Combination anti-HIV antibodies provide sustained virological suppression – Nature

Study design, participants and procedures

The purpose of this clinical trial was to assess the safety, tolerability and virological efficacy of a combination of two bNAbs 3BNC117 and 10-1074 in individuals with HIV. The study consisted of two components: a randomized, double-blind, placebo-controlled trial (group 1) and an open-label, single-arm trial (group 2). The study inclusion criteria for group 1 included start of ART within 12 weeks of being diagnosed with acute or early HIV infection, a CD4⁺ T cell count of >450 cells per µl at screening, continuous ART treatment with suppression of plasma viraemia below the limit of detection for ≥1 year and with general good health. The participants who had previously participated in an ATI study were not excluded. Acute infection was defined as plasma viraemia of greater than 2,000 copies of HIV RNA per ml with a negative HIV-1 enzyme immunoassay (EIA; criterion 1); a positive result from an HIV-1 EIA with a negative or indeterminate HIV-1 western blot that subsequently evolves to a confirmed positive result (criterion 2); or a negative result from an HIV-1 EIA within the past 4 months and plasma viraemia greater than 400,000 copies per ml in the setting of a potential exposure to HIV-1 (criterion 3). Early infection was defined as a negative result from an HIV-1 EIA within 6 months before a positive result from an HIV-1 EIA and confirmatory HIV-1 western blot (criterion 4); a negative result from a rapid HIV-1 test within 1 month before a positive result from an HIV-1 EIA and western blot (criterion 5); or the presence of low level of HIV antibodies as determined by having a positive EIA and western blot with a non-reactive detuned EIA according to a multi-assay testing algorithm for recent infection (criterion 6). The study inclusion criteria for group 2 included no ART within 24 months, plasma viraemia of 200–5,000 copies per ml, and at least two documented instances of plasma viraemia ≥200 copies per ml on at least two occasions in the 12 months before screening. The predetermined primary end point of the study was the rate of occurrence of grade 3 or higher adverse events or serious adverse events that were probably or definitely related to the study antibodies. Adverse events were determined according to the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, Version 2.1. The virological end points were the number of study participants who experienced plasma viral rebound after ATI and who met criteria to restart ART before study week 28 (group 1) or the number of study participants who achieved sustained suppression of plasma viraemia by study week 28 (group 2).

The study participants in group 1 were randomized to receive multiple (up to 8) infusions of 3BNC117 (30 mg kg⁻¹ body weight) and 10-1074 (30 mg kg⁻¹ body weight) or placebo during the 24-week period. One study participant (participant 05) whose regimen contained a non-nucleoside reverse transcriptase inhibitor (NNRTI) was switched to an integrase inhibitor-based regimen 2 weeks before ATI due to the long half-life of NNRTIs. The study participants in group 1 discontinued ART 3 days after the first infusion of 3BNC117 and 10-1074 or placebo. The protocol investigators and study participants in group 1 were blinded to treatment assignments for the duration of study. All of the study participants in group 2 received up to 8 infusions of 3BNC117 (30 mg kg⁻¹ body weight) and 10-1074 (30 mg kg⁻¹ body weight) during the 24-week period. The study participants received sequential 3BNC117 and 10-1074 intravenously according to their body weight over a 60 min period per antibody. Plasma viraemia and CD4⁺ T cell count were monitored every 2 weeks. Plasma viraemia was determined using the Abbott Real-Time HIV-1 assay with a detection limit of 40 copies of HIV RNA per ml. The study participants in group 1 discontinued antibody infusions or placebo and reinitiated ART if they met one of more of the following criteria during the ATI phase: (1) a confirmed >30% decline in baseline CD4⁺ T cell count; (2) an absolute CD4⁺ T cell count of <350 cells per µl; or (3) sustained plasma viraemia of >1,000 copies per ml for greater than 4 weeks. The study participants in group 2 discontinued antibody infusions if their CD4⁺ T cells declined as specified above or they had an increase in baseline plasma viraemia of >0.5 log₁₀.

Blood and leukapheresed products were collected in accordance with protocols approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (ClinicalTrials.gov ID: NCT03571204). All of the participants provided written informed consent.

Measurements of IC₈₀

Near-clonal replication-competent HIV isolates were derived from coculturing enriched CD4⁺ T cells of the study participants with CD8-depleted anti-CD3 stimulated peripheral blood mononuclear cells (PBMCs) from HIV-seronegative donors as described below. The concentration of each infectious viral isolate was initially determined by HIV p24 enzyme-linked immunosorbent assay (ELISA). Each isolate was titrated using a standard TZM-bl target cell assay. To determine the IC₈₀ concentrations of both antibodies against reservoir-derived replication-competent HIV over time, each viral isolate was incubated with serially diluted (40, 10, 2.5, 0.625, 0.156, 0.039 µg ml⁻¹ in duplicate) 3BNC117 and/or 10-1074 for 90 min followed by incubation with TZM-bl cells for 48 h. Cells were then lysed and substrate (Promega) was added to measure the luciferase activity (SparkControl, v.3.1, Tecan).

Pharmacokinetics of 3BNC117 and 10-1074

The levels of 3BNC117 and 10-1074 in the plasma of the study participants were longitudinally measured using a validated luciferase-based neutralization assay in TZM-bl cells as previously described³⁸. In brief, plasma samples were tested in duplicate using a primary 1:20 dilution with a fivefold titration series and tested against the HIV Env pseudotyped viruses Q461.e2 (3BNC117-sensitive/10-1074-resistant) and Du422.1 (10-1074-sensitive/3BNC117-resistant). Pseudotyped virus with murine leukaemia virus (MuLV) Env was used as a negative control. 3BNC117 and 10-1074 clinical drug products were used as positive controls and tested using a primary concentration of 10 μg ml⁻¹ with fivefold serial dilution series. Plasma concentrations (µg ml⁻¹) of 3BNC117 and 10-1074 were determined as follows: plasma 50% inhibitory dilution (ID₅₀) titre (dilution) × IC₅₀ titre (µg ml⁻¹) of 3BNC117 or 10-1074.

Quantification of HIV reservoirs

The frequency of CD4⁺ T cells carrying total HIV DNA was determined by droplet digital PCR as previously described³⁹. In brief, genomic DNA was isolated from highly enriched CD4⁺ T cells using the Puregene DNA extraction kit (Qiagen) and digested with restriction enzyme MscI (New England BioLabs). Subsequently, the digested genomic DNA was analysed using droplet digital PCR (QuantaSoft, v.1.7.4.0917, Bio-Rad) according to the manufacturer’s instructions. The following PCR primers and probe were used for amplification of the 5′ LTR region of HIV DNA: forward primer 5′-GRAACCCACTGCTTAAGCCTCAA-3′ (nucleotides 506–528 in HXB2; GenBank: K03455.1), reverse primer 5′-TGTTCGGGCGCCACTGCTAGAGA-3′ (nucleotides 648–626) and probe 5′-6FAM-AGTAGTGTGTGCCCGTCTGTT-IABkFQ-3′ (nucleotides 552–572). The following PCR primers and probe were used for amplification of the housekeeping gene RPP30: forward primer 5′-GATTTGGACCTGCGAGCG-3′ (nucleotides 29–46; GenBank: NM_001104546.2), reverse primer 5′-GCGGCTGTCTCCACAAGT-3′ (nucleotides 90–73) and probe 5′-HEX-TTCTGACCTGAAGGCTCTGCGC-IABkFQ-3′ (nucleotides 49–70). Copy numbers of HIV DNA were normalized per 1 × 10⁶ CD4⁺ T cells.

The level of CD4⁺ T-cell-associated HIV RNA was determined by PCR with reverse transcription (RT–PCR). Total RNA was isolated from highly enriched CD4⁺ T cells using the RNeasy Mini kit (Qiagen), followed by synthesis of complementary DNA (cDNA) using the qScript XLT cDNA Master Mix (Quanta Biosciences) according to the manufacturer’s instructions. cDNA was analysed using droplet digital PCR (Bio-Rad Laboratories) using the following primers: HIV-specific primers HIV-US-F (5′-TCTCTAGCAGTGGCGCCCGAACA-3′, nucleotides 626–648), HIV-US-R (5′-TCTCCTTCTAGCCTCCGCTAGTC-3′, nucleotides 786–764) and HIV-US-probe (5′-6FAM-CAAGCCGAGTCCTGCGTCGAGAG-IABkFQ-3′, nucleotides 705–683); and TATA-box-binding protein (TBP; housekeeping gene)-specific primers TBP-F (5′-CACGAACCACGGCACTGATT-3′, nucleotides 863–882; GenBank: NM_003194.5) and TBP-R (5′-TTTTCTTGCTGCCAGTCTGGAC-3′, nucleotides 951–930) and TBP-probe (5′-HEX-TGTGCACAGGAGCCAAGAGTGAAGA/3-IABkFQ-3′, nucleotides 902–926). Copy numbers of cell-associated HIV RNA were normalized per 1 × 10⁶ copies of TBP.

The frequency of CD4⁺ T cells carrying intact HIV proviruses was determined using the Intact Proviral DNA Assay (IPDA) as previously described⁴⁰.

The level of CD4⁺ T cells carrying replication-competent HIV was determined by quantitative co-culture assay using serially diluted and replicates of 5 × 10⁶ CD4⁺ T cells as previously described³⁹. Highly enriched CD4⁺ T cells were incubated with anti-CD3 antibodies and irradiated PBMCs from healthy HIV-negative donors. After incubation for 1 day, 1 × 10⁶ CD8-depleted and anti-CD3-stimulated PBMCs from HIV-negative donors were added to each well, followed by periodic removal of cell suspensions and replenishment with fresh medium containing IL-2. Subsequently, HIV p24 ELISA was performed on the culture supernatants to identify wells containing replication-competent HIV. The infectious units per million cells from quantitative coculture assays were determined as previously described⁴¹.

Phenotypic characterization of immune cells using flow cytometry

Cryopreserved PBMCs were thawed, washed and stained with the viability reagent Zombie NIR (BioLegend, 423106) and fluorophore-conjugated antibodies in Brilliant Stain Buffer Plus (BD, 566385). Flow cytometry data were acquired on the Cytek Aurora cytometer using the SpectroFlo Software (Cytek Biosciences) and analysed using FlowJo v.10.7.1 and the OMIQ platform (https://www.omiq.ai/).

High-dimensional data analysis of flow cytometry data

Optimized t-distributed stochastic neighbour embedding (opt-SNE) and FlowSOM analyses were conducted using OMIQ software (https://www.omiq.ai/). Opt-SNE analysis was performed using equal sampling of 100,000 CD3⁺ T cells from each FCS file, with 1,000 iterations, a perplexity of 30 and a theta of 0.5. The following immune markers were used to generate opt-SNE maps: CD4, CD8, CD45RA, CCR7, CD27, CD28, CD38, HLA-DR, CD226, TIGIT, PD-1, 2B4, CD160, CTLA-4, CD96, OX40, CXCR5, ICOS and 4-1BB. The resulting opt-SNE maps were used for the FlowSOM algorithm. The self-organizing map was generated using hierarchical consensus clustering and 15 meta-clusters were identified. The heat map displaying column-scaled z-scores of mean fluorescence intensity for individual FlowSOM clusters was generated using OMIQ platform.

Intracellular cytokine staining assay

The frequency of virus-specific CD8⁺ T cells was assessed by incubating PBMCs with a pool of HIV-1 consensus B Gag overlapping peptides (NIH AIDS Reagent Program) with brefeldin A (Sigma-Aldrich) for 6 h at 37 °C. Unstimulated cells were used as a negative control for background subtraction. Cells were stained with Zombie NIR (BioLegend, 423106) and antibodies to immune markers (Supplementary Table 5). Cells were then fixed with 1× lysing solution (BD Biosciences) and permeabilized with 1× permeabilization solution 2 (BD Biosciences). After washing, cells were stained with the antibodies to the intracellular cytokines and chemokines (Supplementary Table 5 and Supplementary Fig. 1). Data were acquired on the Cytek Aurora cytometer using the SpectroFlo Software (Cytek Biosciences) and analysed using FlowJo v.10.7.1.

TCR repertoire analysis

Complementarity determining regions (CDR) 3 of TCR β-chains present in highly enriched CD8⁺ T cells of the study participants were sequenced in a high-throughput manner using the immunoSEQ assay^42,43 after amplification of the extracted DNA in a bias-controlled multiplex PCR. The resulting CDR3 sequences were collapsed and filtered to quantify the absolute abundance and frequency of each unique TCR β region with immunoSEQ Analyzer (Adaptive Biotechnologies)⁴⁴. TCR repertoire statistics, including gene usage (the fraction of clonotypes in which a given TRBV or TRBJ gene is present), were computed using Immunarch⁴⁵. TCR sequencing data are available online (https://clients.adaptivebiotech.com/login; login: [email protected], password: chun2021review). HIV-specific breadth and depth were computed using the HIV-specific CDR3 sequences previously reported in the literature from four databases, namely the immune epitope database (IEDB)⁴⁶, VDJdb⁴⁷, McPAS-TCR⁴⁸ and the Pan immune repertoire database (PIRD)⁴⁹. The reported breadth values represent the fraction of clonotypes in each repertoire that are HIV-specific, whereas depth calculations take the abundance of each clonotype into account, such that each HIV-specific clonotype affects the overall HIV-specific depth (per sample) with a magnitude proportional to its abundance.

Measurements of biomarkers in plasma

Levels of biomarkers in plasma were determined using the ELLA platform (Simple Plex Runner, v.3.7.2.0, ProteinSimple) according to the manufacturer’s instructions.

Statistical analysis

P values for the virological end points (group 1) were determined using exact log-rank tests. Sensitivity analyses were performed to handle the two study participants (11 and 14) who reinitiated ART before meeting the restart criteria. Two independent analyses, with and without censoring the data from the above study participants, generated the same P values.

P values for paired and unpaired comparisons were computed using Wilcoxon signed-rank tests and Mann–Whitney U-tests, respectively, using Prism v.9.1 (GraphPad). P values computed in the TCR repertoire analysis were determined using Wilcoxon signed rank tests (paired) and Mann–Whitney U-tests (unpaired) using the R package ggpubr⁵⁰. The factoMineR⁵¹ package in R was used to perform PCA with the TCR repertoire data.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.