HIV proviral genetic diversity, compartmentalization and inferred dynamics in lung and blood during long-term suppressive antiretroviral therapy.

The lung is an understudied site of HIV persistence. We isolated 898 subgenomic proviral sequences (nef) by single-genome approaches from blood and lung from nine individuals on long-term suppressive antiretroviral therapy (ART), and characterized genetic diversity and compartmentalization using formal tests. Consistent with clonal expansion as a driver of HIV persistence, identical sequences comprised between 8% to 86% of within-host datasets, though their location (blood vs. lung) followed no consistent pattern. The majority (77%) of participants harboured at least one sequence shared across blood and lung, supporting the migration of clonally-expanded cells between sites. The extent of blood proviral diversity on ART was also a strong indicator of diversity in lung (Spearman's ρ = 0.98, p<0.0001). For three participants, insufficient lung sequences were recovered to reliably investigate genetic compartmentalization. Of the remainder, only two participants showed statistically significant support for compartmentalization when analysis was restricted to distinct proviruses per site, and the extent of compartmentalization was modest in both cases. When all within-host sequences (including duplicates) were considered, the number of compartmentalized datasets increased to four. Thus, while a subset of individuals harbour somewhat distinctive proviral populations in blood and lung, this can simply be due to unequal distributions of clonally-expanded sequences. For two participants, on-ART proviruses were also phylogenetically analyzed in context of plasma HIV RNA populations sampled up to 18 years prior, including pre-ART and during previous treatment interruptions. In both participants, on-ART proviruses represented the most ancestral sequences sampled within-host, confirming that HIV sequences can persist in the body for decades. This analysis also revealed evidence of re-seeding of the reservoir during treatment interruptions. Results highlight the genetic complexity of proviruses persisting in lung and blood during ART, and the uniqueness of each individual's proviral composition. Personalized HIV remission and cure strategies may be needed to overcome these challenges.

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.

Older Adults Mount Less Durable Humoral Responses to Two Doses of COVID-19 mRNA Vaccine but Strong Initial Responses to a Third Dose.

BACKGROUND: Third coronavirus disease 2019 (COVID-19) vaccine doses are broadly recommended, but immunogenicity data remain limited, particularly in older adults. METHODS: We measured circulating antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, ACE2 displacement, and virus neutralization against ancestral and omicron (BA.1) strains from prevaccine up to 1 month following the third dose, in 151 adults aged 24-98 years who received COVID-19 mRNA vaccines. RESULTS: Following 2 vaccine doses, humoral immunity was weaker, less functional, and less durable in older adults, where a higher number of chronic health conditions was a key correlate of weaker responses and poorer durability. One month after the third dose, antibody concentrations and function exceeded post-second-dose levels, and responses in older adults were comparable in magnitude to those in younger adults at this time. Humoral responses against omicron were universally weaker than against the ancestral strain after both the second and third doses. Nevertheless, after 3 doses, anti-omicron responses in older adults reached equivalence to those in younger adults. One month after 3 vaccine doses, the number of chronic health conditions, but not age, was the strongest consistent correlate of weaker humoral responses. CONCLUSIONS: Results underscore the immune benefits of third COVID-19 vaccine doses, particularly in older adults.

Impact of combinations of clinically observed HIV integrase mutations on phenotypic resistance to integrase strand transfer inhibitors (INSTIs): a molecular study.

BACKGROUND: Routine HIV drug resistance genotyping identified an integrase sequence harbouring T97A, E138K, G140S and Q148H, with high predicted resistance to all integrase strand transfer inhibitors (INSTIs). OBJECTIVES: To assess the impact of these substitutions alone and together on phenotypic INSTI susceptibility. METHODS: We constructed recombinant NL4.3 viruses harbouring all mutation combinations in the autologous integrase sequence. Viruses were grown in GFP-reporter CD4+ T-cells in the presence of 0.01-1000 nM raltegravir, elvitegravir, dolutegravir, bictegravir, and cabotegravir. Infection was measured by imaging cytometry. RESULTS: Q148H-containing viruses lacking G140S failed to propagate or mutated in vitro, consistent with fitness costs. Statistically significant reductions in INSTI susceptibility were observed for several mutation combinations, as follows. T97A or G140S alone conferred 3.6- to 5.6-fold decreased susceptibility to raltegravir and elvitegravir. Two-mutation combinations conferred low-to-moderate resistance to raltegravir and elvitegravir only, except G140S/Q148H which eliminated raltegravir and elvitegravir activity and conferred 24.6-, 7.9-, and 107.5-fold reduced susceptibility to dolutegravir, bictegravir and cabotegravir. Addition of E138K to G140S/Q148H conferred 35.5, 11.6 and 208-fold reduced susceptibility to dolutegravir, bictegravir, and cabotegravir, while addition of T97A to G140S/Q148H conferred 318, 121 and >1000-fold reduced susceptibility to these drugs. T97A/E138K/G140S/Q148H in the autologous backbone conferred >300-fold reduced susceptibility to all INSTIs. Notably, bictegravir EC50 was significantly lower when T97A/E138K/G140S/Q148H was introduced into NL4.3, suggesting that other mutations in the autologous sequence enhanced resistance. CONCLUSIONS: High-level dolutegravir, bictegravir and cabotegravir resistance requires multiple integrase substitutions including compensatory mutations. T97A and E138K further enhance the resistance conferred by G140S/Q148H, yielding >300-fold decreased susceptibility to all INSTIs when all four mutations are present.

Rapid Detection of SARS-CoV-2 Variants of Concern, Including B.1.1.28/P.1, British Columbia, Canada.

To screen all severe acute respiratory syndrome coronavirus 2-positive samples in Vancouver, British Columbia, Canada, and determine whether they represented variants of concern, we implemented a real-time reverse transcription PCR-based algorithm. We rapidly identified 77 samples with variants: 57 with B.1.1.7, 7 with B.1.351, and an epidemiologic cluster of 13 with B.1.1.28/P.1.

Suboptimal Biological Sampling as a Probable Cause of False-Negative COVID-19 Diagnostic Test Results.

False-negative severe acute respiratory syndrome coronavirus 2 test results can negatively impact the clinical and public health response to coronavirus disease 2019 (COVID-19). We used droplet digital polymerase chain reaction (ddPCR) to demonstrate that human DNA levels, a stable molecular marker of sampling quality, were significantly lower in samples from 40 confirmed or suspected COVID-19 cases that yielded negative diagnostic test results (ie, suspected false-negative test results) compared with a representative pool of 87 specimens submitted for COVID-19 testing. Our results support suboptimal biological sampling as a contributor to false-negative COVID-19 test results and underscore the importance of proper training and technique in the collection of nasopharyngeal specimens.

Theoretical and experimental assessment of degenerate primer tagging in ultra-deep applications of next-generation sequencing.

Primer IDs (pIDs) are random oligonucleotide tags used in next-generation sequencing to identify sequences that originate from the same template. These tags are produced by degenerate primers during the reverse transcription of RNA molecules into cDNA. The use of pIDs helps to track the number of RNA molecules carried through amplification and sequencing, and allows resolution of inconsistencies between reads sharing a pID. Three potential issues complicate the above applications. First, multiple cDNAs may share a pID by chance; we found that while preventing any cDNAs from sharing a pID may be unfeasible, it is still practical to limit the number of these collisions. Secondly, a pID must be observed in at least three sequences to allow error correction; as such, pIDs observed only one or two times must be rejected. If the sequencing product contains copies from a high number of RT templates but produces few reads, our findings indicate that rejecting such pIDs will discard a great deal of data. Thirdly, the use of pIDs could influence amplification and sequencing. We examined the effects of several intrinsic and extrinsic factors on sequencing reads at both the individual and ensemble level.

Replication fitness of multiple nonnucleoside reverse transcriptase-resistant HIV-1 variants in the presence of etravirine measured by 454 deep sequencing.

We applied an efficient method to characterize the relative fitness levels of multiple nonnucleoside reverse transcriptase (NNRTI)-resistant HIV-1 variants by simultaneous competitive culture and 454 deep sequencing. Using this method, we show that the Y181V mutation in the HIV-1 reverse transcriptase in particular confers a clear selective advantage to the virus over 14 other NNRTI resistance mutations in the presence of etravirine in vitro.

Use of cellular HIV DNA to predict virologic response to maraviroc: performance of population-based and deep sequencing.

BACKGROUND: A tropism test is required before administration of the antiretroviral drug maraviroc. However, plasma RNA testing is not possible in patients with undetectable plasma viral loads. Here we assess genotypic testing of cellular human immunodeficiency virus (HIV) DNA from peripheral blood mononuclear cells (PBMCs) to predict virologic responses in treatment-experienced patients beginning maraviroc-containing regimens. METHODS: PBMC samples from 181 maraviroc recipients at study entry in MOTIVATE or A4001029 (51% R5 by original Trofile). The V3 loop was amplified in triplicate from cellular HIV DNA, and matching plasma RNA (n = 156). Sequencing was performed using standard population-based methods and next-generation deep sequencing, with tropism assessment as previously defined. RESULTS: Genotypic DNA-based tropism testing from the cellular compartment had 78%-81% sensitivity relative to RNA-based Trofile at the same time point. Cell-based genotypic tropism methods and plasma-based phenotypic and genotypic methods were predictive of virologic response. However, when classifications were discordant, the outcomes favored the plasma predictions over the DNA ones. CONCLUSIONS: Genotypic determination of HIV tropism can be performed using cell-derived viral DNA, and is a predictor of virologic success on maraviroc in therapy-experienced patients. However, the PBMC compartment appears to be a suboptimal predictor compared to plasma.

Genotypic analysis of the V3 region of HIV from virologic nonresponders to maraviroc-containing regimens reveals distinct patterns of failure.

Changes in HIV tropism from R5 to non-R5 or development of drug resistance is often associated with virologic failure in patients treated with maraviroc, a CCR5 antagonist. We sought to examine changes in HIV envelope sequences and inferred tropism in patients who did not respond to maraviroc-based regimens. We selected 181 patients who experienced early virologic failure on maraviroc-containing therapy in the MOTIVATE trials. All patients had R5 HIV by the original Trofile assay before entry. We used population-based sequencing methods and the geno2pheno algorithm to examine changes in tropism and V3 sequences at the time of failure. Using deep sequencing, we assessed whether V3 sequences observed at failure emerged from preexisting subpopulations. From population genotyping data at failure, 90 patients had R5 results, and 91 had non-R5 results. Of the latter group, the geno2pheno false-positive rate (FPR) value fell from a median of 20 at screening to 1.1 at failure. By deep sequencing, the median percentage of non-R5 variants in these patients rose from 1.4% to 99.5% after a median of 4 weeks on maraviroc. In 70% of cases, deep sequencing could detect a pretreatment CXCR4-using subpopulation, which emerged at failure. Overall, there were two distinct patterns of failure of maraviroc. Patients failing with R5 generally had few V3 substitutions and low non-R5 prevalence by deep sequencing. Patients with non-R5 HIV who were failing developed very-high-prevalence non-R5 HIV (median, 99%) and had very low geno2pheno values.

Factors influencing the sensitivity and specificity of conventional sequencing in human immunodeficiency virus type 1 tropism testing.

Human immunodeficiency virus type 1 (HIV-1) V3 loop sequence can be used to infer viral coreceptor use. The effect of input copy number on population-based sequencing of the V3 loop of HIV-1 was examined through replicate deep and population-based sequencing of samples with known tropism, a heterogeneous clinical sample (624 population-based sequences and 47 deep-sequencing replicates), and a large cohort of clinical samples from phase III clinical trials of maraviroc including the MOTIVATE/A4001029 studies (n = 1,521). Proviral DNA from two independent samples from each of 101 patients from the MOTIVATE/A4001029 studies was also analyzed. Cumulative technical error occurred at a rate of 3 × 10(-4) mismatches/bp, without observed effect on inferred tropism. Increasing PCR replication increased minority species detection with an ~10% minority population detected in 18% of cases using a single replicate at a viral load of 1,072 copies/ml and in 44% of cases using three replicates. The nucleotide prevalence detected by population-based and deep sequencing were highly correlated (Spearman's ρ, 0.73), and the accuracy increased with increasing input copy number (P < 0.001). Triplicate sequencing was able to predict tropism changes in the MOTIVATE/A4001029 studies for both low (P = 0.05) and high (P = 0.02) viral loads. Sequences derived from independently extracted and processed samples of proviral DNA for the same patient were equivalent to replicates from the same extraction (P = 0.45) and had correlated position-specific scoring matrix scores (Spearman's ρ, 0.75; P << 0.001); however, concordance in tropism inference was only 83%. Input copy number and PCR replication are important factors in minority species detection in samples with significant heterogeneity.

Maraviroc treatment in non-R5-HIV-1-infected patients results in the selection of extreme CXCR4-using variants with limited effect on the total viral setpoint.

OBJECTIVES: Using deep sequencing methods, we intensively investigated the selective pressure of maraviroc on the viral population in four patients with dual/mixed HIV-1 experiencing treatment failure. METHODS: Patients received maraviroc add-on therapy (n = 4). Tropism was determined by Monogram's Trofile assay and/or 'deep' sequencing. Longitudinal 'deep' sequence analysis used triplicate HIV V3 RT-PCR on plasma samples. Sequences were interpreted using the geno2phenocoreceptor algorithm with a 3.5% false-positive rate (FPR) cut-off. RESULTS: Patients had a median viral load of 4.7 log10 HIV RNA copies/mL with a median of 24% chemokine (C-X-C motif) receptor 4 (CXCR4)-using virus at baseline. Following maraviroc exposure, the chemokine (C-C motif) receptor 5 (CCR5)-using virus (R5) plasma viral load decreased by at least 1 log10, and only non-R5 variants with extremely low FPR values predominated after 21 days. Virus with an FPR ≤1.8% accounted for more than 90% of the circulating virus, having expanded to occupy the 'space' left by the suppression of R5 variants. Population genetic estimates of viral fitness in the presence of maraviroc showed a steep rise around an FPR value of 2%. CONCLUSIONS: Longitudinal analysis of independent R5 and non-R5 HIV populations shows that maraviroc selects viruses with an extremely low FPR, implying that the antiviral activity of maraviroc may extend to a broader range of HIV variants than previously suspected.

Detection of inferred CCR5- and CXCR4-using HIV-1 variants and evolutionary intermediates using ultra-deep pyrosequencing.

The emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants is associated with accelerated disease progression. CXCR4-using variants are believed to evolve from CCR5-using variants, but due to the extremely low frequency at which transitional intermediate variants are often present, the kinetics and mutational pathways involved in this process have been difficult to study and are therefore poorly understood. Here, we used ultra-deep sequencing of the V3 loop of the viral envelope in combination with the V3-based coreceptor prediction tools PSSM(NSI/SI) and geno2pheno([coreceptor]) to detect HIV-1 variants during the transition from CCR5- to CXCR4-usage. We analyzed PBMC and serum samples obtained from eight HIV-1-infected individuals at three-month intervals up to one year prior to the first phenotypic detection of CXCR4-using variants in the MT-2 assay. Between 3,482 and 10,521 reads were generated from each sample. In all individuals, V3 sequences of predicted CXCR4-using HIV-1 were detected at least three months prior to phenotypic detection of CXCR4-using variants in the MT-2 assay. Subsequent analysis of the genetic relationships of these V3 sequences using minimum spanning trees revealed that the transition in coreceptor usage followed a stepwise mutational pathway involving sequential intermediate variants, which were generally present at relatively low frequencies compared to the major predicted CCR5- and CXCR4-using variants. In addition, we observed differences between individuals with respect to the number of predicted CXCR4-using variants, the diversity among major predicted CCR5-using variants, and the presence or absence of intermediate variants with discordant phenotype predictions. These results provide the first detailed description of the mutational pathways in V3 during the transition from CCR5- to CXCR4-usage in natural HIV-1 infection.

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).

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.

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.

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.

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.

Increasingly successful highly active antiretroviral therapy delays the emergence of new HLA class I-associated escape mutations in HIV-1.

BACKGROUND: HLA class I-restricted cytotoxic T lymphocytes and highly active antiretroviral therapy (HAART) exert strong selective pressures on human immunodeficiency virus type 1 (HIV-1), leading to escape mutations compromising virologic control. Immune responses continue to shape HIV-1 evolution after HAART initiation, but the extent and rate at which this occurs remain incompletely quantified. Here, we characterize the incidence and clinical correlates of HLA-associated evolution in HIV-1 Pol after HAART initiation in a large, population-based observational cohort. METHODS: British Columbia HAART Observational, Medical Evaluation and Research cohort participants with available HLA class I types and longitudinal posttherapy protease/reverse transcriptase sequences were studied (n = 619; median, 5 samples per patient and 5.2 years of follow-up). HLA-associated polymorphisms were defined according to published reference lists. Rates and correlates of immune-mediated HIV-1 evolution were investigated using multivariate Cox proportional hazard models incorporating baseline and time-dependent plasma viral load and CD4 response data. RESULTS: New HLA-associated escape events were observed in 269 (43%) patients during HAART and occurred at 49 of 63 (78%) investigated immune-associated sites in Pol. In time-dependent analyses adjusting for baseline factors, poorer virologic, but not immunologic, response to HAART was associated with increased risk of immune escape of 1.9-fold per log(10) viral load increment (P < .0001). Reversion of escape mutations following HAART initiation was extremely rare. CONCLUSIONS: HLA-associated HIV-1 evolution continues during HAART to an extent that is inversely related to the virologic success of therapy. Minimizing the degree of immune escape could represent a secondary benefit of effective HAART.