Determinants of protection against SARS-CoV-2 Omicron BA.1 and Delta infections in fully vaccinated outpatients.

We aimed to evaluate the association between the humoral and cellular immune responses and symptomatic SARS-CoV-2 infection with Delta or Omicron BA.1 variants in fully vaccinated outpatients. Anti-receptor binding domain (RBD) IgG levels and interferon-gamma (IFN-γ) release were evaluated at PCR-diagnosis of SARS-CoV-2 in 636 samples from negative and positive patients during Delta and Omicron BA.1 periods. Median levels of anti-RBD IgG in positive patients were significantly lower than in negative patients for both variants (p < 0.05). The frequency of Omicron BA.1 infection in patients with anti-RBD IgG concentrations ≥1000 binding antibody units (BAU)/mL was 51.0% and decreased to 34.4% in patients with concentrations ≥3000 BAU/mL. For Delta infection, the frequency of infection was significantly lower when applying the same anti-RBD IgG thresholds (13.3% and 5.3% respectively, p < 0.05). In addition, individuals in the hybrid immunity group had a 4.5 times lower risk of Delta infection compared to the homologous vaccination group (aOR = 0.22, 95% CI: [0.05-0.64]. No significant decrease in the risk of Omicron BA.1 infection was observed in the hybrid group compared to the homologous group, but the risk decreased within the hybrid group as anti-RBD IgG titers increased (aOR = 0.08, 95% CI: [0.01-0.41], p = 0.008). IFN-γ release post-SARS-CoV-2 peptide stimulation was not different between samples from patients infected (either with Delta or Omicron BA.1 variant) or not (p > 0.05). Our results show that high circulating levels of anti-RBD IgG and hybrid immunity were independently associated with a lower risk of symptomatic SARS-CoV-2 infection in outpatients with differences according to the infecting variant (www.clinicaltrials.gov; ID NCT05060939).

Online self-sampling kits for human immunodeficiency virus and other sexually transmitted infections: Feasibility, positivity rates, and factors associated with infections in France.

BACKGROUND: Men who have sex with men are increasingly diagnosed with sexually transmitted infections (STI) in France. To address this situation, quarterly screening for HIV combined with hepatitis B (HBV) and hepatitis C (HCV), as well as annual screening for C.trachomatis (CT) and N.gonorrhoeae (NG) are recommended. The MemoDepistages program offered an at-home screening solution for these infections. This study describes the feasibility of this screening process, the rate of positive test results, and the factors associated with positivity. METHODS: Participants were recruited online. Laboratories verified the quantity and quality of the samples. Logistic regression was used to determine the associated factors for infection. RESULTS: Overall, 1556 out of 1908 (81.6%) blood samples were tested for at least HIV. A total of eight participants (0.5%) were newly diagnosed with HIV and four with HCV (0.3%). No new infection was confirmed for HBV. Overall positivity was 9.3% for CT and 9.6% for NG. The highest positivity was reported in rectal swabs for CT (7.3%) and in pharyngeal swabs for NG (7.2%). Factors associated with extragenital CT/NG were age under 30 years (for pharyngeal and rectal infections) and having at least 10 partners in the past 6 months (p<0.001) (for pharyngeal infections only). CONCLUSIONS: The self-sampling kit for multiple STIs can perform comprehensive tests and identify new infections in young people, especially in extragenital sites.

Online self-sampling kits to screen multipartner MSM for HIV and other STIs: participant characteristics and factors associated with kit use in the first 3 months of the MemoDepistages programme, France, 2018.

OBJECTIVES: In 2017, to reduce the proportion of men who have sex with men (MSM) in the undiagnosed HIV population in France (38%), HIV screening is advised each 3 months and STI screening is advised each year in multipartner MSM. Despite the range of testing solutions, over 40% of MSM were not tested for HIV and over 50% for STIs in the past year. Based on international experiments that offer screening solutions via online advertising, the French National Health Agency launched a programme (MemoDepistages) to provide a free self-sampling kit (SSK) for HIV and STIs. This article analyses the sociodemographic and behavioural characteristics of MSM in terms of kit acceptance and sample return. METHODS: Participants were registered for the programme online after ordering an SSK. The study included men aged over 18 years, living in one of the four selected French regions, and willing to disclose their postal and email address; they had health insurance, acknowledged more than one male partner in the past year, indicated a seronegative or unknown HIV status and were not taking medically prescribed pre-exposure prophylaxis drugs. Samples were collected by users and posted directly to the laboratory. Characteristics associated with kit acceptance and sample return were analysed using logistic regression. RESULTS: Overall, 7158 eligible MSM were offered to participate in the programme, with 3428 ordering the kit (47.9%) and 1948 returning their sample, leading to a return rate of 56.8% and an overall participation rate of 27.2%. Acceptance and return rates were strongly associated with sociodemographic characteristics, mainly education level but not with behavioural characteristics. Non-college graduates had lower acceptance (44.2%) and return rates (47.7%). CONCLUSION: The programme rapidly recruited a large number of MSM. It removed geographical inequalities related to screening access.

Coronavirus disease 2019 attack rate in HIV-infected patients and in preexposure prophylaxis users.

OBJECTIVE: A new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) emerged in China during late 2019 and resulted in the coronavirus disease 2019 (COVID-19) pandemic which peaked in France in March-April 2020. Immunodeficiency, precariousness and promiscuity could increase the risk of COVID-19 in HIV-infected patients and in preexposure prophylaxis (PrEP) users. No epidemiological data are available in these two populations. We report COVID-19 attack rate in HIV-infected patients and in PrEP users in the Rhône department, France, and compared it with the general population. DESIGN: Retrospective analysis of a laboratory database. METHODS: COVID-19 testing strategy in France was centered on symptomatic infections, hospitalized patients and symptomatic healthcare workers while most asymptomatic cases were not confirmed. SARS-CoV-2 positivity rate on PCR assays and COVID-19 attack rate were determined in HIV-infected patients and in PrEP users. COVID-19 attack rate in the general population was estimated from health authorities' database and demographic data. A corrected attack rate taking into account the laboratory representativeness was calculated. RESULTS: From March to April 2020, 24 860 samples from 19 113 patients (HIV-infected 77, PrEP users 27, others 19 009) were assessed for SARS-CoV-2 PCR assay. The positivity rate appeared similar in HIV-infected patients (15.6%), in PrEP users (14.8%) and in other patients (19.1%). The crude/corrected COVID-19 attack rate appeared similar in HIV-infected patients (0.31/0.38%) and in PrEP users (0.38/0.42%), and of the same order as the estimated attack rate in the general population (0.24%). CONCLUSION: The risk of symptomatic COVID-19 in France appeared similar in HIV-infected patients and in PrEP users compared with the general population.

Comparison of eight commercial, high-throughput, automated or ELISA assays detecting SARS-CoV-2 IgG or total antibody.

BACKGROUND: Many commercial assays, of different designs, detecting SARS-CoV-2-specific antibodies exist but with little experience with them. OBJECTIVES: The aim of this study was to compare the performance of assays detecting IgG or total antibodies to N or S antigens, validated for routine use in France, with samples from subjects with more or less severe SARS-CoV-2 infection. METHODS: Eight assays were used: Abbott Architect, DiaSorin Liaison®, bioMérieux Vidas®, Roche Elecsys Cobas®, Siemens Atellica®, BioRad Platelia ELISA, Epitope Diagnostics ELISA, and Wantai ELISA. The tested population included 86 samples from 40 hospitalized subjects and 28 outpatients at different time from symptom onset. RESULTS: The positivity rate varied depending on the assay but was greater for all assays in hospitalized than non-hospitalized patients. Despite a good correlation between the assays, discrepancies occurred, without a systematic origin, even for samples taken more than 20 days after symptom onset. These discrepancies were linked to low antibody levels in pauci-symptomatic patients. CONCLUSION: Whichever assay is chosen, a false negative result may need to be ruled out with another test in a risk situation.

Comparison of HIV-1 drug-resistance genotyping by ultra-deep sequencing and sanger sequencing using clinical samples.

Sanger population sequencing (SPS) is the reference technique to monitor HIV-1-infected patients' therapy. Ultra-deep sequencing (UDS), which allows quantitative detection of drug resistance mutations, may be an alternative method. The study aimed to compare reproducibility and predictions of UDS versus SPS in a routine setting. A control containing low-abundance variants was repeatedly tested and clinical plasma samples from 100 patients were prospectively assayed by SPS and UDS using the Roche 454 system. Complete analysis by UDS was available for 88% of samples with various viral loads and subtypes. Comparison of detection thresholds found that SPS sensitivity was variable. Variations found by UDS between 5% to >20% were detected by SPS in 25% to more than 80% of samples. At the 5% cut-off, disagreements were rare and in most cases UDS detected an additional protease secondary mutation, suggesting a possible resistance to a protease inhibitor according to the 2015 ANRS algorithm. Mutations found on reverse transcriptase by only UDS were often explained by previous therapy. UDS with a variant detection threshold at 5% might allow therapy management with minimal differences compared to population sequencing while providing additional information for further determination of pertinent cutoff values for specific resistance mutations.

A case of Mayaro virus infection imported from French Guiana.

Emergence of arboviruses is a rising problem in several areas in the world. Here we report a case of Mayaro virus infection that was diagnosed in a French citizen presenting a dengue-like syndrome with prolonged arthralgia following a travel in French Guiana. Diagnosis was based on serological testing, a newly developed specific RT-PCR and sequencing. The real incidence of this viral infection among travelers is poorly known but this case is the first reported in a European area where Aedes albopictus mosquitoes are established, which underscores the necessity to determine the vector competence of the European strain of this mosquito species for Mayaro virus.

Relationship between discordant response to HAART, Tregs, immune activation and low-level viraemia.

INTRODUCTION: The incomplete immune recovery upon effective long-term highly active antiretroviral therapy (HAART) has been associated with increased morbidity and mortality in HIV infected patients [1]. Immune cellular activation, Tregs or very low-level viraemia has been alternatively suspected, but never investigated simultaneously [2]. MATERIALS AND METHODS: We performed a cross-sectional study in 87 aviraemic patients (men=62, mean CD4+T cells=570/mm(3), mean duration of HAART=12 years). Patients with at least 500 CD4+ T cells /mm(3) were classified as complete immunological responders (cIR), whereas remaining patients were classified as inadequate immunological responder (iIR). Tregs were characterized based on CD4+CD25highFoxP3+phenotype using a one-step intracellular staining. Effector Tregs and terminal effectors Tregs were respectively defined as CD4+CD25+FoxP3+CD45RA-, and CD4+CD25+FoxP3+CD45RA-HLADR+phenotypes as recently described [3]. Activated T cells were identified using (i) elevated HLA-DR expression for CD4+T cells, and (ii) increased expressions of HLA-DR, or CD38, or both (HLADR+CD38+cells) for CD8+T cells. Very low-level viraemia was defined as detectable viraemia between 1 and 39 cp/mL. Univariate and multivariate analyses were performed to identify determinants of iIR. RESULTS: Thirty-nine patients were classified as iIR, and 48 as cIR. Patients from the iIR group were significantly older (55 vs 50 years, p=0.027), and had percentages of activated CD4+ T cells, Tregs, effector Tregs and terminal effector Tregs significantly higher (5.3 vs 4%, p=0.014; 9 vs 7.5%, p=0,022; 8 vs 6.3%, p=0.01 and 1.8 vs 1.3%, p=0,033 among CD4+T cells, respectively). Neither the percentage of activated CD8+T cell nor very low-level viraemia were found to be associated with iIR. In the multivariate analysis, nadir of CD4+T cell count and percentage of Tregs were the only two parameters independently associated with iIR (OR=2.339, p=0.001, and OR=0.803, p=0.041, respectively). CONCLUSIONS: We present here the largest study investigating simultaneously immune response to long-term HAART, immune activation of CD4+ and CD8+ T cells, Tregs percentages and very low-level viraemia. Our results highlight the importance of Tregs in CD4 homeostasis. This aspect should now be prospectively explored in a large cohort of patients.

Low-level viremia is associated with non-B subtypes in patients infected with HIV with virological success following HAART introduction.

This prospective study aimed to determine factors associated with detection of very low-level viremia in patients infected with HIV-1 with virological success following HAART introduction. Fifty-seven patients, mostly (n = 51, 89%) treated with a protease inhibitor-based regimen, were included and followed for 2 years. Viral loads were monitored by Abbott m2000 RealTime HIV-1. Patients were classified as (i) HIV-RNA-negative if viral loads remained strictly undetectable (0 copies/ml), or (ii) HIV-RNA-positive if at least one HIV-1 RNA could be detected in 1-49 copies/ml during follow-up. At month 24, 44 patients (77%) were in the HIV-RNA-positive group, whereas 13 (23%) remained without very low-level viremia. Univariate analysis, Kaplan-Meier curves and the Cox proportional hazard model revealed that B subtype was the only predictor of belonging to the HIV-RNA-negative group (HR 3.98; 95% CI 1.08-14.7). This association needs to be confirmed. Further study of the reservoir and the mechanisms of viral latency according to HIV-subtype will also be necessary to develop new therapeutic strategies and eradicate HIV infection.

Standardized one-step real-time reverse transcription-PCR assay for universal detection and quantification of hepatitis delta virus from clinical samples in the presence of a heterologous internal-control RNA.

As for other chronic viral diseases, quantification of hepatitis delta virus (HDV) loads may be useful for patient management. We describe a one-step quantitative reverse transcription-PCR assay that is reliable and automatable and meets the regulatory authorities' standards for accurate quantification of the major HDV genotypes. It includes an internal control and uses in vitro-transcribed RNAs as standards. Its linearity range is 500 to 1.7 × 10(11) copies/ml, its sensitivity is around 150 copies/ml, its repeatability is around 15%, and its reproducibility is below 0.25 log(10) copies/ml.

Discordance in HIV-1 co-receptor use prediction by different genotypic algorithms and phenotype assay: intermediate profile in relation to concordant predictions.

Concordant and discordant genotypic predictions of HIV-1 co-receptor tropism were analyzed. V3 region was sequenced from plasma samples of patients screened for R5 tropism by the Trofile® assay, before CCR5 antagonist prescription. Ten tools including geno2pheno, PSSM, an "11/25" and "net charge" rule, and other published algorithms were used. Patients were grouped according to concordance or discordance between tools and Trofile® result. Trofile® tropism reports from 50 patient samples were R5 in 38 and Dual/Mixed (DM) in 12. Prediction with the genotypic tools were concordant for 23 R5 samples, and discordant for the 15 other ones. From Trofile® DM strains were concordant in 6 and discordant in 6. V3 sequences were not clearly distinct between R5 and DM strains, except a greater diversity in the later. Discordances were found with any tool or combination of them, so that no one can be proposed as better than the others. Predictive values of each algorithm were similar and rather good (efficacy ranged from 74% to 84%), but the rate of non-confirmed prediction is greater when compelling the results of all tools with each individual sample. The mean of quantitative values obtained with one tool when another tool give the opposite prediction were different from those obtained when all tools agree with that prediction. The two discordant groups were often not distinguishable from each other. These results suggest that viruses giving discordant prediction with bioinformatic tools could be functionally distinct and/or in a different evolutionary state compared to those with concordant prediction.

HIV-1 load comparison using four commercial real-time assays.

The HIV-1 RNA viral load is commonly used for the monitoring of disease progression and antiretroviral treatment of HIV-1-infected patients. Since the misestimating of values could lead to inappropriate therapeutical management, the comparative performances, especially the ability to span the genetic diversity of HIV-1, of available automated real-time assays need to be evaluated. We conducted a prospective study with 74 consenting patients enrolled between March 2007 and November 2008. A blood sample was obtained at the time of diagnosis of HIV seropositivity and blindly tested for HIV-1 RNA by at least 4 commercial tests: the Abbott m2000 RealTime HIV-1, bioMérieux NucliSens EasyQ HIV-1, version 1.2 (v1.2), and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) v1.0 and v2.0 assays. The means of difference were null between CAP/CTM v2.0 and Abbott for CRF02_AG subtypes but positive in favor of CAP/CTM v2.0 for genotype B and negative in favor of NucliSens for all genotypes. The standard deviation (SD) of difference ranged from 0.3 to 0.59, depending on the considered couples of assays. Reliabilities of these four tests, appreciated by the standard deviation of difference between the measurement and the estimated "true" viral load and by the coefficient of reliability, were significantly different (P < 10(-4)) among each other. Significant differences were also observed within each group of HIV-1 genotype. The global disparity was higher for CRF02_AG than for B subtypes. This study indicates a risk of viral load misestimating or discrepancies between techniques, depending on the HIV-1 subtype, and speaks in favor of using the same assay for the monitoring of HIV-1-infected patients.

Morphological characterization and fusion properties of triglyceride-rich lipoproteins obtained from cells transduced with hepatitis C virus glycoproteins.

The density of hepatitis C virus (HCV) particles circulating in the blood of chronically infected patients and of cell-culture produced HCV is heterogeneous. Specific infectivity and fusion of low density particles are higher than those of high density particles. We recently characterized hybrid particles produced by Caco-2 colon or Huh-7.5 liver cells transduced with HCV E1 and E2 envelope glycoproteins. Caco-2-derived particles, called empty lipo-viral particles (eLVP), are composed of triglyceride-rich lipoproteins positive for apolipoproteins B (i.e. apoB100 and apoB48) and contain HCV E1 and E2. Here we aimed at characterizing the morphology and in vitro fusion properties of eLVP using electron microscopy and fluorescence spectroscopy. They displayed the aspect of beta-lipoproteins, and immunogold labeling confirmed the presence of apoB and HCV E1 and E2 at their surface. These particles are able to fuse with lipid bilayers (liposomes) in a fusion process leading to the coalescence of internal contents of triglyceride-rich lipoproteins particles and liposomes. Fusion was pH-dependent and could be inhibited by either Z-fFG, a peptide known to inhibit viral fusion, or by monoclonal antibodies directed against HCV E2 or the apolipoprotein moiety of the hybrid particle. Interestingly, particles derived from Huh-7.5 cells failed to display equivalent efficient fusion. Optimal fusion activity is, thus, observed when HCV envelope proteins are associated to apoB-positive hybrid particles. Our results, therefore, point to a crucial role of the E1 and E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of eLVP.

Secretion of hepatitis C virus envelope glycoproteins depends on assembly of apolipoprotein B positive lipoproteins.

The density of circulating hepatitis C virus (HCV) particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL) likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1-E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.

Transactivation of the hepatitis B virus core promoter by the nuclear receptor FXRalpha.

Hepatitis B virus (HBV) core promoter activity is positively and negatively regulated by nuclear receptors, a superfamily of ligand-activated transcription factors, via cis-acting sequences located in the viral genome. In this study, we investigated the role of farnesoid X receptor alpha (FXRalpha) in modulating transcription from the HBV core promoter. FXRalpha is a liver-enriched nuclear receptor activated by bile acids recognizing hormone response elements by forming heterodimers with retinoid X receptor alpha (RXRalpha). Electrophoretic mobility shift assays demonstrated that FXRalpha-RXRalpha heterodimers can bind two motifs on the HBV enhancer II and core promoter regions, presenting high homology to the consensus (AGGTCA) inverted repeat FXRalpha response elements. In transient transfection of the human hepatoma cell line Huh-7, bile acids enhanced the activity of a luciferase reporter containing the HBV enhancer II and core promoter sequences through FXRalpha. Moreover, using a greater-than-genome-length HBV construct, we showed that FXRalpha also increased synthesis of the viral pregenomic RNA and DNA replication intermediates. The data strongly suggest that FXRalpha is another member of the nuclear receptor superfamily implicated in the regulation of HBV core promoter activity and that bile acids could play an important role in the natural history of HBV infection.

Transmission of low-density hepatitis C viral particles during sexually transmitted acute resolving infection.

Hepatitis C viruses in the blood of chronically infected patients are heterogeneous in density with the presence of lipoprotein associated viral particles of lower density than conventional virions. If low-density viral particles have been shown to be infectious in animal models it is currently not known whether these particles display the same infectivity for humans. In a case of sexually transmitted acute resolving infection, all isolated NS3 sequences from the acute-phase isolate clustered with a single sequence from the chronic carrier isolate, suggesting bottlenecking during transmission. To determine the density of the transmitted viruses, viral quasispecies from fractions with density below and above 1.055 g/ml were isolated and prepared from the plasma of the chronically infected sexual partner. Interestingly, the three closest sequences to the recipient consensus sequence were isolated from the low-density fraction. These data suggest that low-density viral particles are infectious for humans as they are for chimpanzees and that they can be transmitted during sexual intercourse.

Enhancement of genotype 1 hepatitis C virus replication by bile acids through FXR.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infected patients with high serum levels of bile acids (BAs) usually fail to respond to antiviral therapy. Besides, BAs are essential factors for replication of the porcine enteric calicivirus by inhibiting interferon signaling. The role of BAs on HCV RNA replication was thus assessed. METHODS: BAs and other compounds were tested using an HCV-replication model containing a luciferase reporter gene. RESULTS: BAs, especially chenodeoxycholate and deoxycholate, up-regulated genotype 1 HCV RNA replication by more than tenfold. Only free but not conjugated BAs were active, suggesting that their effect was mediated by a nuclear receptor. Only farnesoid X receptor (FXR) ligands stimulated HCV replication while FXR silencing and FXR antagonism by guggulsterone blocked the up-regulation induced by BAs. Furthermore, guggulsterone alone inhibited basal level of HCV replication by tenfold. Modulation of HCV replication by FXR ligands occurred in the same proportion in presence or absence of type I interferon, suggesting a mechanism of action independent of this control of viral replication. However, BAs or guggulsterone did not affect replication of genotype 2a-JFH1. CONCLUSIONS: Exposure to routinely measured concentrations of BAs increases HCV replication by a novel mechanism involving activation of the nuclear receptor FXR.

Variants with different mutation patterns persist in the quasispecies of enfuvirtide-resistant HIV-1 population during and after treatment in vivo.

BACKGROUND: Genotypic and phenotypic resistance in 11 HIV-1-infected patients receiving enfuvirtide (ENF), as part of a salvage regimen, has been evaluated. METHODS: Resistance mutations were detected by sequencing the gp41 ectodomain from plasma samples. During treatment, longitudinal samples from 1 patient were sequenced after limiting dilution of complementary DNA to isolate single genomes. Phenotypic resistance was evaluated with a new recombinant virus assay (PHENOSCRIPT; VIRalliance, Paris, France), allowing the determination of coreceptor use. RESULTS: All patients experienced ENF failure. One to 4 mutations in the 36-to-45 gp41 region appeared during ENF therapy in all patients and disappeared after ENF removal. Mixtures of wild type and mutants unexpectedly persisted under ENF treatment, however, despite continued replication, leading to discordant results between genotypic and phenotypic data. Sequencing of isolated genomes from 1 patient confirmed that a wild-type first heptad repeat region (HR1) region was still present at the end of therapy. Several mutated variants coexisted at different time points, despite a tendency toward quasispecies reduction with time. CONCLUSION: Individual variability of the mutation pattern and persistence of strains without mutation in the region mainly targeted by ENF resistance probably reflect the fact that resistance to ENF may rely on regions of gp41 or gp120 other than residues 36 to 45.