gs hiv combo ag ab eia  (Bio-Rad)

Journal: bioRxiv

Article Title: Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

doi: 10.64898/2025.12.07.692769

Figure Lengend Snippet: Longitudinal viral load, CD4⁺ T-cell counts, and sampling overview for all study participants. Individual participant trajectories of plasma HIV-1 RNA (left y-axis, red) and peripheral CD4⁺ T cell counts (right y-axis, gray) are shown from pre-ART baseline through long-term suppressive ART and analytic treatment interruption (ATI). Blue shaded regions denote periods of continuous ART, and yellow shaded regions indicate ATI phases during which plasma viremia was monitored three times per week to capture rebound kinetics with high temporal resolution. Horizontal tracks below each plot depict the timing and source of sample collection, including pre-ART plasma, proviral DNA from resting CD4⁺ T cells, quantitative viral outgrowth assay (QVOA) cultures, and rebound plasma isolates, as indicated by color-coded markers. Dashed vertical lines mark the start and end of ART or ATI periods. Open diamonds indicate the participant-reported date when viral suppression was first achieved if clinical documentation was unavailable. Together, these longitudinal clinical profiles illustrate the diverse treatment histories and rebound kinetics among the 14 participants, providing temporal context for all downstream sequencing, neutralization, and reservoir analyses.

Article Snippet: Qualitative detection of HIV-1 antigen reactivity by purified participant IgG was performed using the GS HIV-1 Western Blot Kit (Bio-Rad Laboratories, #32508) according to the manufacturer’s protocol.

Techniques: Sampling, Clinical Proteomics, Quantitative Viral Outgrowth Assay, Sequencing, Neutralization

Journal: bioRxiv

Article Title: Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

doi: 10.64898/2025.12.07.692769

Figure Lengend Snippet: Longitudinal clinical trajectories for representative participants from each phenotype: (a) standard progressor, (b) viremic controller, and (c) elite controller are shown including plasma HIV-1 RNA (left y-axis, red), CD4⁺ T-cell counts (right y-axis, gray), ART exposure (blue shading), and timepoints for biological sampling for env sequencing, antibody purification, and pseudovirus production are indicated with gray circles in the horizontal time track. Gold shading indicates the analytic treatment interruption (ATI) period. Detailed clinical parameters, including ART regimen, duration, and CD4 nadir, are provided in Supplementary Table 1.

Article Snippet: Qualitative detection of HIV-1 antigen reactivity by purified participant IgG was performed using the GS HIV-1 Western Blot Kit (Bio-Rad Laboratories, #32508) according to the manufacturer’s protocol.

Techniques: Clinical Proteomics, Sampling, Sequencing, Antibody Purification

Journal: bioRxiv

Article Title: Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

doi: 10.64898/2025.12.07.692769

Figure Lengend Snippet: (a) Western blots assessing antibody reactivity to HIV-1 Env proteins (gp41, gp120, and gp160) in plasma from all REBOUND participants at pre-ATI timepoints. Red arrows indicate the positions of gp41-, gp120-, and gp160-reactive bands, to assess HIV-1 Env-specific IgG binding. Qualitative western blots were performed using the GS HIV-1 Western Blot Kit, according to the manufacturer’s protocol. Each blot includes Bio-Rad reference controls: ++ (high positive), + (low positive), and - (negative) controls, used to verify assay performance. Control IgG represents pooled polyclonal IgG purified from HIV-seronegative donors, included to confirm assay specificity. All blots were processed using the same exposure. (b) Comparison of IC 50 values between reservoir viruses from and rebound viruses from , showing only data points from the REBOUND cohort. Values for reservoir pseudoviruses from the REBOUND cohort are on average lower than IC 50 values for the rebound isolates. The difference between the distributions of reservoir and rebound IC 50 values in the REBOUND cohort was significant as assessed by Mann-Whitney test (p = 0.0095). Bars indicate the median and IQR.

Article Snippet: Qualitative detection of HIV-1 antigen reactivity by purified participant IgG was performed using the GS HIV-1 Western Blot Kit (Bio-Rad Laboratories, #32508) according to the manufacturer’s protocol.

Techniques: Western Blot, Clinical Proteomics, Binding Assay, Control, Purification, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

doi: 10.64898/2025.12.07.692769

Figure Lengend Snippet: (a-c) Modified quantitative viral outgrowth assays (mQVOAs) from representative participants showing variable suppression of ex vivo viral outgrowth by pre-ATI autologous IgG (50 µg/mL) compared to control arms with no IgG, or IgG (50 µg/mL) purified from HIV-negative donors. Each circle represents the supernatant p24 level in an individual well seeded with 2X10 5 resting CD4 + T cells isolated from pre-ATI leukapheresis samples. Maximum likelihood IUPM estimates were provided for mQVOAs (see Methods). (d) Percent reduction in infectious units per million cells (IUPM) relative to control IgG across standard progressors (SPs), illustrating wide functional heterogeneity in aNAbs against inducible, infectious reservoir virus; open circles represent values from a previously described cohort . SP206 lacked detectable HIV-specific antibodies due to being treated during hyperacute infection (HAI) and served as a control for study rebound dynamics in the absence of neutralizing antibody responses. Bars indicate median with IQR. (e) Time to rebound was measured as the time in days to ≥200 HIV-1 RNA copies/mL, and was variable across all study participants. The LOD for the VL measurements was 30 HIV-1 RNA copies/mL. (f) Among SPs with measurable viral outgrowth, those with higher aNAb-mediated suppression of outgrowth showed significantly delayed rebound by Kaplan-Meier analysis (p = 0.0025, log-rank test). (g) Plasma HIV-1 RNA rebound trajectories for representative phenotypes (SP with HAI, SP, VC, EC), modeled by exponential growth curves. (h) Correlation of pre-ATI IgG-mediated suppression of ex vivo outgrowth with in vivo plasma viral load exponential growth rate during the ATI for SPs, including SP with HAI. Higher ex vivo suppression of outgrowth by aNAbs correlates with slower viral load exponential growth rates (p=0.0409, simple linear regression). (i) Plasma HIV-1 RNA doubling time during the ATI for representative phenotypes (HAI, SP, VC, EC), modeled by exponential growth curves. (j) Correlation of pre-ATI IgG-mediated suppression of ex vivo outgrowth with in vivo plasma viral load doubling time during the ATI for SPs, including SP with HAI. Higher ex vivo suppression of outgrowth by aNAbs correlates with delayed viral load doubling time during the ATI (p=0.0060, simple linear regression).

Article Snippet: Qualitative detection of HIV-1 antigen reactivity by purified participant IgG was performed using the GS HIV-1 Western Blot Kit (Bio-Rad Laboratories, #32508) according to the manufacturer’s protocol.

Techniques: Modification, Ex Vivo, Control, Purification, Isolation, Functional Assay, Virus, Infection, Clinical Proteomics, In Vivo

Journal: bioRxiv

Article Title: Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

doi: 10.64898/2025.12.07.692769

Figure Lengend Snippet: mQVOAs were performed using purified resting CD4⁺ T cells isolated from pre-ATI leukapheresis samples to assess aNAb-mediated suppression of inducible, replication-competent reservoir virus. Culture wells were activated by PHA and irradiated feeders in the presence of no IgG, control IgG from HIV-seronegative donors (50 µg/mL), or contemporaneous autologous pre-ATI IgG (50 µg/mL). Each circle represents the HIV-1 p24 antigen concentration measured by ELISA in the supernatant of a single well seeded with 2×10 5 resting CD4⁺ T cells. p24 antigen levels are color-coded from white (no detectable p24 antigen) to dark red (high p24 antigen). Replicate well outcomes were used to calculate maximum likelihood estimates of infectious units per million (IUPM) resting CD4 + T cells (including 95% CI) for each experimental condition and participant. Participants are stratified by clinical classification: (a-j) standard progressors (SPs), (k-l) viremic controllers (VCs), and (m-n) elite controllers (ECs), highlighting phenotype-specific differences in inducible reservoir frequency and the extent of suppression mediated by pre-ATI autologous IgG.

Article Snippet: Qualitative detection of HIV-1 antigen reactivity by purified participant IgG was performed using the GS HIV-1 Western Blot Kit (Bio-Rad Laboratories, #32508) according to the manufacturer’s protocol.

Techniques: Purification, Isolation, Virus, Irradiation, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

doi: 10.64898/2025.12.07.692769

Figure Lengend Snippet: Conventional reservoir metrics do not correlate with time to rebound. Simple linear regression analyses between conventional reservoir parameters and time to rebound revealed no significant correlations (all p = ns). (a) Frequency of intact, 3′ defective, and 5′ defective proviruses measured by the intact proviral DNA assay (IPDA) for all participants. (b) Relationship between infectious units per million (IUPM) resting CD4⁺ T cells, determined by quantitative viral outgrowth assay (QVOA), and the number of intact proviruses measured by IPDA. (c) Inducibility index, defined as the ratio of IUPM to intact proviral DNA copies. Correlations of time to rebound vs. (d) intact proviral DNA copies, (e) total HIV DNA copies, (f) IUPM, (g) the inducibility index, (h) CD4⁺ T-cell nadir, (i) median pairwise K2P nucleotide divergence of HIV-1 env sequences, and (j) duration of suppressive ART. (k) Time to rebound stratified by quartiles of ART duration (0-5, 5-9, 9-14, and 14-22 years on ART).

Article Snippet: Qualitative detection of HIV-1 antigen reactivity by purified participant IgG was performed using the GS HIV-1 Western Blot Kit (Bio-Rad Laboratories, #32508) according to the manufacturer’s protocol.

Techniques: Quantitative Viral Outgrowth Assay

Journal: bioRxiv

Article Title: Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

doi: 10.64898/2025.12.07.692769

Figure Lengend Snippet:

Article Snippet: Qualitative detection of HIV-1 antigen reactivity by purified participant IgG was performed using the GS HIV-1 Western Blot Kit (Bio-Rad Laboratories, #32508) according to the manufacturer’s protocol.

Techniques:

Journal: bioRxiv

Article Title: Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

doi: 10.64898/2025.12.07.692769

Figure Lengend Snippet: Schematic illustrating how the potency and breadth of autologous neutralizing antibodies (aNAbs) shape the rebound-competent reservoir during long-term ART. In individuals who initiated therapy during chronic HIV-1 infection, potent aNAbs initially restrict which proviruses are capable of reactivation. Over time, however, continued ART suppresses viral antigen exposure, leading to the gradual waning of aNAb titers and functional activity. As the inhibitory threshold ( IP 10mg/mL ) declines, a progressively larger fraction of archived proviruses previously neutralized can regain rebound-competency. Reservoir proviruses that remain resistant to contemporaneous aNAbs at the time of ATI therefore define the functionally rebound-competent subset. Together, this “moving-target” model illustrates how the effective breadth of the rebound-competent reservoir increases over time as humoral immune pressure decays, linking antibody waning to the reactivation potential of archived proviruses.

Article Snippet: Qualitative detection of HIV-1 antigen reactivity by purified participant IgG was performed using the GS HIV-1 Western Blot Kit (Bio-Rad Laboratories, #32508) according to the manufacturer’s protocol.

Techniques: Infection, Functional Assay, Activity Assay