Ethics statement
The research reported in the manuscript complies with all National Institutes of Health ethical regulations, and it was approved by the Center for Cancer Research non-human primate animal study protocol prospective scientific committee.
Animals, vaccines, and SIVmac251 challenge
The animals enrolled in the study were Indian rhesus macaques (Macaca mulatta). Macaques were provided by Alpha Genesis Inc. (Yemasee, SC) and Primate Products Inc. (Immokalee, FL) and were housed at the National Institutes of Health (Bethesda, MD) and handled in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) in an AAALAC-accredited facility (OLAW, Animal Welfare Assurance A4149-01). Animal care and procedures were performed under animal study protocols approved by the NCI Animal Care and Use Committees (ACUC; Protocol numbers: VB-013, VB026, VB034, VB042, and VB047). Animals were monitored daily for any signs of illness, and appropriate medical care was provided as needed. Animals were socially housed per the approved ACUC protocol and social compatibility, except during the viral challenge phase, when they were individually housed. All clinical procedures, including biopsy collection, administration of anesthetics and analgesics, and euthanasia, were conducted under the direction of a laboratory animal veterinarian.
V1-deleted DNA/ALVAC/gp120/ ALFQA SIV vaccination study
Twelve female macaques with an average age of 4.17 years (Standard deviation 0.51) were included in the ALFQA group. The animals were intramuscularly immunized twice (weeks 0 and 4) with DNA constructs 206S SIV p57gagmac239 (1 mg/dose) and V1-deleted SIVmac251-M766 gp160ΔV1 (2 mg/dose), mixed together and administered concomitantly in the two thighs (half-dose per thigh), as previously done28. All twelve macaques were then boosted twice (weeks 8 and 12) with intramuscular inoculations of 108 plaque-forming units (PFU) of recombinant ALVAC (vCP2432), expressing SIVmac251 gag-pro and gp120TM (Sanofi Pasteur, Bridgewater, NJ). During the second ALVAC boost (week 12) macaques were also administered 400 μg of V1-deleted SIVmac251-M766 gp120ΔV1, adjuvanted in ALFQA (Fig. 1a, ALFQA group). The protein boost was formulated by adsorbing 400 μg SIVmac251-M766 gp120ΔV1 protein diluted in phosphate-buffered saline (PBS) to 850 μg aluminum Al3+ (as aluminum hydroxide fluid gel suspension Rehydragel) for 10 min at room temperature (RT), and on a shaker. The protein adsorbed to rehydragel was then mixed with 200 μg of 3D-PHAD and 100 μg of QS21 and incubated on a shaker for another 10 min at RT. At the time of administration, the vaccine was briefly shaken, loaded into the syringe and promptly injected into the contralateral thigh of each animal.
During the immunization protocol, mucosal rectal biopsies were collected at baseline and 1 week following the ALVAC/protein boost (week 13). At the end of immunization (week 17), the animals were intravaginally challenged with 11 low doses of pathogenic SIVmac251. Challenges were performed using a dilution of 1:25 of a stock of SIVmac251 propagated in macaque cells (QBI#305342b, Quality Biological, Gaithersburg, MD), repeated once a week until confirmation of the infection by viral load performed in the plasma, and by administration of 1 mL of SIVmac251 diluted in RPMI 1640 (Gibco, Waltham, MA) to a final concentration of 4000 TCID50/mL (evaluated in rhesus 221 cells). Thirty-seven naïve rhesus macaques enrolled in prior studies28, that were exposed to intravaginal SIVmac251 challenges, following the same procedures, and using the same viral stock and at the same dilution as described above, were used as historical naïve controls.
V1-deleted DNA/ALVAC/gp120/alum SIV vaccination study
Viral acquisitions and immunological data obtained from thirty female macaques with an average age of 3.24 years (standard deviation 0.61) and immunized in prior studies28,29 were used for comparison (Fig. 1a, alum group). Briefly, thirty female macaques received the same DNA primes (week 0 and 4), and ALVAC boosts (week 8 and 12) administered to the ALFQA group described above. At week 12, the protein boost, consisting of the same SIVmac251-M766 gp120ΔV1 protein, was formulated by adsorbing the protein diluted in PBS to 5000 μg aluminum Al3+ (as alhydrogel adjuvant 2%, Invivogen) for 10 min on a shaker at RT. Five weeks following the last vaccination, the animals were exposed to intravaginal SIVmac251 challenges, following the same procedures, and using the same viral stock and at the same dilution, as described for the ALFQA group above.
V1-deleted DNA/ALVAC/gp120/Alum SIV vaccination study for mucosal samples
Mucosal samples from the rectum were obtained from twelve female macaques with an average age of 3.44 years (standard deviation 1.35) (Supplementary Fig. 4a) and immunized following the exact same vaccination regimen described above for the V1-deleted DNA/ALVAC/gp120/Alum SIV vaccination study. Mucosal samples were collected at baseline and 1 week following the protein boost adjuvanted in Rehydragel, following the same procedures and timeline used for the ALFQA group described above in the V1-deleted DNA/ALVAC/gp120/ ALFQA SIV vaccination study. Rectal mucosa samples were processed as described below. Following the collection of mucosal samples, these twelve animals received unrelated treatments (microbicide), that were not performed in the ALFQA group. However, since the samples were collected before the treatments, it was possible to perform immunological comparisons between the alum and ALFQA groups.
V1-deleted DNA/ALVAC/gp120/Alum and ALFQA HIV vaccination study for mucosal samples
Mucosal samples from the rectum to compare alum and ALFQA groups immunized with HIV immunogens were obtained from twelve macaques with an average age of 3.32 years (standard deviation 0.31) (Supplementary Fig. 4g). Six males and six females, macaques were equally randomized in two groups. All 12 animals were intramuscularly immunized twice (weeks 0 and 4) with DNA constructs clade B-HXB2 p55gag (1 mg/dose) and clade AE-A244 V1-deleted (ΔV1) gp120 (2 mg/dose), mixed together and administered. All macaques were then boosted twice (weeks 8 and 12) with intramuscular inoculations of 108 plaque-forming units (PFU) of recombinant ALVAC (vCP2438), expressing Clade B-IIB gag-pro, clade C ZM96 V1-replete gp120, and clade B-LAI gp120-transmembrane domain (Sanofi Pasteur, Bridgewater, NJ). During the second ALVAC boost (week 12), macaques were also administered 400 μg of clade AE-A244 ΔV1gp120 protein. In six macaques, the protein was adjuvanted with alum rehydragel (Supplementary Fig. 4g, alum group) and the other six with ALFQA (Supplementary Fig. 4g, ALFQA group). Formulation of the immunogens and their administration was performed as described for the SIV vaccination study. Mucosal rectal biopsies were concomitantly collected 1 week following the ALVAC/protein boost (week 13) and then processed as described below.
Viral RNA load
The RNA copies of SIVmac251 in plasma were quantified by digital droplet polymerase chain reaction (ddPCR) as in prior studies45. Briefly, following extraction, nucleic acid samples are prepared and mixed with primers and fluorescent probes, and Supermix (One-Step RT-ddPCR Advanced Kit; Bio-Rad Catalog No. 1864022). The following primers were used: SIV gag forward primer: 5’-GCAGAGGAGGAAATTACCCAGTAC-3’, SIV gag reverse primer: 5’-CAATTTTACCCAGGCATTTAATGTT-3’. The system partitions 20 μL of the RNA extract from plasma into 20,000 nL droplets. Each droplet contained a random distribution of the target and/or background RNA. The inactive reverse transcriptase and Taq DNA polymerase (Bio-Rad one-step ddPCR kit) blend into the droplets and activate due to the temperature increase at 50 °C. In each droplet, double-quenched SIV gag probe: 5’-/56FAM/TGTCCACCTGCCATTAAGCCCGA-3’ were included at the start of the reaction. The plate is then transferred to a beta-prototype droplet reader (optical reader). Poisson statistics are used to quantify the proportion of positive droplets, i.e., the number of target templates from which absolute viral RNA levels can be calculated precisely in copies/µL. Based on fluorescence amplitude, each 1 nL droplet is defined as either positive or negative. The fraction of fluorescent droplets determines the concentration of the target in the sample. Copies/mL were calculated based on copies/µL (Volume of mastermix per well/Volume of template) * plasma dilution factor/(volume of plasma used for extraction/elution volume). The assay’s limit of quantification (LOQ) is set at 50 RNA copies per milliliter of plasma.
Immunoglobulin G plasma titers to gp120
gp120 total IgG antibodies (immunoglobulin G) were measured by ELISA as previously described27. Briefly, ELISA plates were incubated overnight at 4 °C with 50 ng of SIVmac251-M766 gp120 protein/well in 100 µl of 50 mM sodium bicarbonate buffer (pH 9.6). Following coating with the protein, the plates were washed and blocked for 1 h at room temperature (RT) with 200 µl/well PBS Superblock (Thermo Fisher Scientific). Cryopreserved plasma collected at 3 weeks following last immunization (week 15), were thawed and serially diluted with sample diluent (Avioq), added to plates (100 µl/well), and incubated for 1 h at 37 °C. plates were then washed and incubated for 1 h at 37 °C with Horseradish Peroxidase HRP conjugated anti-human antibody (100 µl/well, diluted at 1:120,000 in sample diluent, Avioq). Finally, the plates were washed and developed using K-Blue Aqueous substrate and 2 N Sulfuric acid to block the reaction. Plates were read at 450 nm by using a Molecular Devices E-max plate reader. The titers were calculated as the highest dilution that provided an optical density (OD) value that was double the average value obtained for unvaccinated normal rhesus macaques.
Pepscan
Plasma samples were assayed by PEPSCAN analysis using SIVmac251 gp120 linear 20-mer peptides as previously described27. Cryopreserved plasma samples collected between 2 and 5 weeks following the last immunization (weeks 14–17) were analyzed. Briefly, ELISA plates were incubated overnight at 4 °C with 1000 ng/well of each of the V1 and V2 overlapping peptides (Supplementary Table 1) in 50 mM sodium bicarbonate buffer (pH 9.6). Following incubation, plates were blocked for 1 h at RT with Pierce SuperBlock blocking buffer, loaded with 100 μl/well diluted plasma (1:50 in sample diluent, Avioq), incubated for 1 h at 37 °C, washed, and incubated for 1 h at 37 °C with 100 µl/well anti-human HRP (diluted at 1:120,000 in sample diluent Avioq). The plates were then washed again and developed using 100 µl/well of K-Blue Aqueous substrate (Neogen) and incubating 30 min at RT. Two Normal Sulfuric acid (100 µl/well) was added to the plates to stop the reaction. The plate was read at 450 nm with a Molecular Devices E-max plate reader, and optical density for each sample was used for the analysis.
Antibody avidity
Antibody avidity determinations were conducted using the Biacore 4000 surface plasmon resonance (SPR) system as previously described63,64,65,66. Briefly, the immobilizations were performed using a standard amine-coupling kit. The CM5 sensor chip (Cytiva) surface was activated with a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.1 M N-hydroxysuccinimide (NHS) (Cytiva) for 600 s Protein SIVmac251-M766-ΔV1gp120 (20 μg/mL, spot 1, 2, 4, and 5 in flow cell 1), and Streptavidin (1 μM, spot 1, 2, 4, and 5 in Flow cells 2, 3, and 4) in 10 mM sodium acetate pH 4.5 were immobilized on the CM5 sensor chip. Spot 3 of each flow cell was left unmodified to serve as a reference. The resulting Response Units (RUs) for flow cells 1, 2, 3, and 4, respectively, were as follows: 12542-14330 RU; 7807-8015 RU; 7060-7924 RU; and 7073-7920 RU. Biotinylated cyclic V2 peptide was injected onto the streptavidin immobilized surface (flow cells 2, 3, and 4) for capturing, and the contact time was 240 s. Capture level (RU) of biotinylated cV2 peptide (SIVmac251) was as follows: 1951-2051 RU. Following the surface preparation, heat-inactivated (56 °C for 45 min plasma samples were diluted 1:50 in running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4) and injected onto the protein, and peptide immobilized surface for 250–300 s followed by dissociation for 1300–3000 s. Data for each sample were collected at a rate of 10 Hz, with an analysis temperature of 25 °C. All sample injections were conducted at a flow rate of 10 μL/min. The bound surface was regenerated with 150 mM HCl for 60 s. Data analysis was performed using Biacore 4000 Evaluation software 4.1 with double subtractions for the unmodified surface and buffer blank. Fitting was conducted using the dissociation mode integrated with Evaluation software 4.1. The data were further processed using Microsoft excel (version 16.79.1), and GraphPad Prism (version 10.0.1). The data were shown as an avidity score. Avidity score was calculated as RU/ Kd.
Plasma neutralizing antibodies
The levels of neutralizing antibodies were measured in the plasma of vaccinated animals collected between 2 to 3 weeks following the last immunization as a reduction in luciferase reporter gene expression after a single round of infection in TZM-bl cells. Test samples were serial-diluted (threefold dilution in duplicate) and incubated with 200 TCID50 of virus in a total volume of 150 μl for 1 h at 37 °C in 96-well flat-bottom culture plates. TZM-bl cells were trypsinized and added to each well (10,000 cells in 100 μl of growth medium containing 20 μg/mL DEAE dextran). A set of wells with cells only was used as background control, and another set with cells and virus was used as virus control. After incubation (48 h), the cells were lysed by the addition of Britelite (PerkinElmer Life Sciences, Waltham, MA), and three quarters of the cell lysate were transferred to a 96-well black solid plate (Corning Costar) for luminescence measurement. Neutralization titers are defined as the dilution at which relative luminescence units were reduced by 50% (ID50) or 80% (ID80) compared to that in virus control wells after subtraction of background relative luminescence units. Neutralization was tested against the virus SIVmac251.6 (ID #10848), SIVsmE660/BR-CG7G.IR1 (ID #1370DB2), SIVsmE660/BR-CG7G.IR1 (ID #1634DB2), and SIVmac251 (challenge virus).
gp120-specific IgG antibodies in vaginal secretions
gp120-specific IgG antibodies in vaginal secretions were measured following extraction from the swabs, followed by ELISA.
Vaginal secretion extraction
Extraction Buffer (0.8% sodium chloride, 0.67% Proclin 300 (Sigma, 48912-U), 1% Protease Inhibitor Cocktail (Calbiochem, 539131) in 1x Dulbecco’s phosphate-buffered saline [DPBS]) was prepared and filtered with a 0.22 µm filter unit and stored at 4 °C. Frozen vaginal swabs were thawed at room temperature for 5 min and then placed on ice to completely thaw. Each swab was examined for visible blood. Extraction buffer was added to each tube containing a swab, and the mucosal solution was manually extracted by squeezing the swab with a pipette tip. Tubes were vortexed three times each for 1-min intervals prior to incubation at room temperature for 15 min. The swabs were centrifuged at 4 °C, 16,000×g for 15 min. Supernatant was extracted and replicates were combined. The protein concentration of the mucosal extract was determined by Nanodrop at an absorbance of 280 nm.
Antigen-specific IgG ELISAs
ELISA titers were determined by coating plates with 0.1 µg/well coating antigen (ΔV1 SIVM766 gp120) in 1X DPBS w/o Ca and Mg. Coated plates were incubated overnight at 4 °C. Plates were washed with Wash buffer (0.1% Tween-20 in 1XDPBS), and blocking buffer (5% milk, 0.1% Tween-20 in DPBS) was added to the plates and incubated at room temperature for 2 h. Vaginal mucosal extract was thawed at 4 °C, then diluted to an initial dilution of 1:2.5 in blocking buffer. An NHP sample from a previous study served as a positive control for specific IgG titers. Samples were added to the plate in triplicate and then serially diluted 1:2 down the plate, mixing thoroughly with each dilution. Plates containing the sample were incubated at room temperature for 2 h. Secondary antibody (goat anti-monkey IgG (gamma chain) HRP conjugated, Alpha Diagnostic, 70021) was diluted 1:2000 in blocking buffer. Plates were washed, secondary Ab was added to each plate and incubated at room temperature for 1 h. The plates were washed and substrate [KPL ABTS Peroxidase Soln. A (Fisher Scientific, Cat: 5120-0035), and Peroxidase Soln. B (Fisher Scientific, Cat: 5120-0038) were mixed and added to the plates. The plates were incubated in the dark for 1 h at RT. Stop solution (1% sodium dodecyl sulfate (Invitrogen, 24730-020) in diH2O) was added to each plate, and the plates were read on a Molecular Devices SpectraMax M5E plate reader (405 nm Absorbance). Absorbance outliers were excluded, and the triplicates averaged. The ELISA final titer was defined as the reciprocal of the final dilution at which the sample absorbance was equal to or greater than twice the negative control absorbance. The negative control wells had all the components except the test sample.
Plasma ADCC killing, ADCC titers, and V2-specific ADCC killing measured by F(ab’)2 blocking
ADCC killing, ADCC titers, and V2-specific ADCC assay were measured as previously described27. Cryopreserved plasma samples collected between 2 and 5 weeks following the last immunization (weeks 14–17) were analyzed. Briefly, 106 GFP-expressing EGFP-CEM-NKr-CCR5-SNAP cells were coated with 50 μg of ∆V1gp120 protein for 2 h at 37 °C, washed and then labeled for 30 min at RT with SNAP-Surface® Alexa Fluor® 647 (New England Biolabs, Ipswich, MA). Total ADCC killing and ADCC titers were determined by incubating cells with plasma from each animal in the absence of F(ab’)2. In order to block the binding of plasma anti-V2 antibodies to the gp120, cells were incubated with 1 μg of purified F(ab’)2 fragments from NCI05 monoclonal antibody for 1 h at 37 °C. Plasma were heat-inactivated, diluted 1:100 for V2-specific ADCC or for 7 ten-fold dilutions (starting at 1:10) for total ADCC, and added to target cells. Target cells were then added of human PBMCs to generate an effector/target (E/T) ratio of 50:1. Cocultured cells were incubated at 37 °C for 2 h, washed with PBS and resuspended in 200 μl of a 2% paraformaldehyde in PBS solution. Cells were acquired by flow cytometry using an LSRII cytometer equipped with a high-throughput system (BD Biosciences). Specific killing was calculated as the loss of GFP from the SNAP-Alexa647+ target cells. Target and effector cells cultured in the presence of R10 media were used as background. Normalized percent killing was calculated as: (killing in the presence of plasma or plasma + F(ab’)2 – background)/(killing in the presence of positive control − background) × 100. Total ADCC killing was determined by incubating cells with plasma from each animal in the absence of F(ab’)2. The ADCC endpoint titer was calculated as the dilution at which the % ADCC killing was greater than the mean % killing of the background wells + three standard deviations. The V2-specific ADCC killing was calculated as: ADCC killing in the absence of F(ab’)2–ADCC killing in the presence of NCI05 F(ab’)2.
Trogocytosis
Trogocytosis was evaluated as previously described67 using cryopreserved plasma with plasma collected at baseline and 2- or 3-weeks following vaccination (weeks 14-15). CEM.NKR.CCR5 cells were stained with 2 μM PKH26 (Sigma-Aldrich) in Diluent C for 5 min at room temperature. Cells were then washed once, resuspended in R10 media, and incubated with wild-type gp120 or ΔV1gp120 proteins for 1 h at room temperature. Following gp120 coating, cells were washed, incubated with plasma samples diluted 300-folds. Healthy control PBMCs were used as effector cells. Cryopreserved PBMCS were thawed and cocultured with target cells (E:T ratio 50:1) for 5 h at 37 °C. At the end of the coculturing, cells were washed and incubated with live/dead aqua fixable dye and APC-H7-conjugated anti-CD14 antibody (5μl, clone MΦP9, Cat# 560180, BD Biosciences). Following incubation, cells were washed once and fixed with 4% formaldehyde (Tousimis, Rockville, MD). Cells were analyzed by flow cytometry using an LSRII flow cytometer (BD Biosciences). Trogocytosis score was calculated by calculating the PKH26 mean fluorescence intensity of the live CD14+ cells. Vaccine-induced trogocytosis scores were calculated by subtracting the values obtained for samples collected following the last immunization from those obtained for samples collected at baseline.
Antibody-dependent neutrophil phagocytosis
∆V1gp120 and wt gp120 proteins were biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin and following the manufacturer’s instructions (Cat. # 21338, Thermo Fisher Scientific), and using a biotin to gp120 ratio of 50. The biotin excess was then removed by using Zeba spin desalting columns (Cat. # PI-89883, Thermo Fisher Scientific) following the manufacturer’s instructions. Proteins were subsequently coupled with yellow-green or red NeutrAvidin-fluorescent beads (Cat. # F8776 and F8775, respectively; Life Technologies, Carlsbad, CA, USA) by incubating protein and beads at a 1:1 ratio for 2 h at 37 °C, washed and resuspended with 100x volume in 0.1% bovine serum albumin.
ADNP was assayed as already described68. Briefly, 10 μl of a 100-fold dilution of coupled protein/beads were incubated with 100 μl of 1:100 diluted plasma samples for 2 h at 37 °C. Following incubation, 50,000 cells/well of fresh peripheral blood leukocytes, isolated from one healthy donor, were added to the mix as effector cells, incubated for 1 h at 37 °C, washed, stained and fixed with 4% formaldehyde solution (Tousimis). Staining was conducted using Anti-human CD3 AF700 (5 μl, clone UCHT1, Cat# 557943) and anti-human CD14 APC-Cy7 (5 μl, clone MΦP9, Cat# 557831) antibodies obtained from BD Biosciences, and anti-human CD66b Pacific Blue (5 μl, clone G10F5, Cat# 305112) antibody from BioLegend. Cells were analyzed by flow cytometry using an LSRII flow cytometer (BD Biosciences). The phagocytic score was calculated by multiplying the % of bead-positive neutrophils (SSC high, CD3− CD14– CD66+) by the geometric MFI of the bead-positive cells and dividing by 104. Vaccine-induced ADNP was calculated by subtracting the values obtained for samples collected following the last immunization from those obtained for samples collected at baseline.
Antibody-dependent cell phagocytosis
∆V1gp120 and wt gp120 proteins were biotinylated and coupled with beads as described above for ADNP. ADCP was assayed as already described69. Briefly, 10 μl of a 100-fold dilution of coupled protein/beads were incubated with 100 μl of 1:100 diluted plasma samples for 2 h at 37 °C. Following incubation, 25,000 cells/well of THP-1 cells, were added to the mix as effector cells, incubated for 18 h at 37 °C, washed and fixed with 4% formaldehyde solution (Tousimis). Cells were analyzed by flow cytometry using an LSRII flow cytometer (BD Biosciences). The phagocytic score was calculated by multiplying the % of bead-positive cells by the geometric MFI of the bead-positive cells and dividing by 104. Vaccine-induced ADCP was calculated by subtracting the values obtained for samples collected following the last immunization from those obtained for samples collected at baseline.
Efferocytosis
The frequency of CD14+ cells able to conduct efferocytosis and their capability to engulf apoptotic cells was determined by the Efferocytosis Assay kit (cat. #601770, Cayman Chemical Company, Ann Arbor, MI) as previously described28. Due to the lack of availability of cells collected from vaccinated animals of the alum group, the assay was conducted on n = 6 macaques of the alum group and n = 12 macaques of the ALFQA group.
Effector cells
Isolation of CD14+ cells was performed using non-human primate CD14 MicroBeads (#130-091-097) and AutoMACSpro (Miltenyi Biotec) starting from 10 × 106 cryopreserved PBMCs. Following isolation, cells were stained with CytoTell™ Blue provided in the kit and washed three times.
Target cells
Apoptotic neutrophils collected from a naïve macaque were used as target cells. The isolation of the neutrophils was performed by processing fresh blood collected in EDTA tubes by Ficoll Plaque (GE Healthcare) and subsequent incubation with Dextran. Following isolation, neutrophils were at first stained with CFSE, washed three times with R10 media (RPMI media, 10% FBS and 1X anti-anti), incubated with R10 media containing Staurosporine apoptosis-inducer, and finally washed two times.
CD14+ cells and neutrophils coculture
Effector and target cells were cocultured (ratio 1 effector to 3 target cells) in R10 media at a concentration of 1 × 106 cells/ml and incubated overnight in an incubator at 37 °C. Concomitantly, CFSE-stained apoptotic neutrophils and CytoTell™ Blue-stained CD14+ cells were cultured alone as controls. Following incubation, cells were washed once with PBS and resuspended in 1% paraformaldehyde. All cells were analyzed by flow cytometry, using a FACSymphony A5 and FACSDiva software (BD Biosciences). Further analyses were performed using FlowJo v10.1 (TreeStar, Inc.). Cells were gated as follows: FSC/SSC/Sigle cells/CytoTell™ Blue+/CFSE+. The % of CD14+ efferocytes was calculated as the % of CFSE+ cells in the CytoTell™ Blue+ cells (CD14+ cells), whereas the engulfing capability was determined as the median fluorescence intensity of CFSE in CFSE+ CytoTell™ Blue+ cells. The vaccine-induced % of CD14+ efferocytes and their engulfing capability were calculated by subtracting the values obtained for samples collected 1 week following the last immunization (week 13) from those obtained for samples collected at baseline.
Flow cytometry of rectal mucosal cells
The frequency of natural killer (NK)/Innate lymphoid cells (ILCs), macrophages and dendritic cells were measured in the rectal mucosa of macaques collected at baseline and 1 week following the last vaccination (week 13). Eleven freshly collected rectal biopsies were digested with collagenase (2 mg/ml; Sigma-Aldrich) in RPMI (Gibco) without FBS for 1 h at 37 °C. Following incubation, pinches were mechanically separated by using a 10 ml syringe with a blunt head canula, cells were washed with R10 and passed through a 70-μm cell strainer. Cells were counted and used for the experiment. For each sample, 10–15 million cells were recovered from the pinches.
Phenotyping of innate lymphoid, myeloid and dendritic cells in rectal mucosa
Two million cells were used for phenotype analysis. Cells were stained with Live/Dead blue dye (cat. #L34962, 0.5 μl) from Thermo Fisher, followed by surface staining for 30 min at RT with the following antibodies: BB700 anti-CD14 (M5E2; cat. # 745790, 5 μl), APC anti-CCR2 (48607; cat. # 558406, 5 μl), APC-Cy7 anti-HLA-DR (L243; cat. # 335796, 5 μl), BV480 anti-CD45 (D058-1283; cat. # 566145, 5 μl), BV650 anti-NKp44 (p44-8; cat. # 744302, 5 μl), BV750 anti-CD163 (GHI/61; cat. # 747185, 5 μl), BUV493 anti-CD73 (AD2; cat. # 750061, 5 μl), BUV563 anti-CD184(CXCR4) (12G5; cat. # 741400, 5 μl), BUV661 anti-CD141 (1A4; cat. # 741650, 5 μl), BUV737 anti-CD206 (19.2; cat. # 741860, 5 μl), BUV805 anti-CD3 (SP34-2; cat. # 742053, 5 μl), BUV805 anti-CD20 (2H7; cat. # 612905, 5 μl) from BD Biosciences (San Jose, California, USA); PE-Cy7 anti-NKG2A (Z199; cat. no. B10246, 5 μl) from Beckman Coulter (Brea, California, USA); AF488 anti-CD1a (O10; cat. No. NBP2-34697AF488, 5 μl) from Novus Biologicals (Centennial, Colorado, USA); PE anti-CD33 (AC104.3E3; cat. #130-113-349, 5 μl) from Miltenyi Biotec (Bergisch Gladbach, North Rhine-Westphalia, Germany); and PE/Dazzle594 anti-CD16 (3G8; cat. # 302054, 5 μl), PE-Cy5 anti-CD11c (3.9; cat. # 301610, 5 μl), BV570 anti-CD11b (ICRF44; cat. # 301325, 5 μl), BV605 anti-CD1c (L161; cat. # 331538, 5 μl) from BioLegend (San Diego, California, USA). Samples were acquired on a BD FACSymphony A5 cytometer and analyzed with FlowJo software 10.6.
NKG2A+ NK cells were gated as singlets/live/CD45+/CD3–CD20–/CD14–/NKG2A+NKp44– cells. NKp44+ cells were gated as singlets/live/CD45+/CD3–CD20–/CD14–/NKG2A–NKp44+ cells. NKG2A− NKp44− cells were gated as singlets/live/CD45+/CD3−/CD20−/CD14−/NKG2A−NKp44− cells. CD73+ macrophages were gated as singlets/live/CD45+/CD3–CD20–/CD11b+/HLA-DR+/FSC-AHighSSC-AHigh/CD163+/CD73+ cells and expressed as frequency of parental population. Dendritic DC-10 cells were gated as previously described28 as singlets/SSChighFSChigh/live/CD45+/CD3–CD20–/HLA-DR+/CD1c–/CD11b+/CD11c+/CD14+CD16+/CD163+/CD141+/CD1a– and expressed as frequency of CD45+ cells.
Since the protocol allowed for the collection of a maximum of 11 rectal mucosa biopsies, which usually yield between 10 and 15 million cells, it was not possible to perform concomitant gate validation. Therefore, validation of the gates for NKG2A, Nkp44, CD163, CD141, and CD73 was performed at a later time point and using mucosal samples collected from two animals enrolled in another study. Briefly, following cell isolation, cells were divided into six tubes. One tube was fully stained following the procedure described above, whereas the other five were stained with all the antibodies minus one, constituting the fluorescence minus one (FMO) tubes of NKG2A, Nkp44, CD163, CD141 and CD73 antibodies. Examples of the gating in full-stained and FMO tubes are reported in Supplementary Fig. 3b. Further validation of the gate positioning in the original dataset was performed by gating each marker on live CD45+ cells. Briefly, using the same FCS files used to generate the data reported here, each marker was gated in the total live CD45+ population (Supplementary Fig. 3c). The presence of a high number of positive and negative cells in the CD45+ population allowed an accurate positioning of the gates. The generated gates were then applied in the full gating strategies (Supplementary Fig. 3a). Examples of the gating on live CD45+ cells are reported in Supplementary Fig. 3c.
Vaccine-induced frequencies of cells were calculated by subtracting the values obtained for samples collected following last immunization of those obtained for samples collected at baseline.
Cytokine expression upon stimulation of innate lymphoid cells in rectal mucosa
Two million rectal mucosal cells were stimulated for 2 h at 37 °C with overlapping gp120 peptides encompassing the sequence of SIVmac251 gp120 or HIV-1 A244 gp120 (2 μg/ml), or 1X PMA/Ionomycin (eBioscience cell stimulation cocktail, Cat. #00-4970-93 Invitrogen). Subsequently, GolgiPlug protein transport inhibitor (containing Brefeldin A) (cat. #555029, 1 μl) and GolgiStop protein transport inhibitor (containing Monensin) (cat. #554724, 0.7 μl) were added and culturing continued for 18 hours. Following incubation, cells were stained with Live/Dead blue dye (cat. #L34962, 0.5 μl) from Thermo Fisher, followed by surface staining for 30 min at room temperature. Surface staining was conducted as described above for phenotyping of innate lymphoid, myeloid and dendritic cells in rectal mucosa. Following surface staining, cells were fixed and permeabilized with a FOXP3-transcription buffer set (cat. #00-5523-00) from eBioscience (San Diego, California, USA) according to the manufacturer’s recommendation and subsequently intracellular staining with the following: R718 anti-TNFα (MAb11; cat. #566957, 5 μl), BV711 anti-IL-10 (JES3-9D7; cat. # 564050, 5 μl), BV786 anti-CD107 (H4A3; cat. # 563869, 5 μl), BUV395 anti-IFN-γ (B27; cat. # 563563, 5 μl) from BD Biosciences (San Jose, California, USA); and BV421 anti-IL-17 (BL168; cat. # 512312, 5 μl) from BioLegend (San Diego, California, USA). Samples were acquired on a BD FACSymphony A5 cytometer and analyzed with FlowJo software 10.6. Cytokines were gated on the parent population in NKG2A+ NK cells, NKp44+ and NKG2A– NKp44– cells.
Proximity extension assay
Protein quantification was executed employing the Olink® Target 48 Cytokine panel* (Olink Proteomics AB, Uppsala, Sweden) in accordance with the manufacturer’s protocols. Cryopreserved plasma collected from naïve control animals, and at 24 h or 1 week following the last immunization were analyzed. This method leverages the proximity extension assay (PEA) technology as detailed extensively by ref. 70. This specific PEA methodology enables the concurrent assessment of 45 distinct analytes. Briefly, pairs of oligonucleotide-labeled antibody probes, each tailored to selectively bind to their designated protein targets, were used. Probe pairs mix were incubated with 1 μl of plasma. Probes that encountered their cognate proteins are then in close spatial proximity, and their respective oligonucleotides engage in pair-wise hybridization. A DNA polymerase was used to amplify the hybridized DNA duplex, and to create distinct PCR target sequences. Subsequently these newly formed DNA sequences were detected and quantified through utilization of a microfluidic real-time PCR platform, specifically the Biomark HD system by Fluidigm (Olink Signature Q100 instrument). Data validation to uphold data integrity was conducted with the Olink NPX Signature software, specifically designed for the Olink® analysis: the application was used to import data from the Olink Signature Q100 instrument and process the data. Data normalization procedures were executed employing an internal extension control and calibrators, thereby effectively mitigating any inherent intra-run variability. The ultimate assay output was reported in picograms per milliliter (pg/ml), predicated upon a robust 4-parameter logistic (4-Pl) fit model, thereby ensuring precise absolute quantification. Comprehensive insights into the assay’s validation parameters, encompassing limits of detection, intra- and inter-assay precision data, and related metrics, are available at www.olink.com.
Output from the Olink software was further processed to extrapolate values for samples that were below the lower limit of quantification (LLOQ) and above the upper limit of quantification (annotated as “>ULOQ”). The Olink software interpolates values below the LLOQ through fitting the 4-Pl model to a distinct minimum limit of detection for each plate of samples run, and values below this interpolation range are set to NaN. Since these values are below the limit of detection but not truly missing, for each assay, we determined a universal “below detection” value by taking the mode LLOQ across 22 plates, divided by 10,000 (which was below all interpolated values in this extensive historical dataset) and set all NaN to this assay-specific below detection value. Samples which were above the ULOQ were set to the ULOQ value for the indicated target assay from the plate on which the sample was run. Finally, true missing values annotated as “No Data” were converted to NA to be systematically treated as missing. For Fig. 4c, d, the values plotted on the x-axis were calculated as the log10(abs(MW estimate *100)) * sign of MW estimate. The MW estimate = median of outer differences, and outer differences = difference between all pairs of values in group Alum—group ALFQA (e.g., for A = a1, a2 and B = b1, b2; then the outer differences would be: a1-b1, a2-b1, a1-b2, a2-b2. And the MW estimate is the median of these deltas). For MW estimate >0, ALFQA > ALUM; and for MW estimate 1 (o R 10 does not transform values −1
For Supplementary Fig. 5d, e, mean log10 values for each experimental group were visualized relative to the distribution of historical values for each assay (grey boxplot, with grey triangle designating the mean). Mann–Whitney/Wilcoxon p
Proteome analysis by ingenuity pathway analysis
Qiagen ingenuity pathway analysis application (Version 01-23-01; Qiagen Sciences, Germantown, MD, USA) was used to perfume pathway analysis. Data for analysis was uploaded as fold change of the mean of the ALFQA group compared to the mean of the alum group for each detectable target of the proteome (Fold change = mean ALFQA/mean alum). Each fold change was uploaded on the application in association with the p values and adjusted p values (false discovery rate, Benjamini–Hochberg) of the comparison of mean ALFQA vs mean alum. Data obtained from samples collected at week 12 + 24 h and week 13 were analyzed independently. Only the 36 detectable targets were included in the analysis. For the graphical summary (Fig. 5b, c), nodes were kept unmodified. For pathways (Supplementary Fig. 6a, b), only pathways with significance higher than −log(p value) 10 were exported.
Statistical analysis
GraphPad Prism (Version 10.3.0 (461)) was used to calculate the statistics. The Mann–Whitney–Wilcoxon test was used to perform comparisons of continuous factors between macaques belonging to the alum and ALFQA groups. All scatter plots report the median, except where stated otherwise. Spearman’s rank correlation was used to infer linear relationships between measured variables. The number of challenges is a numeric variable taking integer values between 1 and 11 with right-censoring of higher values, recorded as >11. For graphing, we assigned 12 as the value of the right-censored numbers, but the ranks are the same for any assigned value greater than 11. The exact log-rank Mantel–Cox test of the discrete-time proportional hazards model was used to compare the viral acquisition in animals exposed to SIVmac251. All statistical tests were performed as two-tailed.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
