Impact of SARS-CoV-2 Resistance to Antiviral Monoclonal Antibody Therapy on Neutralizing Antibody Response.

Background: Anti-SARS-CoV-2 monoclonal antibodies (mAbs) have played a key role as an anti-viral against SARS-CoV-2, but there is a potential for resistance to develop. The interplay between host antibody responses and the development of monoclonal antibody (mAb) resistance is a critical area of investigation. In this study, we assessed host neutralizing antibody (nAb) responses against both ancestral virus and those with treatment-emergent E484K bamlanivimab resistance mutations. Methods: Study participants were enrolled in the ACTIV-2/Advancing Clinical Therapeutics Globally (ACTG) A5401 phase 2 randomized, placebo-controlled trial of bamlanivimab 700 mg mAb therapy (NCT04518410). Anterior nasal and nasopharyngeal swabs were collected for SARS-CoV-2 RNA testing and S gene next-generation sequencing to identify the E484K bamlanivimab resistance mutation. Serum nAb titers were assessed by pseudovirus neutralization assays. Results: Higher baseline (pre-treatment) nAb titers against either ancestral or E484K virus was associated with lower baseline viral load. Participants with emerging resistance had low levels of nAb titers against either ancestral or E484K nAb at the time of study entry. Participants with emergent E484K resistance developed significantly higher levels of E484K-specific nAb titers compared to mAb-treated individuals who did not develop resistance. All participants who developed the E484K mAb resistance mutation were eventually able to clear the virus. Conclusion: Emerging drug resistance after SARS-CoV-2-specific mAb therapy led to a heightened host neutralizing antibody response to the mAb-resistant variant that was associated with eventual viral clearance. This demonstrates the interplay between the antiviral treatment-directed viral evolution and subsequent host immune response in viral clearance.

First Case of HIV Seroconversion With Integrase Resistance Mutations on Long-Acting Cabotegravir for Prevention in Routine Care.

Background: Long-acting cabotegravir (CAB-LA) is highly effective for HIV prevention, but delayed HIV diagnoses and integrase strand transfer inhibitor (INSTI) resistance were observed in trials. We report the first case in routine clinical care of HIV infection on CAB-LA with INSTI resistance. Methods: The SeroPrEP study enrolls individuals in the United States who acquire HIV on pre-exposure prophylaxis modalities to assess diagnostics, antiretroviral (ARV) drug levels, resistance, and treatment outcomes. Resistance mutations in full-length HIV-1 integrase were identified by single-genome sequencing (SGS). Cabotegravir concentrations in plasma and hair segments were measured by liquid chromatography-tandem mass spectrometry. Results: A 23-year-old gender-nonbinary person, male at birth, restarted CAB-LA 6 months after discontinuation due to losing insurance. Prior to restart, HIV-1 RNA was not detected, but 20 days elapsed before CAB-LA injection. After the second CAB-LA injection, HIV antigen/antibody returned reactive (HIV-1 RNA 451 copies/mL). SGS of plasma HIV-1 RNA identified INSTI mutation Q148R in 2/24 sequences 2 days postdiagnosis; commercial genotype failed amplification. Cabotegravir hair concentration was 0.190 ng/mg 2 weeks prediagnosis; plasma cabotegravir was high (3.37 µg/mL; ∼20× PA-IC90) 14 days postdiagnosis. Viral suppression was maintained for 6 months on darunavir/cobicistat/emtricitabine/tenofovir alafenamide, then switched to doravirine + emtricitabine/tenofovir alafenamide due to nausea. Conclusions: In this first case of HIV infection on CAB-LA with INSTI resistance in routine care, cabotegravir resistance was detected only with a sensitive research assay. Accelerated pathways to minimize time between HIV testing and CAB-LA initiation are needed to optimize acute HIV detection and mitigate resistance risk. Sustained product access regardless of insurance is imperative to reduce HIV infections on CAB-LA.

Comparison Study of the Bio-Plex and Meso Scale Multiplexed SARS-CoV-2 Serology Assays Reveals Evidence of Diminished Host Antibody Responses to SARS-CoV-2 after Monoclonal Antibody Treatment.

Background: Assessing the breadth and duration of antigen-specific binding antibodies provides valuable information for evaluating interventions to treat or prevent SARS-CoV-2 infection. Multiplex immunoassays are a convenient method for rapid measurement of antibody responses but can sometimes provide discordant results, and antibody positive percent agreement for COVID-19 diagnosis can vary depending on assay type, disease severity, and population sampled. Therefore, we compared two assays marked for research applications, MSD and Bio-Plex Pro, to evaluate qualitative interpretation of serostatus and quantitative detection of antibodies of varying isotypes (IgG, IgM, and IgA) against receptor binding domain (RBD) and nucleocapsid (N) antigens. Methods: Specimens from ACTIV-2/A5401, a placebo-controlled clinical trial of the SARSCoV-2 monoclonal antibody (mAb) bamlanivimab to prevent COVID-19 disease progression, were used to evaluate the concordance of the Bio-Rad Bio-Plex Pro Human SARS-CoV-2 Serology Assay and the Meso Scale Discovery (MSD) V-PLEX COVID-19 Panel 1 serology assay in detecting and quantifying IgG, IgA, and IgM binding anti-SARS-CoV-2 antibody responses against the RBD and N antigens. Data were disaggregated by study arm, bamlanivimab dose, days post-enrollment, and presence of emerging resistance. Results: We observed 90.5% (412 of 455 tests) concordance for anti-RBD IgG and 87% (396 of 455) concordance for anti-N IgG in classifying samples as negative or positive based on assay-defined cutoffs. Antibody levels converted to the WHO standard BAU/mL were significantly correlated for all isotypes (IgG, IgM, and IgA) and SARS-CoV-2 antigen targets (RBD and N) tested that were common between the two assays (Spearman r 0.65 to 0.92, P < 0.0001). Both assays uncovered evidence of diminished host-derived IgG immune responses in participants treated with bamlanivimab compared to placebo. Assessment of immune responses in the four individuals treated with the 700 mg of bamlanivimab with emerging mAb resistance demonstrated a stronger anti-N IgG response (MSD) at day 28 (median 2.18 log BAU/mL) compared to participants treated with bamlanivimab who did not develop resistance (median 1.55 log BAU/mL). Conclusions: These data demonstrate the utility in using multiplex immunoassays for characterizing the immune responses with and without treatment in a study population and provide evidence that monoclonal antibody treatment in acute COVID-19 may have a modest negative impact on development of host IgG responses.

Brief Report: HIV Drug Resistance Assessment Among Women Who Seroconverted During the MTN-025/HOPE Open-Label Extension Dapivirine Vaginal Ring Trial.

BACKGROUND: Clinical trials of dapivirine (DPV) vaginal ring have shown it is safe, effective, and desired by women as an HIV prevention option. The risk of drug resistance is a potential concern for DPV ring users who acquire HIV. We conducted a comprehensive resistance evaluation of plasma samples from the women who seroconverted during the Microbicide Trials Network-025/HIV Open-label Prevention Extension (HOPE) study of DPV ring. METHODS: Plasma collected on the visit at which seroconversion was detected was tested by next-generation sequencing with unique molecular identifiers for non-nucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations (DRM) present at ≥1% frequency. Bulk-cloned plasma-derived recombinant HIV was phenotyped in a TZM-bl-based assay for susceptibility to DPV and other NNRTI. HIV-1 RNA was retrospectively quantified in plasma samples collected before HIV seroconversion. RESULTS: Among 38 participants who seroconverted in HOPE, 7 (18%) had NNRTI DRM detected by next-generation sequencing with unique molecular identifiers including A98G, K103N, V106M, E138A, and V179D. Six of 7 samples with NNRTI DRM had <3-fold reduction in susceptibility to DPV. Only 1 sample with K103N and V179I polymorphism had 9-fold reduction in susceptibility to DPV, but this genotype occurred in an individual who did not use DPV ring, likely indicating transmitted resistance. Detection of NNRTI resistance was not higher in individuals who remained on DPV ring >3 months after acquiring HIV infection. CONCLUSIONS: NNRTI resistance among women who seroconverted during HOPE was infrequent and selection of DPV-specific mutations was not detected. DPV ring is considered a safe and effective option for HIV prevention in women.

Rapid Emergence of Potentially Transmissible Severe Acute Respiratory Syndrome Coronavirus 2 With Resistance to Combination Monoclonal Antibody Therapy.

Prolonged coronavirus disease 2019 may generate new viral variants. We report an immunocompromised patient treated with monoclonal antibodies who experienced rebound of viral RNA and emergence of an antibody-resistant (>1000-fold) variant containing 5 mutations in the spike gene. The mutant virus was isolated from respiratory secretions, suggesting the potential for secondary transmission.

Rapid determination of SARS-CoV-2 antibody neutralization titer using Bio-Rad Bio-Plex correlates strongly with pseudovirus-determined neutralization titer.

Accurate and rapid evaluation of SARS-CoV-2 half-maximal neutralizing antibody (nAb) titer (NT50) is an important research tool for measuring nAb responses after prophylaxis or therapeutics for COVID-19 prevention and management. Compared with ACE2-competitive enzyme immunoassays for nAb detection, pseudovirus assays remain low-throughput and labor intensive. A novel application of the Bio-Rad Bio-Plex Pro Human SARS-CoV-2 D614G S1 Variant nAb Assay was used to determine NT50 from COVID-19-vaccinated individuals and showed strong correlation to a laboratory-developed SARS-CoV-2 pseudovirus nAb assay. The Bio-Plex nAb assay could provide a rapid, high-throughput, culture-free method for NT50 determination in sera.

HIV-1 drug resistance among individuals who seroconverted in the ASPIRE dapivirine ring trial.

INTRODUCTION: A potential concern with the use of dapivirine (DPV) for HIV prevention is the selection of a drug-resistant virus that could spread and reduce the effectiveness of non-nucleoside reverse transcriptase (NNRTI)-based first-line antiretroviral therapy. We evaluated HIV-1 seroconversions in MTN-020/ASPIRE for selection of drug resistance and evaluated the genetic basis for observed reductions in susceptibility to DPV. METHODS: MTN-020/ASPIRE was a placebo-controlled, Phase III safety and effectiveness study of DPV ring for HIV-1 prevention conducted at 15 sites in South Africa, Zimbabwe, Malawi and Uganda between 2012 and 2015. Plasma from individuals who seroconverted in ASPIRE was analysed for HIV-1 drug resistance using both population Sanger sequencing and next-generation sequencing (NGS) with unique molecular identifiers to report mutations at ≥1% frequency. DPV susceptibility of plasma-derived recombinant HIV-1 containing bulk-cloned full-length reverse transcriptase sequences from MTN-020/ASPIRE seroconversions was determined in TZM-bl cells. Statistical significance was calculated using the Fisher's exact test. RESULTS: Plasma from all 168 HIV seroconversions were successfully tested by Sanger sequencing; 57 of 71 DPV arm and 82 of 97 placebo (PLB) arm participants had NGS results at 1% sensitivity. Overall, 18/168 (11%) had NNRTI mutations including K101E, K103N/S, V106M, V108I, E138A/G, V179D/I/T and H221Y. Five samples from both arms had low-frequency NNRTI mutations that were not detected by Sanger sequencing. The frequency of NNRTI mutations from the DPV arm (11%) was not different from the PLB arm (10%; p = 0.80). The E138A mutation was detected in both the DPV (3 of 71 [4.2%]) and PLB arm (5 of 97 [5.2%]) and conferred modest reductions in DPV susceptibility in some reverse transcriptase backgrounds but not others. CONCLUSIONS: HIV-1 drug resistance including NNRTI resistance did not differ between the DPV and placebo arms of the MTN-020/ASPIRE study, indicating that drug resistance was not preferentially acquired or selected by the DPV ring and that the preventive benefit of DPV ring outweighs resistance risk.

Clinical and Virologic Outcomes Following Initiation of Antiretroviral Therapy Among Seroconverters in the Microbicide Trials Network-020 Phase III Trial of the Dapivirine Vaginal Ring.

BACKGROUND: A vaginal ring containing dapivirine, a non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitor (NNRTI), was safe and effective in preventing HIV-1 infection in African women. We examined the impact of dapivirine ring use at the time of HIV-1 acquisition on subsequent HIV-1 disease progression and responses to NNRTI-containing antiretroviral therapy (ART). METHODS: HIV-1 disease progression and virologic failure following initiation of ART were assessed among women who acquired HIV-1 while participating in Microbicide Trials Network-020, a randomized, placebo-controlled trial of a monthly, dapivirine vaginal ring. RESULTS: Among the 158 participants who acquired HIV-1 (65 dapivirine, 93 placebo), no differences between dapivirine and placebo participants were observed in CD4+ cell counts or plasma HIV-1 RNA over the first year after infection (prior to ART). During follow-up, 100/158 (63%) participants initiated NNRTI-containing ART (dapivirine: 39/65; placebo: 61/93); the median time to HIV-1 RNA <200 copies/ml was approximately 90 days for both dapivirine and placebo ring recipients (log-rank P = .40). Among the 81 participants with at least 6 months of post-ART follow-up, 19 (24%) experienced virologic failure (dapivirine: 6/32, 19%; placebo: 13/39, 27%; P = .42). CONCLUSIONS: The acquisition of HIV-1 infection during dapivirine or placebo treatment in ASPIRE did not lead to differences in HIV-1 disease progression. After the initiation of NNRTI-containing ART, dapivirine and placebo participants had similar times to virologic suppression and risks of virologic failure. These results provide reassurance that NNRTI-based ART regimens are effective among women who acquired HIV-1 while receiving the dapivirine vaginal ring. CLINICAL TRIALS REGISTRATION: NCT016170096 and NCT00514098.