Method details

SHCS project no 839 approval was obtained from the local ethics committees of all study sites of the Swiss HIV Cohort Study (Kantonale Ethikkommission Bern, Ethikkommission beider Basel, Ethikkommission des Kantons St. Gallen, Kantonale Ethikkommission Zurich, Comitato etico cantonale del Ticino, Commission d’ethique de la recherche clinique de la faculte de biologie et de medicine de l’universite de Lausanne and Comite d’ethique du department de medicine des hopitaux universitaires de Geneve), and written consent from all participating patients is available.For the study samples from 2 female and 10 male cohort individuals were included. The median age at infection was 45 ± 11.7 years, with a median baseline viral load of 24,200 copies/mL. For further details please see Table S2.

HUT4-3 cells are HUT78 (M. Martin, NIAID, Bethesda, MD, USA) derivatives, constitutively expressing infectious HIV-1. In brief, they were derived after infection with the clonal HIV-1 isolate NL4-3 (M. Martin); after in vitro infection surviving cells were repeatedly passaged until stable, CD4-negative cells emerged. Cells were cloned by limiting dilution and characterized for high virus production (laboratory of T. Klimkait, University of Basel, Switzerland). SupT1 hu R5 cells were from Monique Nijhuis, UMC Utrecht, NL. TZM-bl cells were obtained through the NIBSC center for AIDS reagents,⁴³ Ramos cells were obtained from Gertrud Steger (University of Cologne, GER).

HUT4-3 cells, Ramos cells and SupT1 cells were maintained in RPMI 1640 medium supplement with 10% FBS, penicillin (100 U/mL) and streptomycin (100 U/mL) (Bioconept, Allschwil, CH). TZM-bl and SXR-5 cells were maintained in DMEM medium supplement with 10% FBS, penicillin (100U/mL), and streptomycin (100U/mL). Cultures were maintained in 5% CO₂ at 37°C.

Up to 40 mL of fresh blood was drawn from consenting, recently diagnosed HIV-1 positive individuals. PBMC were isolated by density gradient centrifugation using Histopaque 1.077 g/mol (Sigma) on the day of harvest or on day 1 and were either stained directly or, for storage, frozen in FBS with 10% DMSO.

SupT1 (unstimulated) or HIV negative PBMC (2 days PHA pre-stimulated) were used for spinoculation: Viral stocks (NL4-3 X4-tropic, LAI X4-tropic, Ala-1 X4-tropic, Mal-2 R5-tropic) were spun onto target cells for 90 min at 800xg in minimal volume of culture media in a 24-well plate. Fresh RPMI 1640 supplemented with 10% FBS and Pen/Strep was then added to each well and cells were kept for 2 days at 37°C, 5% CO₂. Cells were washed extensively with PBS and cultured in fresh medium for an additional 3 days. Cells were now used for FACS and GERDA analysis. Viral infectivity in the supernatant was evaluated by using the SXR5 cell line containing an LTR-lacZ reporter (see below).

The proviral load was measured as described by others.²² In brief, primer and probe pairs targeting either HIV-1 LTR⁶⁴ or CCR5 were used in a single-plex real-time PCR approach. HUT4-3 gDNA was used as standard (2 HIV-1 copies per cell). HIV-1 DNA were extracted by Maxwell RSC Cultured Cells DNA Kit (Promega, Madison, USA). The GoTaq Probe qPCR Master Mix (Promega, Madison, USA) was used for quantification, cycling conditions were as follows: 1 min at 95°C, 45 cycles of alternating 15 s at 95°C and 1 min at 60°C. We performed the assay and analyzed the data with 7500 Fast Real-time PCR System (Themo Fisher, Walthan, MA, USA).

Intracellular poly-A HIV-1 transcripts were measured according to a published method.²⁶ Briefly, intracellular total nucleic acids from frozen PBMC were isolated with Promega Maxwell RSC simply RNA cells kit (Promega, Madison, USA), and aliquots immediately frozen at −20°C or −80°C. HIV-1 poly-A transcripts were quantified by One-step RT qPCR using standards of inhouse plasmid pMVQA; reads were normalized to cell count using CCR5 as described elsewhere.²² The qRT-PCR Brilliant III probe master mix kit (Agilent, Santa Clara, USA) was used for the quantification of HIV-1 RNA transcripts, qPCR was performed using the 7500 Fast Real-time PCR System (Themo Fisher, Walthan, MA, USA) with the following cycling conditions: 50°C for 10 min, 95°C for 3 min, 45 cycles of 95°C 15 s, 60°C 30 s.

Viral RNA was extracted from culture supernatant using Maxwell RSC miRNA Plasma and Serum Kit (Promega, Madison, WI, USA), which included a DNaseI digestion step to have only RNA present. SGA of 3′ half genome sequences was performed as previously described.⁶⁵ Complementary DNA was synthesized using the SuperScript IV First-strand Synthesis System (Invitrogen, Carlsbad, CA) with primer R-9632 5′-ACTACTTGAAGCACTCAAGGCAAGCTTTATTG-3′. cDNA was serially diluted with the goal of less than 30% positive amplification by nested PCR using Herculase II Fusion DNA Polymerase (Agilent, California, USA). At this dilution, most positive wells contain amplicons derived from a single cDNA molecule. First round PCR included sense primer F-4875 5′-CAAATTAYAAAAATTCAAAATTTTCGGGTTTATTACAG-3′ and antisense primer R-9626 5′- TGAAGCACTCAAGGCAAGCTTTATTGAGGC-3′. Following PCR conditions were used: 94°C for 2 min followed by 35 cycles of 94°C 15 s, 58°C 30 s, 68°C 4 min, with a final extension of 68°C for 10 min. The second PCR included sense primer F-4900 5′-GGGTTTATTACAGGGACAGCAGAG-3′ and antisense primer R-9602 5′-TGAGGCTTAAGCAGTGGGTTCC-3′. PCR conditions were used: 94°C for 2min followed by 45 cycles of 94°C 15 s, 58°C 30 s, 68°C 4 min, with a final extension of 68°C for 10 min. All PCR products are verified on a 1% agarose gel and sent directly for sequencing.

For amplification a nested PCR approach was chosen. The Herculase II Fusion DNA Polymerase Kit (Agilent Technologies) was used in a 25 μL reaction volume. 24 μL of Master Mix were mixed with 1 μL of extracted patient gDNA or purified first PCR. Each PCR was carried out in triplicates to normalize for PCR amplification bias. First PCR included sense primer F-6553 5′- ATGGGATCAAAGCCTAAAGCCATGTG-3′ and R-7801 5′- AGTGCTTCCTGCTGCTCCCAAGAACCCAAG-3′. Following cycling conditions were used: initial 3 min at 95°C, 30 cycles of denaturation for 15 s at 95°C, annealing for 20 s at 60°C and extension for 45 s at 72°C. Final extension was done for 3 min at 72°C.

The second PCR included sense primer F-6848 (5′- AGGCCTGTCCAAAGGTATCCTTTGA-3′; second PCR) and antisense primer R- 7371 (5′- TTACAGTAGAAAAATTCCCCTCCACAATTAAA-3′; second PCR). Cycling conditions for the second PCR were the same as for the first PCR except an annealing temperature of 56°C for 20 s.

For DNA quantification of NGS samples the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) was used according to protocol. DNA concentration was adjusted to 0.2 ng/μL and the Nextera XT DNA Library Preparation Kit (Illumina) was used to prepare the library according manufacturer’s protocol. Sequencing was performed with an Illumina MiSeq Benchtop sequencer with 2 × 250bp reads.

PCR amplicons were sequenced by the Sanger sequencing on an ABI 3730xl sequencer or by NGS with an Illumina MiSeq Benchtop sequencer. The full-length envelope sequences were assembled and aligned using Genious Prime2021.0.3 version. Phylogenetic trees were based on nucleotide sequences and constructed using the neighbor-joining method. Highlighter plots were compiled at https://www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter_top.html.

Sorted cell fractions were plated in 96-well format. Per well 1–3X10E5 patient’s cells were plated with addition of 10E4 SupT1 hu R5 in 50% ImmunoCult-XF T cell Expansion Medium, 50% conditioned medium, 100IU IL-2/mL and anti-human CD3/CD28/CD2 Antibody stimulation cocktail (Stem Cell Technologies). After 3 days fresh medium with 50 IU IL-2 was added and every 2–3 days medium was exchanged. On days 7 and 14 cells were re-stimulated. From the seventh day onward, viral outgrowth was measured by X-Gal stain to follow viral propagation over time.

The reporter assay for HIV-induction is based on the induction of an integrated LTR-lacZ gene in the stable reporter line SXR5, which is driven by an HIV-1 infection in vitro. The assay was performed as described.⁴² SXR5 (or TZM-bl) cells were used to detect HIV-1 infection events after in vitro infection by the induction of HIV-1 LTR dependent β-Galactosidase activity. 2–3X10E6 SXR5 or TZM-bl cells were plated in a 24- or 48-well plate the day before co-culture. Cells were co-cultured for 2 days after addition of putatively infected cells; alternatively, viral supernatants were spinoculated as described above. At harvest, cells were fixed with 2% formaldehyde and X-Gal was added for readout. X-Gal stains were imaged on a CTL Immunospot S6 Ultimate UV Image analyzer (CTL). A true infection event was considered when blue colored cell event consisted of more than 1 cell or if clear syncytia had formed (viral X4 tropism).

For titration, infectious HUT4-3 cells were pre-stained with CellTrace Far Red Cell Proliferation Kit (Invitrogen) at 37°C. Then, HUT4-3 cells were spiked into Ramos (B-)cells and stained for live/dead cell exclusion (Viobility 405/452, Miltenyi). FcR was blocked for 5–10 min at RT and respective cell surface markers were stained as described below. For all experiments with PBMCs obtained from HIV+ individuals, cells were kept at 37°C during all PBS washing steps. FcR was blocked for 5–10 min at RT, followed by 15 min Live/Dead (Zombie NIR, Biolegend) and CCR7 staining. Subsequently, all other surface markers were incubated for additional 20 min at RT. Cells were washed in all subsequent steps in staining buffer (PBS, 2 mM EDTA, 1% human serum). For FACS analysis, the following antibody panel was used: CD3 (BV510, Biolegend) CD4 (BV650, Biolegend), CD8 (A700, Biolegend), CD14/CD19 (APC-Cy7, Biolegend), CD25 (BV605, Biolegend), CD28 (PE-Dazzle, Biolegend), CD45RA (BV711, Biolegend), CCR6(PerCP-Cy5.5, Biolegend), CCR7 (APC, Biolegend), CLA (PerCP-Cy5.5, Biolegend), Integrin β7 (PE-Cy7, Biolegend), PD1 (BV421, Biolegend), HIV-1 Gag (PE, Beckman Coulter). An LSR Fortessa (BD Bioscience) was used for cell analysis. For sorts, a FITC-labeled alternative antibody against CD4 (Biolegend) was used.

Up to 40 mL of whole blood was drawn from HIV-1 positive individuals on cART, and CD4 cells were isolated by whole blood untouched selection (Miltenyi). Preselected cells were cultured overnight in ImmunoCult-XF T cell Expansion Medium (Stem Cell Technologies), 1 μM AMD, 1 μM MVC, 0.5 μM EFV and 50 IU IL-2/mL (Miltenyi) at 37°C 5% CO2. The next day cells were harvested, washed and stained. Cells were strictly kept on ice until sorting. Cells were sorted on a FACS AriaIII (BD Bioscience) cell sorter under BSL-3 conditions.

Flow cytometry data were analyzed using FlowJo v10.6.1 software (TreeStar). Cell populations of interest (viable CD3⁺CD4+/−CD8⁻CD14⁻CD19⁻) were exported in CSV format for tSNE downstream applications.

All bNAbs (3BNC117 (CD4bs), 10–1074 (V3-loop), PG16 (V1V2-loop), 35O22 (gp120-gp41 interface), 10E8 (MPER gp41) were biotinylated with EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher). Briefly, a total of 2.5 μg bNAb mix of equal quantities was biotinylated with a 30-fold molar excess of Sulfo-NHS-LC-Biotin in 500 μL volume for 60 min at room temperature (RT). To remove excess Biotin, biotinylated bNAbs were purified over a Zeba Spin Desalting Column. Biotinylated bNAbs were stored at 4°C until further use.

A CD4-untouched pre-selection step was performed to enrich for CD4⁺ cells. Selected cells were then stimulated in 50% ImmunoCult-XF T cell Expansion Medium (Stem Cell Technologies), 50% conditioned medium, 2.5 μg/mL PHA (Sigma), 5 μM AMD3100 (Selleckchem), 5 μM MVC (Selleckchem), 1 μM EFV (Selleckchem), 5 μM Z-VAD-FMK (Selleckchem), 100 IU IL-2/mL (Miltenyi) for 36-40h to reactivate latent proviruses. HUT4-3 cells, as positive control, were stimulated with PHA 24h prior to staining. For optimized conditions ≥2.5X10E6 Ramos cells (CD19⁺CD3⁻) were added as carrier cells. Subsequently, cells were surface stained for immunological characterization or for assessing Env expression. To detect Env, we used 5 μg/mL biotinylated bNAb mix (3BNC117, 10–1074, PG16, 35O22, 10E8 all provided by F. Klein) which was targeted by an Anti-Biotin-VioBright 515 (Miltenyi) secondary antibody. Cells were fixed for 30 min at RT in PBS 2% PFA. Cells were permeabilized twice with ICS Permeabilization Wash buffer (Biolegend) labeled intracellularly with anti-HIV Gag (KC-57PE, Beckman Coulter). ICS labeled samples were washed twice in perm-wash buffer and acquired on an LSRFortessa (BD Bioscience).

For titration, the infectious cell clone HUT4-3⁴¹ was spiked into the HIV-1 non-susceptible B-cell line (Ramos) to estimate infectious cells per million (quantities of either 100,000, 2,250, 225, 45, 9, 3, 1, 0.3 presumed infectious HUT4-3 cells). Each dilution was examined by qPCR for HIV-1 DNA load, RT-qPCR for poly-A transcripts, GERDA for Gag+/Env+ translation-competent cells and HIV-1 LTR driven X-Gal readout for HIV-1 infection events.