Peter Cheung

ABSTRACTBackgroundLonger-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.MethodsWe 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.ResultsThough 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 substantial...

ABSTRACTHumoral 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/mm3. Nadir CD4+ T-cell counts ranged as low as <10 (median 280; IQR 120-490) cells/mm3. After adjustment for sociodemographic, health and vaccine-related variables, HIV infection was significantly associated with 0.2 log10 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 respons...

ABSTRACTBackgroundmRNA vaccines reduce COVID-19 incidence and severity, but the durability of vaccine-induced immune responses, particularly among the elderly, remains incompletely characterized.MethodsAnti-spike RBD antibody titers, ACE2 competition and virus neutralizing activities were longitudinally assessed in 151 healthcare workers and older adults (overall aged 24-98 years) up to three months after vaccination.ResultsOlder adults exhibited lower antibody responses after one and two vaccine doses for all measures. In multivariable analyses correcting for sociodemographic, chronic health and vaccine-related variables, age remained independently associated with all response outcomes. The number of chronic health conditions was additionally associated with lower binding antibody responses after two doses, and male sex with lower ACE2 competition activity after one dose. Responses waned universally at three months after the second dose, but binding antibodies, ACE2 competition and...

ABSTRACTPhenotypic resistance data is relatively sparse for the newest HIV-1 integrase strand transfer inhibitors (INSTIs), dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB). In this study, we report the phenotypic susceptibility of a large panel of oligo-clonal patient-derived HIV-1 integrase viruses. Representative clinical samples (N=141) were selected from a large database (N=17,197) of clinically-derived HIV integrase sequences, based on the presence of permutations of substitutions at 27 pre-defined positions in integrase (N=288). HIV-1 RNA was extracted from patient samples and diluted to approximately 500 HIV RNA copies/mL. Using an “oligo-clonal” amplification approach to achieve single-copy amplification, these dilutions were subjected to 12 parallel RT-PCR reactions to amplify integrase. Confirmed clonal amplicons were co-transfected with linearized pNL4.3∆int into CEM-GXR cells. In total, 162 HIV-1 viruses that carried no mixtures and had a unique sequence we...

ABSTRACTSARS-CoV-2 Omicron infections are common among individuals who are vaccinated or have recovered from prior variant infection, but few reports have documented serial Omicron infections. We characterized SARS-CoV-2 humoral responses in a healthy young person who acquired laboratory-confirmed Omicron BA.1.15 ten weeks after a third dose of BNT162b2, and BA.2 thirteen weeks later. Responses were compared to those of 124 COVID-19 naive vaccinees. One month after the second and third vaccine doses, the participant’s wild-type and BA.1-specific IgG, ACE2 competition and virus neutralization activities were average for a COVID-19 naive triple-vaccinated individual. BA.1 infection boosted the participant’s responses to the cohort ≥95th percentile, but even this strong “hybrid” immunity failed to protect against BA.2. Moreover, reinfection increased BA.1 and BA.2-specific responses only modestly. Results illustrate the risk of Omicron infection in fully vaccinated individuals and high...

ABSTRACTBackgroundLonger-term immune response data after three doses of COVID-19 mRNA vaccine remain limited, particularly among older adults and following Omicron breakthrough infection.MethodsWe quantified wild-type- and Omicron-specific serum IgG levels, ACE2 displacement activities and live virus neutralization up to six months post-third dose in 116 adults aged 24-98 years who remained COVID-19-naïve or experienced their first SARS-CoV-2 infection during this time.ResultsAmong 78 participants who remained COVID-19-naïve throughout follow-up, wild-type- and Omicron BA.1-specific IgG concentrations were comparable between younger and older adults, though 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 among COVID-19-naïve younger and older adults, with median half-lives ranging from 69-78 days. Antiviral antibody function declined substantially...

ABSTRACTBackgroundThird COVID-19 vaccine doses are broadly recommended, but immunogenicity data remain limited, particularly in older adults.MethodsWe measured circulating antibodies against the SARS-CoV-2 spike protein receptor-binding domain, ACE2 displacement, and virus neutralization against ancestral and Omicron (BA.1) strains from pre-vaccine up to one month following the third dose, in 151 adults aged 24-98 years who received COVID-19 mRNA vaccines.ResultsFollowing two 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. Third doses boosted antibody binding and function to higher levels than second-doses, and induced responses in older adults that were comparable in magnitude to those in younger adults. Humoral responses against Omicron were universally weaker than against the ancestral strain after both second and third do...

Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC50 ~ 0.25 µM), and alterations in the production of unspliced, singly spliced, and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and t...

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.

Initial in vitro studies of bevirimat resistance failed to observe mutations in the clinically significant QVT motif in SP1 of HIV-1 gag . This study presents a novel screening method involving mixed, clinically derived gag-protease recombinant HIV-1 samples to more accurately mimic the selection of resistance seen in vivo . Bevirimat resistance was investigated via population-based sequencing performed with a large, initially antiretroviral-naïve cohort before ( n = 805) and after ( n = 355) standard HIV therapy (without bevirimat). The prevalence of any polymorphism in the motif comprising Q, V, and T was ∼6%, 29%, and 12%, respectively, and did not change appreciably over the course of therapy. From these samples, three groups of 10 samples whose bulk sequences were wild type at the QVT motif were used to generate gag-protease recombinant viruses that captured the existing diversity. Groups were mixed and passaged with various bevirimat concentrations for 9 weeks. gag variations...

In the majority of cases, the mechanism underlying the resistance to acyclovir (ACV) of herpes simplex viruses (HSVs) is thymidine kinase (TK) deficiency. Plaque isolates from eight ACV-resistant (ACVr) clinical isolates from AIDS patients, of which five reactivated, were sequenced to determine the genetic lesion within the tk gene conferring resistance and whether this may have correlated with reactivation potential. Mutations were clustered within two homopolymer nucleotide stretches. Three plaque isolates (1737-14, 90-150-3, and 89-650-5) had insertion mutations within a stretch of 7 guanosines, while two isolates (89-063-1 and 89-353-1) had frameshift mutations within a stretch of 6 cytosines (a deletion and an insertion, respectively). Mutations resulted in premature termination codons, and the predicted 28- and 32-kDa truncated TK products were detected by Western blot analysis of virus-infected cell extracts. The repair of one homopolymer frameshift mutation (in isolate 1737-...

Bictegravir (BIC) and cabotegravir (CAB) are the latest available HIV integrase inhibitors in clinical trials. The combination of major integrase inhibitor substitutions G140S/Q148H has been shown to confer high-level resistance to the approved integrase inhibitors raltegravir (RAL) and elvitegravir (EVG) but not necessarily dolutegravir (DTG). We assayed recombinant viruses made from patient-derived RNA extracts for resistance phenotype for a panel of viruses containing G140S/Q148H with additional accessory substitutions. The accumulation of multiple integrase substitutions confers high-level resistance to all five integrase inhibitors. There is extensive cross-resistance between DTG, BIC, and CAB (r = 0.96 - 0.97).

We recently identified the 4-pyridinone-benzisothiazole carboxamide compound 1C8 as displaying strong anti-HIV-1 potency against a variety of clinical strains in vitro Here we show that 1C8 decreases the expression of HIV-1 and alters splicing events involved in the production of HIV-1 mRNAs. Although 1C8 was designed to be a structural mimic of the fused tetracyclic indole compound IDC16 that targets SRSF1, it did not affect the splice site shifting activity of SRSF1. Instead, 1C8 altered splicing regulation mediated by SRSF10. Depleting SRSF10 by RNA interference affected viral splicing and, like 1C8, decreased expression of Tat, Gag and Env. Incubating cells with 1C8 promoted the dephosphorylation of SRSF10 and increased its interaction with hTra2β, a protein previously implicated in the control of HIV-1 RNA splicing. While 1C8 affects the alternative splicing of cellular transcripts controlled by SRSF10 and hTra2β, concentrations greater than those needed to inhibit HIV-1 replic...

Two recombinant phenotypic assays for human immunodeficiency virus (HIV) coreceptor usage and an HIV envelope genotypic predictor were employed on a set of clinically derived HIV type 1 (HIV-1) samples in order to evaluate the concordance between measures. Previously genotyped HIV-1 samples derived from antiretroviral-naïve individuals were tested for coreceptor usage using two independent phenotyping methods. Phenotypes were determined by validated recombinant assays that incorporate either an ∼2,500-bp (“Trofile” assay) or an ∼900-bp (TRT assay) fragment of the HIV envelope gp120. Population-based HIV envelope V3 loop sequences (∼105 bp) were derived by automated sequence analysis. Genotypic coreceptor predictions were performed using a support vector machine model trained on a separate genotype-Trofile phenotype data set. HIV coreceptor usage was obtained from both phenotypic assays for 74 samples, with an overall 85.1% concordance. There was no evidence of a difference in sensi...