controls. RESULTS: Although humoral responses naturally decline after 2-dose vaccination, we found no evidence of lower antibody concentrations or faster rates of antibody decline in PLWH compared with controls after accounting for sociodemographic, health, and vaccine-related factors. We also found no evidence of poorer viral neutralization in PLWH after 2 doses, nor evidence that a low nadir CD4+ T-cell count compromised responses. Post-third-dose humoral responses substantially exceeded post-second-dose levels, though Omicron-specific responses were consistently weaker than responses against wild-type virus. Nevertheless, post-third-dose responses in PLWH were comparable to or higher than controls. An mRNA-1273 third dose was the strongest consistent correlate of higher post-third-dose responses. CONCLUSION: PLWH receiving suppressive antiretroviral therapy mount strong antibody responses after 2- and 3-dose COVID-19 vaccination. Results underscore the immune benefits of third doses in light of Omicron.

Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus.

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.

A simple phylogenetic approach to analyze hypermutated HIV proviruses reveals insights into their dynamics and persistence during antiretroviral therapy.

Hypermutated proviruses, which arise in a single HIV replication cycle when host antiviral APOBEC3 proteins introduce extensive G-to-A mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). But, the within-host evolutionary origins of hypermutated sequences are incompletely understood because phylogenetic inference algorithms, which assume that mutations gradually accumulate over generations, incorrectly reconstruct their ancestor-descendant relationships. Using >1400 longitudinal single-genome-amplified HIV env-gp120 sequences isolated from six women over a median 18 years of follow-up - including plasma HIV RNA sequences collected over a median 9 years between seroconversion and ART initiation, and >500 proviruses isolated over a median 9 years on ART - we evaluated three approaches for removing hypermutation from nucleotide alignments. Our goals were to 1) reconstruct accurate phylogenies that can be used for molecular dating and 2) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the tested approaches (stripping all positions containing putative APOBEC3 mutations from the alignment, or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought env -intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for env -intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, indicating that the corrected trees can be used for molecular dating. Use of these trees to infer the integration dates of hypermutated proviruses persisting during ART revealed that these spanned a wide age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection, and can persist for decades. In two of the six participants, hypermutated proviruses differed from env -intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus' evolutionary histories, allow insights into their in vivo origins and longevity, towards a more comprehensive understanding of HIV persistence during ART.

HIV-1 subtype A1, D, and recombinant proviral genome landscapes during long-term suppressive therapy.

The primary obstacle to curing HIV-1 is a reservoir of CD4+ cells that contain stably integrated provirus. Previous studies characterizing the proviral landscape, which have been predominantly conducted in males in the United States and Europe living with HIV-1 subtype B, have revealed that most proviruses that persist during antiretroviral therapy (ART) are defective. In contrast, less is known about proviral landscapes in females with non-B subtypes, which represents the largest group of individuals living with HIV-1. Here, we analyze genomic DNA from resting CD4+ T-cells from 16 female and seven male Ugandans with HIV-1 receiving suppressive ART (n = 23). We perform near-full-length proviral sequencing at limiting dilution to examine the proviral genetic landscape, yielding 607 HIV-1 subtype A1, D, and recombinant proviral sequences (mean 26/person). We observe that intact genomes are relatively rare and clonal expansion occurs in both intact and defective genomes. Our modification of the primers and probes of the Intact Proviral DNA Assay (IPDA), developed for subtype B, rescues intact provirus detection in Ugandan samples for which the original IPDA fails. This work will facilitate research on HIV-1 persistence and cure strategies in Africa, where the burden of HIV-1 is heaviest.

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

OBJECTIVE: The immunogenic nature of coronavirus disease 2019 (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: Sixty-two participants were sampled prevaccination, 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 (lower limit of quantification: 20 copies/ml). The province-wide analysis included all 290 401 pVL performed in British Columbia, Canada between 2012 and 2022. RESULTS: Prevaccination, the median intact reservoir size was 77 [interquartile range (IQR): 20-204] HIV copies/million CD4 + T-cells, compared to 74 (IQR: 27-212) and 65 (IQR: 22-174) postfirst and -second dose, respectively (all comparisons P > 0.07). Prevaccination, 82% of participants had pVL <20 copies/ml (max: 110 copies/ml), compared to 79% postfirst dose (max: 183 copies/ml) and 85% postsecond 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.

T-Cell Responses to COVID-19 Vaccines and Breakthrough Infection in People Living with HIV Receiving Antiretroviral Therapy.

People living with HIV (PLWH) can exhibit impaired immune responses to vaccines. Accumulating evidence indicates that PLWH, particularly those receiving antiretroviral therapy, mount strong antibody responses to COVID-19 vaccines, but fewer studies have examined cellular immune responses to the vaccinations. Here, we used an activation-induced marker (AIM) assay to quantify SARS-CoV-2 spike-specific CD4+ and CD8+ T cells generated by two and three doses of COVID-19 vaccines in 50 PLWH receiving antiretroviral therapy, compared to 87 control participants without HIV. In a subset of PLWH, T-cell responses were also assessed after post-vaccine breakthrough infections and/or receipt of a fourth vaccine dose. All participants remained SARS-CoV-2 infection-naive until at least one month after their third vaccine dose. SARS-CoV-2 infection was determined by seroconversion to a Nucleocapsid (N) antigen, which occurred in 21 PLWH and 38 control participants after the third vaccine dose. Multivariable regression analyses were used to investigate the relationships between sociodemographic, health- and vaccine-related variables, vaccine-induced T-cell responses, and breakthrough infection risk. We observed that a third vaccine dose boosted spike-specific CD4+ and CD8+ T-cell frequencies significantly above those measured after the second dose (all p < 0.0001). Median T-cell frequencies did not differ between PLWH and controls after the second dose (p > 0.1), but CD8+ T-cell responses were modestly lower in PLWH after the third dose (p = 0.02), an observation that remained significant after adjusting for sociodemographic, health- and vaccine-related variables (p = 0.045). In PLWH who experienced a breakthrough infection, median T-cell frequencies increased even higher than those observed after three vaccine doses (p < 0.03), and CD8+ T-cell responses in this group remained higher even after a fourth vaccine dose (p = 0.03). In multivariable analyses, the only factor associated with an increased breakthrough infection risk was younger age, which is consistent with the rapid increase in SARS-CoV-2 seropositivity that was seen among younger adults in Canada after the initial appearance of the Omicron variant. These results indicate that PLWH receiving antiretroviral therapy mount strong T-cell responses to COVID-19 vaccines that can be enhanced by booster doses or breakthrough infection.

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.

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.

Antibody response durability following three-dose coronavirus disease 2019 vaccination in people with HIV receiving suppressive antiretroviral therapy.

BACKGROUND: Limited data exist regarding longer term antibody responses following three-dose coronavirus disease 2019 (COVID-19) vaccination, and the impact of a first SARS-CoV-2 infection during this time, in people with HIV (PWH) receiving suppressive antiretroviral therapy (ART). We quantified wild-type-specific, Omicron BA.1-specific and Omicron BA.5-specific responses up to 6 months post-third dose in 64 PWH and 117 controls who remained COVID-19-naive or experienced their first SARS-CoV-2 infection during this time. DESIGN: Longitudinal observational cohort. METHODS: We quantified wild-type-specific and Omicron-specific anti-Spike receptor-binding domain IgG concentrations, ACE2 displacement activities and live virus neutralization at 1, 3 and 6 months post-third vaccine dose. RESULTS: Third doses boosted all antibody measures above two-dose levels, but BA.1-specific responses remained significantly lower than wild-type-specific ones, with BA.5-specific responses lower still. Serum IgG concentrations declined at similar rates in COVID-19-naive PWH and controls post-third dose (median wild-type-specific and BA.1-specific half-lives were between 66 and 74 days for both groups). Antibody function also declined significantly yet comparably between groups: 6 months post-third dose, BA.1-specific neutralization was undetectable in more than 80% of COVID-19 naive PWH and more than 90% of controls. Breakthrough SARS-CoV-2 infection boosted antibody concentrations and function significantly above vaccine-induced levels in both PWH and controls, though BA.5-specific neutralization remained significantly poorer than BA.1 even post-breakthrough. CONCLUSION: Following three-dose COVID-19 vaccination, antibody response durability in PWH receiving ART is comparable with controls. PWH also mounted strong responses to breakthrough infection. Due to temporal response declines, however, COVID-19-naive individuals, regardless of HIV status, would benefit from a fourth dose within 6 months of their third.

SARS CoV-2 mRNA vaccination exposes latent HIV to Nef-specific CD8+ T-cells.

Efforts to cure HIV have focused on reactivating latent proviruses to enable elimination by CD8+ cytotoxic T-cells. Clinical studies of latency reversing agents (LRA) in antiretroviral therapy (ART)-treated individuals have shown increases in HIV transcription, but without reductions in virologic measures, or evidence that HIV-specific CD8+ T-cells were productively engaged. Here, we show that the SARS-CoV-2 mRNA vaccine BNT162b2 activates the RIG-I/TLR - TNF - NFκb axis, resulting in transcription of HIV proviruses with minimal perturbations of T-cell activation and host transcription. T-cells specific for the early gene-product HIV-Nef uniquely increased in frequency and acquired effector function (granzyme-B) in ART-treated individuals following SARS-CoV-2 mRNA vaccination. These parameters of CD8+ T-cell induction correlated with significant decreases in cell-associated HIV mRNA, suggesting killing or suppression of cells transcribing HIV. Thus, we report the observation of an intervention-induced reduction in a measure of HIV persistence, accompanied by precise immune correlates, in ART-suppressed individuals. However, we did not observe significant depletions of intact proviruses, underscoring challenges to achieving (or measuring) HIV reservoir reductions. Overall, our results support prioritizing the measurement of granzyme-B-producing Nef-specific responses in latency reversal studies and add impetus to developing HIV-targeted mRNA therapeutic vaccines that leverage built-in LRA activity.

Humoral immune responses to COVID-19 vaccination in people living with HIV receiving suppressive antiretroviral therapy.

Humoral responses to COVID-19 vaccines in people living with HIV (PLWH) remain incompletely characterized. We measured circulating antibodies against the SARS-CoV-2 spike protein receptor-binding domain (RBD), ACE2 displacement and viral neutralization activities one month following the first and second COVID-19 vaccine doses, and again 3 months following the second dose, in 100 adult PLWH and 152 controls. All PLWH were receiving suppressive antiretroviral therapy, with median CD4+ T-cell counts of 710 (IQR 525-935) cells/mm3, though nadir CD4+ T-cell counts ranged as low as <10 cells/mm3. After adjustment for sociodemographic, health and vaccine-related variables, HIV infection was associated with lower anti-RBD antibody concentrations and ACE2 displacement activity after one vaccine dose. Following two doses however, HIV was not significantly associated with the magnitude of any humoral response after multivariable adjustment. Rather, older age, a higher burden of chronic health conditions, and dual ChAdOx1 vaccination were associated with lower responses after two vaccine doses. No significant correlation was observed between recent or nadir CD4+ T-cell counts and responses to two vaccine doses in PLWH. These results indicate that PLWH with well-controlled viral loads and CD4+ T-cell counts in a healthy range generally mount strong initial humoral responses to dual COVID-19 vaccination. Factors including age, co-morbidities, vaccine brand, response durability and the rise of new SARS-CoV-2 variants will influence when PLWH will benefit from additional doses. Further studies of PLWH who are not receiving antiretroviral treatment or who have low CD4+ T-cell counts are needed, as are longer-term assessments of response durability.

People with HIV receiving suppressive antiretroviral therapy show typical antibody durability after dual COVID-19 vaccination, and strong third dose responses.

Background: Longer-term humoral responses to two-dose COVID-19 vaccines remain incompletely characterized in people living with HIV (PLWH), as do initial responses to a third dose. Methods: We measured antibodies against the SARS-CoV-2 spike protein receptor-binding domain, ACE2 displacement and viral neutralization against wild-type and Omicron strains up to six months following two-dose vaccination, and one month following the third dose, in 99 PLWH receiving suppressive antiretroviral therapy, and 152 controls. Results: Though humoral responses naturally decline following two-dose vaccination, we found no evidence of lower antibody concentrations nor faster rates of antibody decline in PLWH compared to controls after accounting for sociodemographic, health and vaccine-related factors. We also found no evidence of poorer viral neutralization in PLWH after two doses, nor evidence that a low nadir CD4+ T-cell count compromised responses. Post-third-dose humoral responses substantially exceeded post-second-dose levels, though anti-Omicron responses were consistently weaker than against wild-type.Nevertheless, post-third-dose responses in PLWH were comparable to or higher than controls. An mRNA-1273 third dose was the strongest consistent correlate of higher post-third-dose responses. Conclusion: PLWH receiving suppressive antiretroviral therapy mount strong antibody responses after two- and three-dose COVID-19 vaccination. Results underscore the immune benefits of third doses in light of Omicron.

Humoral immune responses to COVID-19 vaccination in people living with HIV receiving suppressive antiretroviral therapy.

Humoral responses to COVID-19 vaccines in people living with HIV (PLWH) remain incompletely understood. We measured circulating antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, ACE2 displacement and live viral neutralization activities one month following the first and second COVID-19 vaccine doses in 100 adult PLWH and 152 controls. All PLWH were receiving suppressive antiretroviral therapy, with median CD4+ T-cell counts of 710 (IQR 525-935) cells/mm 3 . Nadir CD4+ T-cell counts ranged as low as <10 (median 280; IQR 120-490) cells/mm 3 . After adjustment for sociodemographic, health and vaccine-related variables, HIV infection was significantly associated with 0.2 log 10 lower anti-RBD antibody concentrations (p=0.03) and ∻11% lower ACE2 displacement activity (p=0.02), but not lower viral neutralization (p=0.1) after one vaccine dose. Following two doses however, HIV was no longer significantly associated with the magnitude of any response measured. Rather, older age, a higher burden of chronic health conditions, and having received two ChAdOx1 doses (versus a heterologous or dual mRNA vaccine regimen) were independently associated with lower responses. After two vaccine doses, no significant correlation was observed between the most recent or nadir CD4+ T-cell counts and vaccine responses in PLWH. These results suggest that PLWH with well-controlled viral loads on antiretroviral therapy and CD4+ T-cell counts in a healthy range will generally not require a third COVID-19 vaccine dose as part of their initial immunization series, though other factors such as older age, co-morbidities, vaccine regimen type, and durability of vaccine responses will influence when this group may benefit from additional doses. Further studies of PLWH who are not receiving antiretroviral treatment and/or who have low CD4+ T-cell counts are needed.

Put a cork in it: Plugging the M2 viral ion channel to sink influenza.

The ongoing threat of seasonal and pandemic influenza to human health requires antivirals that can effectively supplement existing vaccination strategies. The M2 protein of influenza A virus (IAV) is a proton-gated, proton-selective ion channel that is required for virus replication and is an established antiviral target. While licensed adamantane-based M2 antivirals have been historically used, M2 mutations that confer major adamantane resistance are now so prevalent in circulating virus strains that these drugs are no longer recommended. Here we review the current understanding of IAV M2 structure and function, mechanisms of inhibition, the rise of drug resistance mutations, and ongoing efforts to develop new antivirals that target resistant forms of M2.