Search

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.

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.

BackgroundLonger-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.MethodsWe 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.ResultsAmong 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.ConclusionsResults 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.

BackgroundLimited data exist regarding longer-term antibody responses following three-dose COVID-19 vaccination, and the impact of a first SARS-CoV-2 infection during this time, in people living with HIV (PLWH) receiving suppressive antiretroviral therapy (ART). We quantified wild-type-(WT), Omicron BA.1- and Omicron BA.5-specific responses up to six months post-third dose in 64 PLWH and 117 controls who remained COVID-19-naive or experienced their first SARS-CoV-2 infection during this time.DesignLongitudinal observational cohort.MethodsWe quantified WT- and Omicron-specific Anti-Spike receptor-binding domain IgG concentrations, ACE2 displacement activities and live virus neutralization at one, three and six months post-third vaccine dose.ResultsThird doses boosted all antibody measures above two-dose levels, but BA.1-specific responses remained significantly lower than WT-specific ones, with BA.5-specific responses lower still. Serum IgG concentrations declined at similar rates in COVID-19-naive PLWH and controls post-third dose (median WT- and BA.1-specific half-lives were between 66-74 days for both groups). Antibody function also declined significantly yet comparably between groups: six months post-third dose, BA.1-specific neutralization was undetectable in >80% of COVID-19 naive PLWH and >90% of controls. Breakthrough SARS-CoV-2 infection boosted antibody concentrations and function significantly above vaccine-induced levels in both PLWH and controls, though BA.5-specific neutralization remained significantly poorer than BA.1 even post-breakthrough.ConclusionsFollowing three-dose COVID-19 vaccination, antibody response durability in PLWH receiving ART is comparable to controls. PLWH 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.

BackgroundLonger-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 over time in COVID-19-naïve individuals, particularly older adults: by six months, BA.1-specific neutralization was undetectable in 96% of older adults, versus 56% of younger adults. SARS-CoV-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.ConclusionsOur findings underscore the immune benefits of third COVID-19 mRNA vaccine doses in adults of all ages, but rapid decline of Omicron-specific neutralization activity in COVID-19-naïve individuals, particularly among older adults, demonstrates the need for fourth doses within 3-6 months to maintain systemic responses. Individuals who experienced SARS-CoV-2 breakthrough infection post-third vaccine dose however can likely delay a fourth dose beyond this timeframe.

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.

BackgroundLonger-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 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.ConclusionPLWH 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.

Background. Two-dose mRNA vaccines reduce COVID-19 related hospitalization and mortality, but immune protection declines over time. As such, third vaccine doses are now recommended, particularly for older adults. We examined immune response durability up to six months after two vaccine doses, and immunogenicity after a third vaccine dose, in 151 adults ranging in age from 24 to 98 years. Methods. Specimens were collected from 81 healthcare workers (median age 41 years), 56 older adults (median 78 years) and 14 COVID-19 convalescent individuals (median 48 years), at one, three and six months following the second vaccine dose, and from 15 HCW, 28 older adults and 3 convalescent individuals at one month following a third vaccine dose. Binding antibodies to the SARS-CoV-2 spike receptor binding domain were quantified using a commercial immunoassay. Virus neutralizing activity was assessed using a live SARS-CoV-2 infection assay. Results. Compared to healthcare workers, older adults displayed ~0.3 log10 lower peak binding antibodies one month after the second vaccine dose (p

Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1–6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving &gt, 100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load &gt, 5 log10 IU/mL. This genotype-independent sequencing method resulted in &gt, 99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.

In 2018, the World Health Organization identified the Zika virus (ZIKV) as a pathogen that should be prioritized for public health research due to its epidemic potential. In this study, whole-genome sequencing (WGS) of travel-acquired ZIKV infections was used to examine the limitations of phylogenetic analysis. WGS and phylogenetic analysis were performed to investigate geographic clustering of samples from five Canadians with travel-acquired ZIKV infections and to assess the limitations of phylogenetic analysis of ZIKV sequences using a phylogenetic cluster approach. Genomic variability of ZIKV samples was assessed and for context, compared with hepatitis C virus (HCV) samples. Phylogenetic analysis confirmed the suspected region of ZIKV infection for one of five samples and one sample failed to cluster with sequences from its suspected country of infection. Travel-acquired ZIKV samples depicted low genomic variability relative to HCV samples. A floating patristic distance threshold classified all pre-2000 ZIKV sequences into separate clusters, while only Cambodian, Peruvian, Malaysian, and South Korean sequences were similarly classifiable. While phylogenetic analysis of ZIKV data can identify the broad geographical region of ZIKV infection, ZIKV’s low genomic variability is likely to limit precise interpretations of phylogenetic analysis of the origins of travel-related cases.

Hepatitis C virus (HCV) mixed genotype infections can affect treatment outcomes and may have implications for vaccine design and disease progression. Previous studies demonstrate 0–39% of high-risk, HCV-infected individuals harbor mixed genotypes however standardized, sensitive methods of detection are lacking. This study compared PCR amplicon, random primer (RP), and probe enrichment (PE)-based deep sequencing methods coupled with a custom sequence analysis pipeline to detect multiple HCV genotypes. Mixed infection cutoff values, based on HCV read depth and coverage, were identified using receiver operating characteristic curve analysis. The methodology was validated using artificially mixed genotype samples and then applied to two clinical trials of HCV treatment in high-risk individuals (ACTIVATE, 114 samples from 90 individuals; DARE-C II, 26 samples from 18 individuals) and a cohort of HIV/HCV co-infected individuals (Canadian Coinfection Cohort (CCC), 3 samples from 2 individuals with suspected mixed genotype infections). Amplification bias of genotype (G)1b, G2, G3 and G5 was observed in artificially mixed samples using the PCR method while no genotype bias was observed using RP and PE. RP and PE sequencing of 140 ACTIVATE and DARE-C II samples identified the following primary genotypes: 15% (n = 21) G1a, 76% (n = 106) G3, and 9% (n = 13) G2. Sequencing of ACTIVATE and DARE-C II demonstrated, on average, 2% and 1% of HCV reads mapping to a second genotype using RP and PE, respectively, however none passed the mixed infection cutoff criteria and phylogenetics confirmed no mixed infections. From CCC, one mixed infection was confirmed while the other was determined to be a recombinant genotype. This study underlines the risk for false identification of mixed HCV infections and stresses the need for standardized methods to improve prevalence estimates and to understand the impact of mixed infections for management and elimination of HCV.

Most individuals chronically infected with hepatitis C virus (HCV) are asymptomatic during the initial stages of infection and therefore the precise timing of infection is often unknown. Retrospective estimation of infection duration would improve existing surveillance data and help guide treatment. While intra-host viral diversity quantifications such as Shannon entropy have previously been utilized for estimating duration of infection, these studies characterize the viral population from only a relatively short segment of the HCV genome. In this study intra-host diversities were examined across the HCV genome in order to identify the region most reflective of time and the degree to which these estimates are influenced by high-risk activities including those associated with HCV acquisition. Shannon diversities were calculated for all regions of HCV from 78 longitudinally sampled individuals with known seroconversion timeframes. While the region of the HCV genome most accurately reflecting time resided within the NS3 gene, the gene region with the highest capacity to differentiate acute from chronic infections was identified within the NS5b region. Multivariate models predicting duration of infection from viral diversity significantly improved upon incorporation of variables associated with recent public, unsupervised drug use. These results could assist the development of strategic population treatment guidelines for high-risk individuals infected with HCV and offer insights into variables associated with a likelihood of transmission.

Quantitative viral load assays have transformed our understanding of – and ability to manage − viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time reverse transcriptase-polymerase chain reaction (RT-PCR), yield semi-quantitative results only. Reverse transcriptase droplet digital PCR (RT-ddPCR), a technology that partitions each reaction into 20,000 nanolitre-sized droplets prior to amplification, offers an attractive platform for SARS-CoV-2 RNA quantification. We evaluated eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests for use in RT-ddPCR, and identified three (Charité-Berlin E-Sarbeco and Pasteur Institute IP2 and IP4) as the most efficient, precise and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. Analytical efficiency of the E-Sarbeco primer/probe set, for example, was ~83%, while assay precision, as measured by the coefficient of variation, was ~2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. We further calibrated RT-ddPCR-derived SARS-CoV-2 E gene copy numbers against cycle threshold (Ct) values from a commercial real-time RT-PCR diagnostic platform. The resulting log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from Ct values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.

Using a real-time RT-PCR-based algorithm to detect SARS-CoV-2 variants of concern, we rapidly identified 77 variants (57-B.1.1.7, 7-B.1.351, and 13-B.1.1.28/P.1). This protocol enabled our laboratory to screen all SARS-CoV-2 positive samples for variants, and identified a cluster of B.1.1.28/P.1 cases, a variant not previously known to circulate in British Columbia.

The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials., The intact proviral DNA assay quantifies the genomically intact HIV reservoir, but assay failure due to HIV-1 polymorphism has been observed. Here, the authors report a 28% failure rate in a cohort of people with HIV-1, and note within-host HIV-1 diversity as a further challenge to IPDA accuracy.

McCann et al. describe the development and characterization of a new mouse model for studying HIV-specific T cell responses and testing various immunotherapeutic strategies, which they validate by demonstrating enhanced therapeutic effects of autologous HIV-specific T cells augmented with cytokine-loaded nanogels., HIV-specific CD8+ T cells partially control viral replication and delay disease progression, but they rarely provide lasting protection, largely due to immune escape. Here, we show that engrafting mice with memory CD4+ T cells from HIV+ donors uniquely allows for the in vivo evaluation of autologous T cell responses while avoiding graft-versus-host disease and the need for human fetal tissues that limit other models. Treating HIV-infected mice with clinically relevant HIV-specific T cell products resulted in substantial reductions in viremia. In vivo activity was significantly enhanced when T cells were engineered with surface-conjugated nanogels carrying an IL-15 superagonist, but it was ultimately limited by the pervasive selection of a diverse array of escape mutations, recapitulating patterns seen in humans. By applying mathematical modeling, we show that the kinetics of the CD8+ T cell response have a profound impact on the emergence and persistence of escape mutations. This “participant-derived xenograft” model of HIV provides a powerful tool for studying HIV-specific immunological responses and facilitating the development of effective cell-based therapies.

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.

Opening Paragraph (serves as abstract for submission) and BodyThe Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a precise and scalable method for intact HIV reservoir quantification1. This duplexed droplet digital PCR (ddPCR) assay simultaneously targets the HIV Packaging Signal (Ψ) and the Rev Responsive Element (RRE) within Envelope (env) to distinguish genomically intact proviruses against a large background of defective ones2. The IPDA requires less time, resources, and biological material than the gold standard for replication-competent HIV reservoir measurement, the Quantitative Viral Outgrowth Assay (QVOA)3, and is being adopted in research and clinical studies4–7. In our cohort of HIV-1 subtype B-infected individuals from North America however, the IPDA yielded a 28% failure rate due to HIV polymorphism. We further demonstrate that within-host HIV diversity can lead the IPDA to underestimate intact HIV reservoir size, which could negatively impact clinical trial results interpretation. While the IPDA represents an important methodological advance, HIV diversity should be addressed before its widespread adoption.

Persons with hepatitis C virus (HCV) genotype 1a (GT1a) infections harboring a baseline Q80K polymorphism in nonstructural protein 3 (NS3) have a reduced virologic response to simeprevir in combination with pegylated interferon-alfa and ribavirin. We aimed to develop, validate, and freely disseminate an NS3 clinical sequencing assay to detect the Q80K polymorphism and potentially other HCV NS3 drug resistance mutations. HCV RNA was extracted from frozen plasma using a NucliSENS easyMAG automated nucleic acid extractor, amplified by nested reverse transcription-PCR, and sequenced using Sanger and/or next-generation (MiSeq) methods. Sanger chromatograms were analyzed using in-house software (RECall), and nucleotide mixtures were called automatically. MiSeq reads were iteratively mapped to the H77 reference genome, and consensus NS3 sequences were generated with nucleotides present at >20% called as mixtures. The accuracy, precision, and sensitivity for detecting the Q80K polymorphism were assessed in 70 samples previously sequenced by an external laboratory. A comparison of the sequences generated by the Sanger and MiSeq methods with those determined by an external lab revealed >98.5% nucleotide sequence concordance and zero discordant calls of the Q80K polymorphism. The results were both highly repeatable and reproducible (>99.7% nucleotide concordance and 100% Q80K concordance). The limits of detection (>2 and ∼5 log 10 IU/ml for the Sanger and MiSeq assays, respectively) are sufficiently low to allow genotyping in nearly all chronically infected treatment-naive persons. No systematic bias in the under- or overamplification of minority variants was observed. Coinfection with other viruses (e.g., HIV and hepatitis B virus [HBV]) did not affect the assay results. The two independent HCV NS3 sequencing assays with the automated analysis procedures described here are useful tools to screen for the Q80K polymorphism and other HCV protease inhibitor drug resistance mutations.

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.

HIV-1 tropism can be predicted using V3 genotypic algorithms. The performance of these prediction algorithms for non-B subtypes is poorly characterized. Here, we use these genotypic algorithms to predict viral tropism of HIV-1 subtype A, B, C, and D to find apparent sensitivity, specificity, and concordance against a recombinant phenotypic assay, the original Trofile assay. This is a substudy of an epidemiological study (Pfizer A4001064). Plasma samples were selected to represent a large number of DM/X4 and R5 viruses. The HIV-1 env gene V3 loop was genotyped by Sanger sequencing (N=260) or 454 "deep" sequencing (N=280). Sequences were scored with g2p[coreceptor], PSSM X4/R5, PSSM SI/NSI, and PSSM subtype C matrices. Overall, non-B subtypes tropism prediction had similar concordance and apparent sensitivity and specificity as subtype B in predicting Trofile's results in both population sequencing (81.3%, 65.6%, and 90.5% versus 84.2%, 78.5%, and 88.2%) and 454 "deep" sequencing (82.3%, 80.0%, and 83.6% versus 86.8%, 92.0%, and 82.6%) using g2p[coreceptor]. By population sequencing, subtype A had lower sensitivity, whereas subtype D had lower specificity for non-R5 predictions, both in comparison to subtype B. 454 "deep" sequencing improved subtype A sensitivity but not subtype D. Subtype C had greater concordance than subtype B regardless of sequencing methods. In conclusion, genotypic tropism prediction algorithms may be applied to non-B HIV-1 subtypes with caution. Collective analysis of non-B subtypes revealed a performance similar to subtype B, whereas a subtype-specific analysis revealed overestimation (subtype D) or underestimation (subtype A).

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.

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 5 619; 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 log10 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.

Rare individuals homozygous for a naturally-occurring 32 base pair deletion in the CCR5 gene (CCR5∆32/∆32) are resistant to infection by CCR5-using (“R5”) HIV-1 strains but remain susceptible to less common CXCR4-using (“X4”) strains. The evolutionary dynamics of X4 infections however, remain incompletely understood. We identified two individuals, one CCR5wt/wt and one CCR5∆32/∆32, within the Vancouver Injection Drug Users Study who were infected with a genetically similar X4 HIV-1 strain. While early-stage plasma viral loads were comparable in the two individuals (~4.5–5 log10 HIV-1 RNA copies/ml), CD4 counts in the CCR5wt/wt individual reached a nadir of 3 within 17 months but remained >250 cells/mm3 in the CCR5∆32/∆32 individual. Ancestral phylogenetic reconstructions using longitudinal envelope-V3 deep sequences suggested that both individuals were infected by a single transmitted/founder (T/F) X4 virus that differed at only one V3 site (codon 24). While substantial within-host HIV-1 V3 diversification was observed in plasma and PBMC in both individuals, the CCR5wt/wt individual’s HIV-1 population gradually reverted from 100% X4 to ~60% R5 over ~4 years whereas the CCR5∆32/∆32 individual’s remained consistently X4. Our observations illuminate early dynamics of X4 HIV-1 infections and underscore the influence of CCR5 genotype on HIV-1 V3 evolution.

The MOTIVATE-1 and 2 studies compared maraviroc (MVC) along with optimized background therapy (OBT) vs. placebo along with OBT in treatment-experienced patients screened as having R5-HIV (original Monogram Trofile). A subset screened with non-R5 HIV were treated with MVC or placebo along with OBT in a sister safety trial, A4001029. This analysis retrospectively examined the performance of population-based sequence analysis of HIV-1 env V3-loop to predict coreceptor tropism.Triplicate V3-loop sequences were generated using stored screening plasma samples and data was processed using custom software ('ReCall'), blinded to clinical response. Tropism was inferred using geno2pheno ('g2p'; 5% false positive rate). Primary outcomes were viral load changes after starting maraviroc; and concordance with prior screening Trofile results.Genotype and Trofile results were available for 1164 individuals with virological outcome data (N = 169 non-R5 by Trofile). Compared with Trofile, V3 genotyping had a specificity of 92.6% and a sensitivity of 67.4% for detecting non-R5 virus. However, when compared with clinical outcome, virological responses were consistently similar between Trofile and V3 genotype at weeks 8 and 24 following the initiation of therapy for patients categorized as R5.Despite differences in sensitivity for predicting non-R5 HIV, week 8 and 24 week virological responses were similar in this treatment-experienced population. These findings suggest the potential utility of V3 genotyping as an accessible assay to select patients who may benefit from maraviroc treatment. Optimization of the predictive tropism algorithm may lead to further improvement in the clinical utility of HIV genotypic tropism assays.

Background The main determinants of HIV-1 coreceptor usage are located in the V3-loop of gp120, although mutations in V2 and gp41 are also known. Incorporation of V2 is known to improve prediction algorithms; however, this has not been confirmed for gp41 mutations. Methods Samples with V3 and gp41 genotypes and Trofile assay (Monogram Biosciences, South San Francisco, CA, USA) results were taken from the HOMER cohort ( n=444) and from patients screened for the MOTIVATE studies ( n=1,916; 859 with maraviroc outcome data). Correlations of mutations with tropism were assessed using Fisher's exact test and prediction models trained using support vector machines. Models were validated by cross-validation, by testing models from one dataset on the other, and by analysing virological outcome. Results Several mutations within gp41 were highly significant for CXCR4 usage; most strikingly an insertion occurring in 7.7% of HOMER-R5 and 46.3% of HOM-ER-X4 samples (MOTIVATE 5.7% and 25.2%, respectively). Models trained on gp41 sequence alone achieved relatively high areas under the receiver- operating characteristic curve (AUCs; HOMER 0.713 and MOTIVATE 0.736) that were almost as good as V3 models (0.773 and 0.884, respectively). However, combining the two regions improved predictions only marginally (0.813 and 0.902, respectively). Similar results were found when models were trained on HOMER and validated on MOTIVATE or vice versa. The difference in median log viral load decrease at week 24 between patients with R5 and X4 virus was 1.65 (HOMER 2.45 and MOTIVATE 0.79) for V3 models, 1.59 for gp41-models (2.42 and 0.83, respectively) and 1.58 for the combined predictor (2.44 and 0.86, respectively). Conclusions Several mutations within gp41 showed strong correlation with tropism in two independent datasets. However, incorporating gp41 mutations into prediction models is not mandatory because they do not improve substantially on models trained on V3 sequences alone.

Rapidly evolving viruses such as HIV-1 display extensive sequence variation in response to host-specific selection, while simultaneously maintaining functions that are critical to replication and infectivity. This apparent conflict between diversifying and purifying selection may be resolved by an abundance of epistatic interactions such that the same functional requirements can be met by highly divergent sequences. We investigate this hypothesis by conducting an extensive characterization of sequence variation in the HIV-1 nef gene that encodes a highly variable multifunctional protein. Population-based sequences were obtained from 686 patients enrolled in the HOMER cohort in British Columbia, Canada, from which the distribution of nonsynonymous substitutions in the phylogeny was reconstructed by maximum likelihood. We used a phylogenetic comparative method on these data to identify putative epistatic interactions between residues. Two interactions (Y120/Q125 and N157/S169) were chosen to further investigate within-host evolution using HIV-1 RNA extractions from plasma samples from eight patients. Clonal sequencing confirmed strong linkage between polymorphisms at these sites in every case. We used massively parallel pyrosequencing (MPP) to reconstruct within-host evolution in these patients. Experimental error associated with MPP was quantified by performing replicates at two different stages of the protocol, which were pooled prior to analysis to reduce this source of variation. Phylogenetic reconstruction from these data revealed correlated substitutions at Y120/Q125 or N157/S169 repeated across multiple lineages in every host, indicating convergent within-host evolution shaped by epistatic interactions.

We investigated the associations between coreceptor use, V3 loop sequence, and CD4 count in a cross-sectional analysis of a large cohort of chronically HIV-infected, treatment-naive patients. HIV coreceptor usage was determined in the last pretherapy plasma sample for 977 individuals initiating HAART in British Columbia, Canada using the Monogram Trofile Tropism assay. Relative light unit (RLU) readouts from the Trofile assay, as well as HIV V3 loop sequence data, were examined as a function of baseline CD4 cell count for 953 (97%) samples with both phenotype and genotype data available. Median CCR5 RLUs were high for both R5 and X4-capable samples, while CXCR4 RLUs were orders of magnitude lower for X4 samples (p < 0.001). CCR5 RLUs in R5 samples (N = 799) increased with decreasing CD4 count (p < 0.001), but did not vary with plasma viral load (pVL) (p = 0.74). In X4 samples (N = 178), CCR5 RLUs decreased with decreasing CD4 count (p = 0.046) and decreasing pVL (p = 0.097), while CXCR4 RLUs increased with decreasing pVL (p = 0.0008) but did not vary with CD4 (p = 0.96). RLUs varied with the presence of substitutions at V3 loop positions 11, 25, and 6-8. The prevalence and impact of substitutions at codons 25 and 6-8 were CD4 dependent as was the presence of amino acid mixtures in the V3; substitutions at position 11 were CD4 independent. Assay RLU measures predictably vary with both immunological and virological parameters. The ability to predict X4 virus using genotypic determinants at positions 25 and 6-8 of the V3 loop is CD4 dependent, while position 11 appears to be CD4 independent.

Objective To describe a suspected case of intrafamilial transmission of drug-resistant HIV-1 from an infected individual to his caregiver. Methods HIV-1 protease, reverse transcriptase, nef and gp41 DNA sequences were determined from plasma samples after RNA extraction and nested RT-PCR. Phylogenetic trees were generated using neighbour-joining methods. Results HIV-1 infection was diagnosed in an individual (index) following the death of her HIV-infected adopted son (source). The index case cared for her son in the 9 months prior to his death. No obvious instances of contact with bodily secretions or other traditional risk factors for HIV transmission were identified, although the index case had mild eczema. At time of death, the source case's HIV plasma viral load was >1,000,000 HIV copies/ml. Genotyping of the protease and reverse transcriptase of both the source and index samples revealed similar drug-resistance mutations despite the index case being antiretroviral-naive. Both source and index V3 genotypes were consistent with syncytium-inducing virus. The mean pairwise amino acid identity between the source and index samples was 97.0%, 99.2%, 92.6% and 94.6% in protease, reverse transcriptase, nef and gp41, respectively. The source and index sequences clustered together on phylogenetic trees when compared with 50 randomly selected sequences from British Columbia. Conclusion Although exceptionally rare, our case demonstrates transmission of drug-resistant HIV-1 from an infected individual to his caregiver in the absence of traditional risk factors. It should be noted that the source had a very high viral load during the terminal phase of his illness.

OBJECTIVE Integrating CCR5 antagonists into clinical practice would benefit from accurate assays of co-receptor usage (CCR5 versus CXCR4) with fast turnaround and low cost. DESIGN Published HIV V3-loop based predictors of co-receptor usage were compared with actual phenotypic tropism results in a large cohort of antiretroviral naive individuals to determine accuracy on clinical samples and identify areas for improvement. METHODS Aligned HIV envelope V3 loop sequences (n = 977), derived by bulk sequencing were analyzed by six methods: the 11/25 rule; a neural network (NN), two support vector machines, and two subtype-B position specific scoring matrices (PSSM). Co-receptor phenotype results (Trofile Co-receptor Phenotype Assay; Monogram Biosciences) were stratified by CXCR4 relative light unit (RLU) readout and CD4 cell count. RESULTS Co-receptor phenotype was available for 920 clinical samples with V3 genotypes having fewer than seven amino acid mixtures (n = 769 R5; n = 151 X4-capable). Sensitivity and specificity for predicting X4 capacity were evaluated for the 11/25 rule (30% sensitivity/93% specificity), NN (44%/88%), PSSM(sinsi) (34%/96%), PSSM(x4r5) (24%/97%), SVMgenomiac (22%/90%) and SVMgeno2pheno (50%/89%). Quantitative increases in sensitivity could be obtained by optimizing the cut-off for methods with continuous output (PSSM methods), and/or integrating clinical data (CD4%). Sensitivity was directly proportional to strength of X4 signal in the phenotype assay (P < 0.05). CONCLUSIONS Current default implementations of co-receptor prediction algorithms are inadequate for predicting HIV X4 co-receptor usage in clinical samples, particularly those X4 phenotypes with low CXCR4 RLU signals. Significant improvements can be made to genotypic predictors, including training on clinical samples, using additional data to improve predictions and optimizing cutoffs and increasing genotype sensitivity.

Limited access to HIV drug resistance testing in low- and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n= 759) and from a Ugandan study cohort (n= 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of 10HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex “wide” sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.

Background: There exist concerns regarding the potential for elevated rates of anti-retroviral resistance among HIV-infected injection drug users (IDUs) prescribed highly active antiretroviral therapy (HAART), however, no population-based study has examined if IDUs have elevated rates of antiretroviral resistance in comparison to non-IDUs. Objective: To evaluate the time to the development of antiretroviral resistance among antiretroviral-naive patients with and without a history of injection drug use. Methods: In British Columbia there is a province-wide HIV/AIDS treatment program that provides antiretrovirals free of charge. We examined all antiretroviral-naive patients initiating HAART between 1 August 1996 and 30 September 2000 and who were followed to 31 March 2002. The main outcome measure was the time to class-specific antiretroviral resistance. Cumulative antiretroviral resistance rates among IDUs and non-IDUs were evaluated using Kaplan-Meier methods and relative hazards were estimated using Cox regression. Results: Overall, 1191 antiretroviral-naive patients initiated HAART during the study period. Resistance mutations were observed in 298 (25%) subjects during the first 30 months of HAART. In comparison with non-IDUs, the risk of protease inhibitor resistance [relative hazard (RH), 0.9; 95% confidence interval (Cl), 0.5-1.6] and non-nucleoside reverse transcriptase inhibitor resistance (RH, 1.5; 95% Cl, 1.0-2.2) were similar among IDUs, and there were no differences in the rates of resistance to the sub-classes of nucleoside reverse transcriptase inhibitors. Conclusions: Resistance to all major classes of antiretrovirals were similar among IDUs and non-IDUs after 30 months of follow-up. These findings should help to allay fears that prescribing HAART to IDUs may result in elevated rates of resistance.