Infectious Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Virus in Symptomatic Coronavirus Disease 2019 (COVID-19) Outpatients: Host, Disease, and Viral Correlates.

BACKGROUND: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectious virus isolation in outpatients with coronavirus disease 2019 (COVID-19) has been associated with viral RNA levels and symptom duration, little is known about the host, disease, and viral determinants of infectious virus detection. METHODS: COVID-19 adult outpatients were enrolled within 7 days of symptom onset. Clinical symptoms were recorded via patient diary. Nasopharyngeal swabs were collected to quantitate SARS-CoV-2 RNA by reverse transcriptase polymerase chain reaction and for infectious virus isolation in Vero E6-cells. SARS-CoV-2 antibodies were measured in serum using a validated ELISA assay. RESULTS: Among 204 participants with mild-to-moderate symptomatic COVID-19, the median nasopharyngeal viral RNA was 6.5 (interquartile range [IQR] 4.7-7.6 log10 copies/mL), and 26% had detectable SARS-CoV-2 antibodies (immunoglobulin (Ig)A, IgM, IgG, and/or total Ig) at baseline. Infectious virus was recovered in 7% of participants with SARS-CoV-2 antibodies compared to 58% of participants without antibodies (prevalence ratio [PR] = 0.12, 95% confidence interval [CI]: .04, .36; P = .00016). Infectious virus isolation was also associated with higher levels of viral RNA (mean RNA difference +2.6 log10, 95% CI: 2.2, 3.0; P < .0001) and fewer days since symptom onset (PR = 0.79, 95% CI: .71, .88 per day; P < .0001). CONCLUSIONS: The presence of SARS-CoV-2 antibodies is strongly associated with clearance of infectious virus. Seropositivity and viral RNA levels are likely more reliable markers of infectious virus clearance than subjective measure of COVID-19 symptom duration. Virus-targeted treatment and prevention strategies should be administered as early as possible and ideally before seroconversion. CLINICAL TRIALS REGISTRATION: NCT04405570.

A Phase 1/2 Randomized, Placebo-Controlled Trial of Romidespin in Persons With HIV-1 on Suppressive Antiretroviral Therapy.

BACKGROUND: Romidepsin (RMD) is a histone deacetylase inhibitor reported to reverse HIV-1 latency. We sought to identify doses of RMD that were safe and induced HIV-1 expression. METHODS: Enrollees had HIV-1 RNA <40 copies/mL on antiretroviral therapy. Measurements included RMD levels, plasma viremia by single-copy HIV-1 RNA assay, HIV-1 DNA, cell-associated unspliced HIV-1 RNA (CA-RNA), acetylation of histone H3-lysine-9 (H3K9ac+), and phosphorylation of transcription factor P-TEFb. Wilcoxon tests were used for comparison. RESULTS: In the single-dose cohorts 1-3, 43 participants enrolled (36 participants 0.5, 2, 5 mg/m 2 RMD; 7 placebo) and 16 enrolled in the multidose cohort 4 (13 participants 5 mg/m 2 RMD; 3 placebo). One grade 3 event (neutropenia) was possibly treatment related. No significant changes in viremia were observed in cohorts 1-4 compared to placebo. In cohort 4, pharmacodynamic effects of RMD were reduced proportions of CD4+ T cells 24 hours after infusions 2-4 (median, -3.5% to -4.5%) vs placebo (median, 0.5% to 1%; P ≤ .022), and increased H3K9ac+ and phosphorylated P-TEFb in CD4 + T cells vs placebo (P ≤ .02). CONCLUSIONS: RMD infusions were safe but did not increase plasma viremia or unspliced CA-RNA despite pharmacodynamic effects on CD4 + T cells. CLINICAL TRIALS REGISTRATION: NCT01933594.

Novel Criteria for Diagnosing Acute and Early Human Immunodeficiency Virus Infection in a Multinational Study of Early Antiretroviral Therapy Initiation.

BACKGROUND: Antiretroviral therapy (ART) initiation during acute and early human immunodeficiency virus infection (AEHI) limits HIV reservoir formation and may facilitate post-ART control but is logistically challenging. We evaluated the performance of AEHI diagnostic criteria from a prospective study of early ART initiation. METHODS: AIDS Clinical Trials Group A 5354 enrolled adults at 30 sites in the Americas, Africa, and Asia who met any 1 of 6 criteria based on combinations of results of HIV RNA, HIV antibody, Western blot or Geenius assay, and/or the signal-to-cutoff (S/CO) ratio of the ARCHITECT HIV Ag/Ab Combo or GS HIV Combo Ag/Ab EIA. HIV status and Fiebig stage were confirmed by centralized testing. RESULTS: From 2017 through 2019, 195 participants were enrolled with median age of 27 years (interquartile range, 23-39). Thirty (15.4%) were female. ART was started by 171 (87.7%) on the day of enrollment and 24 (12.3%) the next day. AEHI was confirmed in 188 (96.4%) participants after centralized testing, 4 (2.0%) participants were found to have chronic infection, and 3 (1.5%) found not to have HIV discontinued ART and were withdrawn. Retrospectively, a nonreactive or indeterminate HIV antibody on the Geenius assay combined with ARCHITECT S/CO ≥10 correctly identified 99 of 122 (81.2%) Fiebig II-IV AEHI cases with no false-positive results. CONCLUSIONS: Novel AEHI criteria that incorporate ARCHITECT S/CO facilitated rapid and efficient ART initiation without waiting for an HIV RNA result. These criteria may facilitate AEHI diagnosis, staging, and immediate ART initiation in future research studies and clinical practice. CLINICAL TRIALS REGISTRATION: NCT02859558.

Subclinical Genital Herpes Shedding in HIV/Herpes Simplex Virus 2-Coinfected Women during Antiretroviral Therapy Is Associated with an Increase in HIV Tissue Reservoirs and Potentially Promotes HIV Evolution.

Antigen (Ag)-specific immune responses to chronic infections, such as herpes simplex virus type 2 (HSV-2) in HIV/HSV-coinfected persons, may sustain HIV tissue reservoirs by promoting T-cell proliferation but are poorly studied in women on antiretroviral therapy (ART). Mixed anogenital swabs and cervical secretions were self-collected by nine HIV/HSV-2-coinfected women during ART for 28 days to establish subclinical HSV DNA shedding rates and detection of HIV RNA by real-time PCR. Typical herpes lesion site biopsy (TLSB) and cervical biopsy specimens were collected at the end of the daily sampling period. Nucleic acids (NA) isolated from biopsy specimens had HIV quantified and HIV envC2-V5 single-genome amplification (SGA) and T-cell receptor (TCR) repertoires assessed. Women had a median CD4 count of 537 cells/µl (IQR: 483 to 741) at enrollment and HIV plasma viral loads of <40 copies/ml. HSV DNA was detected on 12% of days (IQR: 2 to 25%) from anogenital specimens. Frequent subclinical HSV DNA shedding was associated with increased HIV DNA tissue concentrations and increased divergence from the most recent common ancestor (MRCA), an indicator of HIV replication. Distinct predominant TCR clones were detected in cervical and TLSB specimens in a woman with frequent HSV DNA shedding, with mixing of minor variants between her tissues. In contrast, more limited TCR repertoire mixing was observed in two women with less frequent subclinical HSV DNA shedding. Subclinical HSV shedding in HIV/HSV-coinfected women during ART may sustain HIV tissue reservoirs via Ag exposure or HIV replication. This study provides evidence supporting further study of interventions targeting suppression of Ag-specific immune responses as a component of HIV cure strategies.IMPORTANCE Persons with HIV infection are frequently coinfected with chronic herpesviruses, which periodically replicate and produce viable herpes virions, particularly in anogenital and cervical tissues. Persistent protein expression results in proliferation of CD8+ and CD4+ T cells, and the latter could potentially expand and sustain HIV tissue reservoirs. We found HSV genital shedding rates were positively correlated with HIV DNA concentrations and HIV divergence from ancestral sequences in tissues. Our work suggests that immune responses to common coinfections, such as herpesviruses, may sustain HIV tissue reservoirs during suppressive ART, suggesting future cure strategies should study interventions to suppress replication or reactivation of chronic herpes infections.

Genital Shedding of Human Immunodeficiency Virus Type-1 (HIV) When Antiretroviral Therapy Suppresses HIV Replication in the Plasma.

BACKGROUND: During antiretroviral treatment (ART) with plasma HIV RNA below the limit of quantification, HIV RNA can be detected in genital or rectal secretions, termed discordant shedding (DS). We hypothesized that proliferating cells produce virions without HIV replication. METHODS: ART-naive Peruvians initiating ART were observed for DS over 2 years. HIV env and pol genomes were amplified from DS. Antiretrovirals and cytokines/chemokines concentrations were compared at DS and control time points. RESULTS: Eighty-two participants had ART suppression. DS was detected in 24/82 (29%) participants: 13/253 (5%) cervicovaginal lavages, 20/322 (6%) seminal plasmas, and 6/85 (7%) rectal secretions. HIV RNA in DS specimens was near the limit of quantification and not reproducible. HIV DNA was detected in 6/13 (46%) DS cervicovaginal lavages at low levels. Following DNase treatment, 5/39 DS specimens yielded HIV sequences, all without increased genetic distances. Women with and without DS had similar plasma antiretroviral levels and DS in 1 woman was associated with inflammation. CONCLUSIONS: HIV RNA and DNA sequences and therapeutic antiretroviral plasma levels did not support HIV replication as the cause of DS from the genital tract. Rather, our findings infer that HIV RNA is shed due to proliferation of infected cells with virion production.

A Randomized, Placebo-Controlled Trial of the Quadrivalent Human Papillomavirus Vaccine in Human Immunodeficiency Virus-Infected Adults Aged 27 Years or Older: AIDS Clinical Trials Group Protocol A5298.

Background: Adults living with human immunodeficiency virus (HIV) are at increased risk for anal and oropharyngeal cancer caused by human papillomavirus (HPV). The efficacy of HPV vaccines in this population is unknown. Methods: In this phase 3, double-blind, randomized, controlled trial, we assigned HIV-infected adults aged ≥27 years to the quadrivalent HPV (types 6, 11, 16, 18) vaccine or placebo (1:1) stratified by sex and presence of anal high-grade squamous intraepithelial lesions on biopsy (bHSIL). The primary endpoint was vaccine efficacy against incident persistent anal infection with quadrivalent vaccine types or single detection at the final visit that were not present at baseline. Secondary endpoints included vaccine efficacy for anal bHSIL after week 52, persistent oral HPV infection. Results: A total of 575 participants were randomized. The Data and Safety Monitoring Board stopped the study early due to futility. Vaccine efficacy was 22% (95.1% confidence interval [CI], -31%, 53%) for prevention of persistent anal infection or single detection at the final visit, 0% (95% CI -44%, 31%) for improving bHSIL outcomes and 88% (95.1% CI 2%, 98%) for preventing persistent oral HPV infection, but was 32% (95.1% CI -80%, 74%) for 6-month persistent oral HPV infection or single detection at the final visit. Conclusions: These results do not support HPV vaccination of HIV-infected adults aged ≥27 years to prevent new anal HPV infections or to improve anal HSIL outcomes. However, our data suggest a role for prevention of oral HPV infections, but this finding should be confirmed in future studies. Clinical Trials Registration: NCT01461096.

High Baseline Anal Human Papillomavirus and Abnormal Anal Cytology in a Phase 3 Trial of the Quadrivalent Human Papillomavirus Vaccine in Human Immunodeficiency Virus-Infected Individuals Older Than 26 Years: ACTG 5298.

BACKGROUND: The quadrivalent human papillomavirus (HPV) vaccine (qHPV; types 6, 11, 16, 18) is indicated for men and women aged 9 to 26 years to prevent HPV associated anogenital high-grade squamous intraepithelial lesions (HSIL) and cancer. ACTG 5298 was a randomized placebo controlled Phase 3 study in human immunodeficiency virus (HIV)-infected men who have sex with men, and women of qHPV to prevent persistent anal HPV infection. Baseline data are presented here. METHODS: Human immunodeficiency virus-infected men who have sex with men, and women 27 years or older without previous anogenital or oral cancer were enrolled. Baseline anal cytology, high-resolution anoscopy and collection of anal, oral, and vaginal specimens for HPV genotyping were performed and acceptability assessed. RESULTS: Five hundred seventy-five (575) participants were enrolled (82% men and 18% women). Median age was 47 years. Race/ethnicity was 46% white, 31% black, and 20% Hispanic. Plasma HIV-1 RNA was less than 50 copies/mL in 83% and median CD4 T count was 602 cells/µL. Abnormal anal cytology was detected in 62%, with corresponding HSIL on biopsy (bHSIL) in 33%. Anal HPV 6, 11, 16, and 18 were detected in 25%, 13%, 32%, and 18% of the participants, respectively. Prevalence of 0, 1, 2, 3, and 4 qHPV types was 40%, 38%, 17%, 4%, and 1%, respectively. Oral infection with 1 or more qHPV type was detected in 10% of the participants. Study procedures were generally acceptable. CONCLUSIONS: At study baseline, there was a high prevalence of abnormal anal cytology, bHSIL, and HPV infection. Sixty percent of the participants had anal infection with preventable qHPV types.

Human immunodeficiency viruses appear compartmentalized to the female genital tract in cross-sectional analyses but genital lineages do not persist over time.

BACKGROUND: Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood. METHODS: Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests. RESULTS: In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences. CONCLUSIONS: The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood.

Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile®.

HIV-1 typically infects cells via the CD4 receptor and CCR5 or CXCR4 co-receptors. Maraviroc is a CCR5-specific viral entry inhibitor; knowledge of viral co-receptor specificity is important prior to usage. We developed and validated an economical V3-env Illumina-based assay to detect and quantify the frequency of viruses utilizing each co-receptor. Plasma from 54 HIV+ participants (subtype B) was tested. The viral template cDNA was generated from plasma RNA with unique molecular identifiers (UMIs). The sequences were aligned and collapsed by the UMIs with a custom bioinformatics pipeline. Co-receptor usage, determined by codon analysis and online phenotype predictors PSSM and Geno2pheno, were compared to existing Trofile® data. The cost of V3-UMI was tallied. The sequences interpreted by Geno2pheno using the most conservative cut-off, a 2% false-positive-rate (FPR), predicted CXCR4 usage with the greatest sensitivity (76%) and specificity (100%); PSSM and codon analysis had similar sensitivity and lower specificity. Discordant Trofile® and genotypic results were more common when participants had specimens from different dates analyzed by either assay. V3-UMI reagents cost USD$62/specimen. A batch of ≤20 specimens required 5 h of technical time across 1.5 days. V3-UMI predicts HIV tropism at a sensitivity and specificity similar to those of Trofile®, is relatively inexpensive, and could be performed by most central laboratories. The adoption of V3-UMI could expand HIV drug therapeutic options in lower-resource settings that currently do not have access to phenotypic HIV tropism testing.

Changes in HIV-1 subtypes B and C genital tract RNA in women and men after initiation of antiretroviral therapy.

BACKGROUND: Combination antiretroviral therapy (cART) reduces genital tract human immunodeficiency virus type 1 (HIV-1) load and reduces the risk of sexual transmission, but little is known about the efficacy of cART for decreasing genital tract viral load (GTVL) and differences in sex or HIV-1 subtype. METHODS: HIV-1 RNA from blood plasma, seminal plasma, or cervical wicks was quantified at baseline and at weeks 48 and 96 after entry in a randomized clinical trial of 3 cART regimens. RESULTS: One hundred fifty-eight men and 170 women from 7 countries were studied (men: 55% subtype B and 45% subtype C; women: 24% subtype B and 76% subtype C). Despite similar baseline CD4(+) cell counts and blood plasma viral loads, women with subtype C had the highest GTVL (median, 5.1 log10 copies/mL) compared to women with subtype B and men with subtype C or B (4.0, 4.0, and 3.8 log10 copies/mL, respectively; P < .001). The proportion of participants with a GTVL below the lower limit of quantification (LLQ) at week 48 (90%) and week 96 (90%) was increased compared to baseline (16%; P < .001 at both times). Women were significantly less likely to have GTVL below the LLQ compared to men (84% vs 94% at week 48, P = .006; 84% vs 97% at week 96, P = .002), despite a more sensitive assay for seminal plasma than for cervical wicks. No difference in GTVL response across the 3 cART regimens was detected. CONCLUSIONS: The female genital tract may serve as a reservoir of persistent HIV-1 replication during cART and affect the use of cART to prevent sexual and perinatal transmission of HIV-1.

Emergence of SARS-CoV-2 escape mutations during Bamlanivimab therapy in a phase II randomized clinical trial.

SARS-CoV-2 mutations that cause resistance to monoclonal antibody (mAb) therapy have been reported. However, it remains unclear whether in vivo emergence of SARS-CoV-2 resistance mutations alters viral replication dynamics or therapeutic efficacy in the immune-competent population. As part of the ACTIV-2/A5401 randomized clinical trial (NCT04518410), non-hospitalized participants with symptomatic SARS-CoV-2 infection were given bamlanivimab (700 mg or 7,000 mg) or placebo treatment. Here¸ we report that treatment-emergent resistance mutations [detected through targeted Spike (S) gene next-generation sequencing] were significantly more likely to be detected after bamlanivimab 700 mg treatment compared with the placebo group (7% of 111 vs 0% of 112 participants, P = 0.003). No treatment-emergent resistance mutations among the 48 participants who received 7,000 mg bamlanivimab were recorded. Participants in which emerging mAb resistant virus mutations were identified showed significantly higher pretreatment nasopharyngeal and anterior nasal viral loads. Daily respiratory tract viral sampling through study day 14 showed the dynamic nature of in vivo SARS-CoV-2 infection and indicated a rapid and sustained viral rebound after the emergence of resistance mutations. Participants with emerging bamlanivimab resistance often accumulated additional polymorphisms found in current variants of concern/interest that are associated with immune escape. These results highlight the potential for rapid emergence of resistance during mAb monotherapy treatment that results in prolonged high-level respiratory tract viral loads. Assessment of viral resistance should be prioritized during the development and clinical implementation of antiviral treatments for COVID-19.

Monoclonal antibody treatment drives rapid culture conversion in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs) are among the treatments recommended for high-risk ambulatory persons with coronavirus 2019 (COVID-19). Here, we study viral culture dynamics post-treatment in a subset of participants receiving the mAb bamlanivimab in the ACTIV-2 trial (ClinicalTrials.gov: NCT04518410). Viral load by qPCR and viral culture are performed from anterior nasal swabs collected on study days 0 (day of treatment), 1, 2, 3, and 7. Treatment with mAbs results in rapid clearance of culturable virus. One day after treatment, 0 of 28 (0%) participants receiving mAbs and 16 of 39 (41%) receiving placebo still have culturable virus (p < 0.0001). Recrudescence of culturable virus is detected in three participants with emerging mAb resistance and viral RNA rebound. While further studies are necessary to fully define the relationship between shed culturable virus and transmission, these results raise the possibility that mAbs may offer immediate (household) and public-health benefits by reducing onward transmission.

Post-prostatic massage fluid/urine as an alternative to semen for studying male genitourinary HIV-1 shedding.

OBJECTIVES: Genitourinary tract samples are required to investigate male HIV-1 infectivity. Because semen collection is often impractical, the acceptability, feasibility and validity of post-prostatic massage fluid/urine (post-PMF/U) was evaluated for studying male genitourinary HIV-1 shedding. METHODS: HIV-1-seropositive men were evaluated after 48 h of sexual abstinence. At each visit, a clinician performed prostatic massage, then post-PMF/U and blood were collected. Participants provided semen specimens 1 week later. An audio computer-assisted self-interview (ACASI) administered after each specimen collection evaluated acceptability, adherence to instructions and recent genitourinary symptoms. HIV-1 RNA was quantified using a real-time PCR assay. Detection and quantitation of HIV-1 RNA and stability over visits were compared for semen, post-PMF/U and blood. RESULTS: Post-PMF/U was successfully obtained at 106 visits (64%) and semen at 136 visits (81%, p<0.001). In ACASI, discomfort was rated higher for post-PMF/U collection (p=0.003), but there was no significant difference in acceptability. Detection of HIV-1 RNA in post-PMF/U was associated with detection in semen (p=0.02). Semen and post-PMF/U HIV-1-RNA levels were correlated (ρ=0.657, p<0.001). Concordance of results at repeat visits was 78.9% for post-PMF/U (κ=0.519, p=0.02) and 89.5% for both blood and semen (κ=0.774, p=0.001). CONCLUSIONS: Although semen collections were more successful, both post-PMF/U and semen collections were acceptable to many participants. HIV-1 RNA detection and levels were closely associated in semen and post-PMF/U, and results were relatively stable across visits. To assess male HIV-1 infectivity, post-PMF/U may represent a valid alternative when semen cannot be obtained.

HIV-1 Nucleic Acids Identify Rectal HIV Exposures in Self-Collected Rectal Swabs, Whereas Y-Chromosome Single Tandem Repeat Mixtures Are Not Reliable Biomarkers of Condomless Receptive Anal Intercourse.

BACKGROUND: To focus interventions, biomarkers of HIV-1 exposure could help in identifying subpopulations at highest risk of acquisition. We assessed whether Y-chromosome single tandem repeat (YSTR) mixtures obtained from rectal swabs could serve as a biomarker of condomless receptive anal intercourse (CRAI) among men who have sex with men and transgender women and evaluated the feasibility of detecting HIV-1 virions to assess exposures. METHODS: Twenty-nine sexually active HIV-seronegative men who have sex with men and one transgender woman from New York City answered on-site and mobile app sexual behavior questionnaires. They were randomized to collecting self-administered rectal swabs every morning or after receptive anal intercourse (RAI). YSTR profiles were assessed from blood sample and swabs; HIV-1 exposure was measured by conducting quantitative polymerase chain reaction in swabs. RESULTS: After 2 months, the daily mobile survey had 135%-201% more instances of anal sex acts and 170%-193% more RAI than on-site surveys. Daily mobile reporting had 11%-35% less CRAI events than those reported on-site (Pdaily = 0.001; Pper-sex = 0.047). The daily swabbing arm reported less RAI (P < 0.001) and CRAI (P < 0.038) and had 2.95 lower odds of detecting YSTR mixtures (P = 0.021) than the per-sex-event arm. Surprisingly, YSTR detection was not significantly modified by report of bowel movements and lubricant, enema, or condom use. No participant became HIV-1 infected, yet HIV-1 total nucleic acids were detected in 6 independent episodes of CRAI in 2 participants taking pre-exposure prophylaxis. CONCLUSIONS: YSTR mixtures demonstrated 80% specificity but only 30% sensitivity as a biomarker of CRAI in self-collected rectal swabs. However, detection of HIV-1 exposures in self-collected swabs may help in identifying those needing further HIV risk reduction strategies.

Poststudy Point-of-Care Oral Fluid Testing in Human Immunodeficiency Virus-1 Vaccinees.

BACKGROUND: Experimental human immunodeficiency virus (HIV)-1 vaccines frequently elicit antibodies against HIV-1 that may react with commonly used HIV diagnostic tests, a phenomenon known as vaccine-induced seropositivity/seroreactivity (VISP/VISR). We sought to determine, under clinic conditions, whether a patient-controlled HIV test, OraQuick ADVANCE Rapid HIV-1/2 Antibody Test, detected HIV-1 vaccine-induced antibodies. METHODS: Plasma assessment of HIV-1 cross-reactivity was examined in end-of-study samples from 57 healthy, HIV-uninfected participants who received a candidate vaccine that has entered Phase 2B and 3 testing. We also screened 120 healthy, HIV-uninfected, unblinded HIV-1 vaccine participants with VISP/VISR for an assessment using saliva. These participants came from 21 different parent vaccine protocols representing 17 different vaccine regimens, all of which contained an HIV-1 envelope immunogen. OraQuick ADVANCE was compared with results from concurrent blood samples using a series of commercial HIV screening immunoassays. RESULTS: Fifty-seven unique participant plasma samples were assayed in vitro, and only 1 (1.8%) was reactive by OraQuick ADVANCE. None of the 120 clinic participants (0%; 95% confidence interval, 0% to 3.7%) tested positive by OraQuick ADVANCE, and all were confirmed to be uninfected by HIV-1 viral ribonucleic acid testing. One hundred eighteen of the 120 (98.3%) participants had a reactive HIV test for VISP/VISR: 77 (64%) had at least 1 reactive fourth-generation HIV-1 diagnostic test (P < .0001 vs no reactive OraQuick ADVANCE results), and 41 (34%) only had a reactive test by the less specific third-generation Abbott Prism assay. CONCLUSIONS: These data suggest that this widely available patient-controlled test has limited reactivity to HIV-1 antibodies elicited by these candidate HIV-1 vaccines.

Infectious SARS-CoV-2 Virus in Symptomatic COVID-19 Outpatients: Host, Disease, and Viral Correlates.

BACKGROUND: While SARS-CoV-2 infectious virus isolation in outpatients with COVID-19 has been associated with viral RNA levels and symptom duration, little is known about the host, disease and viral determinants of infectious virus detection. METHODS: COVID-19 adult outpatients were enrolled within 7 days of symptom onset. Clinical symptoms were recorded via patient diary. Nasopharyngeal swabs were collected to quantitate SARS-CoV-2 RNA by reverse transcriptase polymerase chain reaction and for infectious virus isolation in Vero E6-cells. SARS-CoV-2 antibodies were measured in serum using a validated ELISA assay. RESULTS: Among 204 participants with mild-to-moderate symptomatic COVID19, the median nasopharyngeal viral RNA was 6.5 (IQR 4.7-7.6 log10 copies/mL), and 26% had detectable SARS-CoV-2 antibodies (IgA, IgM, IgG, and/or total Ig) at baseline. Infectious virus was recovered in 7% of participants with SARS-CoV-2 antibodies compared to 58% of participants without antibodies (probability ratio (PR)=0.12, 95% CI: 0.04, 0.36; p=0.00016). Infectious virus isolation was also associated with higher levels of viral RNA (mean RNA difference +2.6 log10, 95% CI: 2.2, 3.0; p<0.0001) and fewer days since symptom onset (PR=0.79, 95% CI: 0.71, 0.88 per day; p<0.0001). CONCLUSIONS: The presence of SARS-CoV-2 antibodies is strongly associated with clearance of infectious virus isolation. Seropositivity and viral RNA levels are likely more reliable markers of infectious virus clearance than subjective measure of COVID-19 symptom duration. Virus-targeted treatment and prevention strategies should be administered as early as possible and ideally before seroconversion. CLINICALTRIALSGOV IDENTIFIER: NCT04405570.

Monoclonal antibody treatment drives rapid culture conversion in SARS-CoV-2 infection.

Monoclonal antibodies (mAbs) are the treatment of choice for high-risk ambulatory persons with mild to moderate COVID-19. We studied viral culture dynamics post-treatment in a subset of participants receiving the mAb bamlanivimab in the ACTIV-2 trial. Viral load by qPCR and viral culture were performed from anterior nasal swabs collected on study days 0 (day of treatment), 1, 2, 3, and 7. Treatment with mAb resulted in rapid clearance of culturable virus in participants without treatment-emergent resistance. One day after treatment, 0 of 28 (0%) participants receiving mAb and 16 of 39 (41%) receiving placebo still had culturable virus (p <0.0001); nasal viral loads were only modestly lower in the mAb-treated group at days 2 and 3. Recrudescence of culturable virus was detected in three participants with emerging mAb resistance and viral load rebound. The rapid reduction in shedding of viable SARS-CoV-2 after mAb treatment highlights the potential role of mAbs in preventing disease transmission.

Emergence of SARS-CoV-2 Resistance with Monoclonal Antibody Therapy.

Resistance mutations to monoclonal antibody (mAb) therapy has been reported, but in the non-immunosuppressed population, it is unclear if in vivo emergence of SARS-CoV-2 resistance mutations alters either viral replication dynamics or therapeutic efficacy. In ACTIV-2/A5401, non-hospitalized participants with symptomatic SARS-CoV-2 infection were randomized to bamlanivimab (700mg or 7000mg) or placebo. Treatment-emergent resistance mutations were significantly more likely detected after bamlanivimab 700mg treatment than placebo (7% of 111 vs 0% of 112 participants, P=0.003). There were no treatment-emergent resistance mutations among the 48 participants who received bamlanivimab 7000mg. Participants with emerging mAb resistant virus had significantly higher pre-treatment nasopharyngeal and anterior nasal viral load. Intensive respiratory tract viral sampling revealed the dynamic nature of SARS-CoV-2 evolution, with evidence of rapid and sustained viral rebound after emergence of resistance mutations, and worsened symptom severity. Participants with emerging bamlanivimab resistance often accumulated additional polymorphisms found in current variants of concern/interest and associated with immune escape. These results highlight the potential for rapid emergence of resistance during mAb monotherapy treatment, resulting in prolonged high level respiratory tract viral loads and clinical worsening. Careful virologic assessment should be prioritized during the development and clinical implementation of antiviral treatments for COVID-19.

Monotypic human immunodeficiency virus type 1 genotypes across the uterine cervix and in blood suggest proliferation of cells with provirus.

Understanding the dynamics and spread of human immunodeficiency virus type 1 (HIV-1) within the body, including within the female genital tract with its central role in heterosexual and peripartum transmission, has important implications for treatment and vaccine development. To study HIV-1 populations within tissues, we compared viruses from across the cervix to those in peripheral blood mononuclear cells (PBMC) during effective and failing antiretroviral therapy (ART) and in patients not receiving ART. Single-genome sequences of the C2-V5 region of HIV-1 env were derived from PBMC and three cervical biopsies per subject. Maximum-likelihood phylogenies were evaluated for differences in genetic diversity and compartmentalization within and between cervical biopsies and PBMC. All subjects had one or more clades with genetically identical HIV-1 env sequences derived from single-genome sequencing. These sequences were from noncontiguous cervical biopsies or from the cervix and circulating PBMC in seven of eight subjects. Compartmentalization of virus between genital tract and blood was observed by statistical methods and tree topologies in six of eight subjects, and potential genital lineages were observed in two of eight subjects. The detection of monotypic sequences across the cervix and blood, especially during effective ART, suggests that cells with provirus undergo clonal expansion. Compartmentalization of viruses within the cervix appears in part due to viruses homing to and/or expanding within the cervix and is rarely due to unique viruses evolving within the genital tract. Further studies are warranted to investigate mechanisms producing monotypic viruses across tissues and, importantly, to determine whether the proliferation of cells with provirus sustain HIV-1 persistence in spite of effective ART.

Vaccine-induced HIV seropositivity/reactivity in noninfected HIV vaccine recipients.

CONTEXT: Induction of protective anti-human immunodeficiency virus (HIV) immune responses is the goal of an HIV vaccine. However, this may cause a reactive result in routine HIV testing in the absence of HIV infection. OBJECTIVE: To evaluate the frequency of vaccine-induced seropositivity/reactivity (VISP) in HIV vaccine trial participants. DESIGN, SETTING, AND PARTICIPANTS: Three common US Food and Drug Administration-approved enzyme immunoassay (EIA) HIV antibody kits were used to determine VISP, and a routine diagnostic HIV algorithm was used to evaluate VISP frequency in healthy, HIV-seronegative adults who completed phase 1 (n = 25) and phase 2a (n = 2) vaccine trials conducted from 2000-2010 in the United States, South America, Thailand, and Africa. MAIN OUTCOME MEASURE: Vaccine-induced seropositivity/reactivity, defined as reactive on 1 or more EIA tests and either Western blot-negative or Western blot-indeterminate/atypical positive (profile consistent with vaccine product) and HIV-1-negative by nucleic acid testing. RESULTS: Among 2176 participants free of HIV infection who received a vaccine product, 908 (41.7%; 95% confidence interval [CI], 39.6%-43.8%) had VISP, but the occurrence of VISP varied substantially across different HIV vaccine product types: 399 of 460 (86.7%; 95% CI, 83.3%-89.7%) adenovirus 5 product recipients, 295 of 552 (53.4%; 95% CI, 49.2%-57.7%) recipients of poxvirus alone or as a boost, and 35 of 555 (6.3%; 95% CI, 4.4%-8.7%) of DNA-alone product recipients developed VISP. Overall, the highest proportion of VISP (891/2176 tested [40.9%]) occurred with the HIV 1/2 (rDNA) EIA kit compared with the rLAV EIA (150/700 tested [21.4%]), HIV-1 Plus O Microelisa System (193/1309 tested [14.7%]), and HIV 1/2 Peptide and HIV 1/2 Plus O (189/2150 tested [8.8%]) kits. Only 17 of the 908 participants (1.9%) with VISP tested nonreactive using the HIV 1/2 (rDNA) kit. All recipients of a glycoprotein 140 vaccine (n = 70) had VISP, with 94.3% testing reactive with all 3 EIA kits tested. Among 901 participants with VISP and a Western blot result, 92 (10.2%) had a positive Western blot result (displaying an atypical pattern consistent with vaccine product), and 592 (65.7%) had an indeterminate result. Only 8 participants with VISP received a vaccine not containing an envelope insert. CONCLUSIONS: The induction of VISP in HIV vaccine recipients is common, especially with vaccines containing both the HIV-1 envelope and group-specific core antigen gene proteins. Development and detection of VISP appear to be associated with the immunogenicity of the vaccine and the EIA assay used.