Rising prevalence of non-B HIV-1 subtypes in North Carolina and evidence for local onward transmission.

HIV-1 diversity is increasing in North American and European cohorts which may have public health implications. However, little is known about non-B subtype diversity in the southern United States, despite the region being the epicenter of the nation's epidemic. We characterized HIV-1 diversity and transmission clusters to identify the extent to which non-B strains are transmitted locally. We conducted cross-sectional analyses of HIV-1 partial pol sequences collected from 1997 to 2014 from adults accessing routine clinical care in North Carolina (NC). Subtypes were evaluated using COMET and phylogenetic analysis. Putative transmission clusters were identified using maximum-likelihood trees. Clusters involving non-B strains were confirmed and their dates of origin were estimated using Bayesian phylogenetics. Data were combined with demographic information collected at the time of sample collection and country of origin for a subset of patients. Among 24,972 sequences from 15,246 persons, the non-B subtype prevalence increased from 0% to 3.46% over the study period. Of 325 persons with non-B subtypes, diversity was high with over 15 pure subtypes and recombinants; subtype C (28.9%) and CRF02_AG (24.0%) were most common. While identification of transmission clusters was lower for persons with non-B versus B subtypes, several local transmission clusters (≥3 persons) involving non-B subtypes were identified and all were presumably due to heterosexual transmission. Prevalence of non-B subtype diversity remains low in NC but a statistically significant rise was identified over time which likely reflects multiple importation. However, the combined phylogenetic clustering analysis reveals evidence for local onward transmission. Detection of these non-B clusters suggests heterosexual transmission and may guide diagnostic and prevention interventions

Preservation of tetherin and CD4 counter-activities in circulating Vpu alleles despite extensive sequence variation within HIV-1 infected individuals.

The HIV-1 Vpu protein is expressed from a bi-cistronic message late in the viral life cycle. It functions during viral assembly to maximise infectious virus release by targeting CD4 for proteosomal degradation and counteracting the antiviral protein tetherin (BST2/CD317). Single genome analysis of vpu repertoires throughout infection in 14 individuals infected with HIV-1 clade B revealed extensive amino acid diversity of the Vpu protein. For the most part, this variation in Vpu increases over the course of infection and is associated with predicted epitopes of the individual's MHC class I haplotype, suggesting CD8+ T cell pressure is the major driver of Vpu sequence diversity within the host. Despite this variability, the Vpu functions of targeting CD4 and counteracting both physical virus restriction and NF-κB activation by tetherin are rigorously maintained throughout HIV-1 infection. Only a minority of circulating alleles bear lesions in either of these activities at any given time, suggesting functional Vpu mutants are heavily selected against even at later stages of infection. Comparison of Vpu proteins defective for one or several functions reveals novel determinants of CD4 downregulation, counteraction of tetherin restriction, and inhibition of NF-κB signalling. These data affirm the importance of Vpu functions for in vivo persistence of HIV-1 within infected individuals, not simply for transmission, and highlight its potential as a target for antiviral therapy

Intrapatient Evolutionary Dynamics of Human Immunodeficiency Virus Type 1 in Individuals Undergoing Alternative Treatment Strategies with Reverse Transcriptase Inhibitors.

Structured treatment interruption (STI) has been trialed as an alternative to lifelong antiretroviral therapy (ART). We retrospectively performed single genome sequencing of the HIV-1 pol region from three patients representing different scenarios. They were either failing on continuous therapy (CT-F), failing STI (STI-F), or suppressing on STI (STI-S). Over 460 genomes were generated from three to five different time points over a 2-year period. We found multiple-linked-resistant mutations in both treatment failures. However, the CT-F patient showed a stepwise accumulation of diverse, linked mutations whereas the STI-F patient had lineage turnover between treatment periods with recirculation of wild-type and resistant variants from reservoirs. The STI-F patient showed a 7-fold increase in the third codon position substitution rate relative to the first and second positions compared to a 2-fold increase for CT-F and increased purifying selection in the pol gene (62 vs. 22 sites, respectively). An understanding of intrapatient viral dynamics could guide the future direction of treatment interruption strategies

Prevalence and Transmission Dynamics of HIV-1 Transmitted Drug Resistance in a Southeastern Cohort.

BACKGROUND: Transmitted drug resistance (TDR) compromises clinical management and outcomes. Transmitted drug resistance surveillance and identification of growing transmission clusters are needed in the Southeast, the epicenter of the US HIV epidemic. Our study investigated prevalence and transmission dynamics in North Carolina. METHODS: We analyzed surveillance drug resistance mutations (SDRMs) using partial pol sequences from patients presenting to 2 large HIV outpatient clinics from 1997 to 2014. Transmitted drug resistance prevalence was defined as ≥1 SDRMs among antiretroviral therapy (ART)-naïve patients. Binomial regression was used to characterize prevalence by calendar year, drug class, and demographic and clinical factors. We assessed the transmission networks of patients with TDR with maximum likelihood trees and Bayesian methods including background pol sequences (n = 15 246). RESULTS: Among 1658 patients with pretherapy resistance testing, ≥1 SDRMs was identified in 199 patients, with an aggregate TDR prevalence of 12% (95% confidence interval, 10% to 14%) increasing over time (P = .02). Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs; 8%) was common, followed by nucleoside reverse transcriptase inhibitors (4%) and protease inhibitors (2%). Factors associated with TDR were being a man reporting sex with men, white race, young age, higher CD4 cell count, and being a member of a transmission cluster. Transmitted drug resistance was identified in 106 clusters ranging from 2 to 26 members. Cluster resistance was primarily NNRTI and dominated by ART-naïve patients or those with unknown ART initiation. CONCLUSIONS: Moderate TDR prevalence persists in North Carolina, predominantly driven by NNRTI resistance. Most TDR cases were identified in transmission clusters, signifying multiple local transmission networks and TDR circulation among ART-naïve persons. Transmitted drug resistance surveillance can detect transmission networks and identify patients for enhanced services to promote early treatment

Effectiveness of BNT162b2 and ChAdOx1 against SARS-CoV-2 household transmission: a prospective cohort study in England

Background: The ability of SARS-CoV-2 vaccines to protect against infection and onward transmission determines whether immunisation can control global circulation. We estimated the effectiveness of Pfizer-BioNTech mRNA vaccine (BNT162b2) and Oxford AstraZeneca adenovirus vector vaccine (ChAdOx1) vaccines against acquisition and transmission of the Alpha and Delta variants in a prospective household study in England. Methods: Households were recruited based on adult purported index cases testing positive after reverse transcription-quantitative (RT-q)PCR testing of oral-nasal swabs. Purported index cases and their household contacts took oral-nasal swabs on days 1, 3 and 7 after enrolment and a subset of the PCR-positive swabs underwent genomic sequencing conducted on a subset. We used Bayesian logistic regression to infer vaccine effectiveness against acquisition and transmission, adjusted for age, vaccination history and variant. Results: Between 2 February 2021 and 10 September 2021, 213 index cases and 312 contacts were followed up. After excluding households lacking genomic proximity (N=2) or with unlikely serial intervals (N=16), 195 households with 278 contacts remained, of whom 113 (41%) became PCR positive. Delta lineages had 1.53 times the risk (95% Credible Interval: 1.04 – 2.20) of transmission than Alpha; contacts older than 18 years old were 1.48 (1.20 – 1.91) and 1.02 (0.93 – 1.16) times more likely to acquire an Alpha or Delta infection than children. Effectiveness of two doses of BNT162b2 against transmission of Delta was 36% (-1%, 66%) and 49% (18%, 73%) for ChAdOx1, similar to their effectiveness for Alpha. Protection against infection with Alpha was higher than for Delta, 69% (9%, 95%) vs. 18% (-11%, 59%), respectively, for BNT162b2 and 24% (-41%, 72%) vs. 9% (-15%, 42%), respectively, for ChAdOx1.Conclusions: BNT162b2 and ChAdOx1 reduce transmission of the Delta variant from breakthrough infections in the household setting, although their protection against infection within this setting is low

G2/M cell cycle arrest correlates with primate lentiviral Vpr interaction with the SLX4 complex

UNLABELLED: The accessory gene vpr, common to all primate lentiviruses, induces potent G2/M arrest in cycling cells. A recent study showed that human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi anemia DNA repair pathway. To confirm these observations, we have examined the G2/M arrest phenotypes of a panel of simian immunodeficiency virus (SIV) Vpr proteins. We show that SIV Vpr proteins differ in their ability to promote cell cycle arrest in human cells. While this is dependent on the DCAF1/DDB1/CUL4 ubiquitin ligase complex, interaction with human DCAF1 does not predict G2/M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with human SLX4 (huSLX4) and could be abolished by small interfering RNA (siRNA) depletion of any member of the SLX4 complex. In contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G2/M arrest in grivet cells. Correspondingly, here cell cycle arrest correlated with interaction with the grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G2/M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins and that the ability to dysregulate the Fanconi anemia DNA repair pathway is an essential function of Vpr in vivo. IMPORTANCE: The Vpr protein of HIV-1 and related viruses is essential for the virus in vivo. The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data have shown that HIV-1 Vpr targets the Fanconi anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA cross-links. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. The results of these studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins and that the ability to dysregulate members of the Fanconi anemia DNA repair pathway is essential for HIV/SIV replication in vivo

<i>In vivo</i> variation of HIV-1 clade B <i>vpu.</i>

<p>Unrooted maximum likelihood phylogeny of 851 <i>vpu</i> nucleotide sequences derived from 25 samples from 14 HIV-1-infected individuals. Individuals were classified according to time from seroconversion to progression to AIDS: 5 rapid progressors (RPs), 4 normal progressors (NPs) and 5 long-term non-progressors (LTNPs) with sequences from each individual coloured and labelled accordingly. Bootstrap supports (% confidence) are shown at the base of the branch for each individual. Branch lengths indicate the number of nucleotide substitutions per site.</p

T cell epitopes and variation. Full analysis of the Vpu repertoire of one infected individual.

<p>(<b>A</b>) A phylogenetic tree of <i>vpu</i> sequences obtained from individual LTNP 1 over 3 different time points: 1.1 (light blue branches), 4.4 (dark blue branches) and 10.4 years post-seroconversion (purple branches), rooted with both NL4.3 and consensus B <i>vpu</i> sequences. Squares represent unique amino acid sequences, with the area of the square proportional to the number of Vpus with that specific sequence. The colour of the square is determined by the ability of the Vpu to counteract tetherin, based on ‘traffic light’ formatting: red being completely unable to counteract tetherin (0%), yellow having intermediate activity (75%), and green being highly active (150%). Note that in this individual most Vpu proteins had more than intermediate activity, therefore most squares are shades of green, with the darker the green the more active the protein. Branch support is indicated by one (>70%) or two (>90%) asterisks. Branch length represents the number of nucleotide substitutions per site. (<b>B</b>) An alignment of all of the unique amino acid sequences obtained from LTNP 1 and shown in (A), ordered from top to bottom according to their phylogenetic relationship. Sequence 1, to which all subsequent Vpus are compared, is that closest to the root of the tree shown in (A). The position of each sequence in the tree is indicated on the left of the alignment. Anti-tetherin activity is indicated by colour, as shown in (A). Substitutions from the ancestral sequence are highlighted in the alignment in purple, with the tally of cumulative mutations indicated on the right of the alignment. Predicted high affinity (0–50 nM) T cell epitopes, tailored to the HLA haplotype of the infected individual, are indicated above the alignment. Positions undergoing positive selection are denoted by a “P” above the alignment, with residues undergoing episodic selection (indicative of immune pressure) indicated by “E”.</p

Ability of Vpu to suppress NF-κB activation.

<p>(<b>A</b>) NL4.3 Vpu, NL4.3 A14L and S52,56A mutants, and the highly active patient-derived Vpu RP2v16_2_87 were tested at a range of concentrations for their ability to counteract tetherin-mediated NF-κB activation (left panel) and MAVS-mediated NF-κB activation (right panel). Transient NF-κB activation assays were performed by transfecting 293 cells with 50 ng pCR3.1 human tetherin or 10 ng pCR3.1 MAVS alongside 0, 5, 10, 25, 50 and 100 ng of each pCRVI-Vpu. Fold activation of a luciferase NF-κB reporter gene is calculated relative to a GFP control in the presence of increasing concentrations of Vpu, and results are presented relative to the mean signal obtained in the absence of Vpu (% max). (<b>B</b>) As for (A), but with patient-derived Vpus with defects in tetherin signalling suppression but not promotion of virus release (G59R, E62G); patient-derived Vpu with defects in promotion of virus release but not suppression of signalling (E29K); and patient-derived Vpu with defects in both tetherin signalling suppression and promotion of virus release (A19), all identified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003895#ppat-1003895-g007" target="_blank"><b>Figure 7</b></a>.</p

Analysis of CD4 downregulation and tetherin antagonism of 304 natural Vpu alleles.

<p>Representatives of all amino acid sequences obtained through SGA were cloned and tested in standardised assays for the two major functions of Vpu: (<b>A</b>) cell surface CD4 downregulation and (<b>B</b>) tetherin counteraction. The time point of each sample, in years post-seroconversion, is indicated beneath the graph with the patient identification and progression group. The function of each Vpu is represented relative to the prototypical Vpu from NL4.3, as indicated by a dashed line at 100%, and the functions of all other Vpus are represented as a percentage thereof. The NL4.3 S52,56A Vpu mutant, defective for both CD4 downregulation and tetherin counteraction activity, is indicated by a dashed line (at 0% on the CD4 downregulation graph and 13% on the tetherin counteraction graph). Each symbol represents the average of a minimum of three independent experiments, weighted to represent the number of sequences obtained per sample with that particular amino acid sequence (allele frequency), to give an overall proportional representation of function per time point. Means for overall Vpu function for each time point are shown as short horizontal lines. Significant differences between time points from each individual are indicated by asterisks. Briefly, the assays were performed as follows: (<b>A</b>) HeLa-TZMbl cells were co-transfected with 100 ng of pCRVI-Vpu or empty vector (EV) plasmid and 100 ng of pCR3.1-eGFP. 24 hours later cell surface CD4 levels were analysed by flow cytometry. CD4 downregulation was determined by comparing median fluorescent intensities of CD4 in the presence and absence of Vpu, with the downregulation achieved by NL4.3 Vpu set at 100%. Note that the absolute value of CD4 reduction achieved by NL4.3 was 80%±6. (<b>B</b>) 293T cells were transfected with a fixed dose (50 ng) of pCR3.1-hu-tetherin in combination with 500 ng of NL4.3-del Vpu proviral plasmid and 25 ng of pCRVI-Vpu. 48 hours later the supernatants are removed from the cells and assayed on Hela-TZMbl cells for the quantity of infectious virus. The 100% line represents the amount of infectious virus released in the presence of NL4.3, to allow direct comparisons between the CD4 downregulation and tetherin counteraction assays. 25 ng of pCRVI-Vpu was used as this quantity produced the same amount of Vpu protein as that of the full-length NL4.3 molecular clone, as determined by Western blot analysis.</p