Vector design, propagation, and characterization

artLCMV and artPICV vectors expressing SIVSME543 immunogens were generated by cDNA synthesis of the transgene sequences (SIVSME543 Gag, Env, Pol1, Pol2) by Genscript and subsequent cloning into two plasmids, both encoding the S-Segment of LCMV (based on clone 13) and PICV (strain 18), respectively. S-Segment #1 encodes LCMV or PICV NP, and S-Segment #2 encodes LCMV GP (derived from LCMV strain WE) or PICV GP. Vectors were generated by transient transfection of BHK21 cells stably expressing the LCMV GP. Briefly, cells were transfected with five plasmids encoding: S-Segment #1, S-Segment #2, L-Segment, and two expression plasmids encoding LCMV or PICV NP and L, respectively. Three days post-transfection, cells were propagated. On Day 6 post-transfection, virus-containing supernatant was harvested and subsequently titrated by Focus Forming Assay (FFA) on HEK293 cells to determine replication-competent virus titers (RCV FFU). To this end, monolayers of adherent HEK293 seeded in 24-well plates were infected with serial dilutions of the virus, incubated for 48 h, and subsequently fixed and stained with anti-LCMV-NP or anti-PICV-NP antibody (LCMV: VL-4; Bio X Cell, Lebanon, NH; PICV: purified E4–2 hybridoma supernatant). The number of foci (clusters of infected cells) was determined, and the virus titer was calculated. To generate fetal calf serum (FCS)-free vector stock, suspension HEK293 cells were infected with the respective artLCMV or artPICV vectors at a defined MOI of 0.001 and incubated for 2 (PICV vectors) or 4 (LCMV vectors) days with shaking. Nascent viruses were harvested after low-speed centrifugation and frozen below −65 °C. Vector stocks were titrated by FFA and characterized for antigen insertion, antigen expression, and growth properties. Vector stocks matching pre-set quality criteria were further propagated and subsequently characterized to generate vaccine material for the treatment of Indian-origin rhesus macaques. Vaccine material was tested for the absence of fungal and bacterial contaminants, endotoxins, and mycoplasma.

Animals and artPICV/artLCMV vaccination

Forty outbred adult (median age 4.8 years) male and female Indian-origin rhesus macaques (Maccaca mulatta) were included in this study and maintained at the animal facility of BIOQUAL, Inc. (Rockville, MD, USA). Animals were stratified into two groups based on body weight, age, and sex: artPICV/artLCMV vaccination (n = 24) and placebo (n = 8). Engineered arenavirus-based vectors, artPICV- and artLCMV-expressing SIVSME543 Gag and Env were administered in the left quadricep, and the vectors expressing SIVSME543 Pol antigen were administered in the right quadriceps. Animals were intramuscularly administered 1 × 106 replication-competent virus particles (RCV) of artPICV Gag, Env and Pol vectors at weeks 0 and 20, and 4 × 106 RCV of artLCMV Gag and Env vectors, and 2 × 106 RCV of artLCMV Pol vectors at weeks 12 and 28. Animals in the placebo group were administered placebo buffer solution at weeks 0, 12, 20, and 28. The sample size of the study was determined to detect a 1-log10 difference in setpoint viral loads23 with an estimated 80% power. Animals were anesthetized with Ketamine HCL at a dose prescribed by the veterinarian via the intramuscular route. If euthanasia was required as determined by the veterinarian, animals were sedated, and pentobarbital was administered at a dose prescribed by the veterinarian via intravenous route following American Veterinary Medical Association Guidelines on Euthanasia (2020 Edition). Death was verified and is defined as the cessation of respiration and/or a heartbeat. The experimental endpoint was 10 months after the SIVMAC251 challenge, defined as week 72 of the study. All animal studies were approved by the Institutional Animal Care and Use Committee at BIOQUAL.

Serum cytokine/chemokine analysis

The Cytokine & Chemokine 30-Plex NHP ProcartaPlex™ Panel (Thermo Fisher Scientific Inc., Waltham, MA, USA) was utilized to determine serum levels of 30 cytokines and chemokines (macrophage inflammatory protein-1α and 1β, stromal cell-derived factor-1α, CXCL13, IL-1β, IL-2, IL-4, IL-5, IFN-γ-induced protein-10, IL-6, IL-7, IL-8, IL-10, eotaxin, IL-12p70, IL-13, IL-17A, IL-1Ra, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IFN-α, IFN-γ, TNF-α, ITAC, MCP1, CXCL9, IL-23, IL-15, IL-18, and CD40 ligand) per manufacturer’s instructions. Samples were read in duplicates on FLEXMAP 3D® and analyzed using xPONENT® software (Luminex Corporation, Austin, TX, USA).

IFN-γ ELISpot

We determined T-cell responses to SIV peptide stimulation through IFN-γ ELISpot. The PBMCs were isolated from 5 to 10 mL of whole blood by density gradient centrifugation. ELISpot plates precoated with an anti-IFN-γ capture antibody (ELISpot Plus: Monkey IFN-γ [horseradish peroxidase (HRP)], Mabtech, Nacka Strand, Sweden) and ELISpot testing was performed on freshly isolated samples according to the manufacturer’s instructions. Peptide pools for stimulation included SIVSME543 Gag, Env, Pol, and vector-specific NP (LCMV and PICV) added at a final peptide concentration of 1 µg/mL. Phytohemagglutinin at a final concentration of 5 µg/mL served as a positive control. Dimethyl sulfoxide (DMSO) served as a negative control at a final concentration identical to the DMSO in the peptide stimulations. For measurement of cellular immune breadth, SIVSME543 Gag, Env, and Pol subpools were used (10 peptides/pool; 51 pools in total, including 12 for Gag, 16 for Env, and 23 for Pol; each peptide was a 15-mer with an 11 amino-acid overlap). The plates were incubated at 37 °C, 5% carbon dioxide (CO2) for 20–24 h before development of IFN-γ spots. Spots were visualized through a two-step binding process using an anti-IFN-γ-biotin detection antibody, followed by a tertiary streptavidin-HRP; IFN-γ spot-forming units were visualized using a chromogenic HRP substrate.

Intracellular cytokine staining multiparameter flow cytometry

Intracellular staining for flow cytometry was performed using predetermined concentrations of antibodies per manufacturer’s recommendations for CD3 (Alexa Fluor® 700, SP34–2, BD Biosciences, Franklin Lakes, NJ, USA), CD4 (OKT4, Brilliant Violet 605™, BioLegend, San Diego, CA, USA), CD8 (RPA-T8, Brilliant Violet 650, BioLegend), CD45RA (5H9, PE-Cy™7, BD Biosciences), CCR7 (G043H7, Brilliant Violet 785, BioLegend), CD27 (O323, Brilliant Violet 711, BioLegend), IFN-γ (B27, PE-CF594, BD Biosciences), IL-2 (MQ1–17H12, PE, BD Biosciences), TNFα (MAB11, PerCP-Cy™5.5, BD Biosciences) and CD107a (H4A3, APC, BioLegend), as previously described37. Freshly isolated PBMCs were plated at 500,000 cells/well in a 96-well plate and stimulated with SIV peptide pools at 2 µg/mL, phorbol-12-myristate-13-acetate (50 ng/mL)/ionomycin (500 ng/mL) or DMSO control. Cells were stained for cell surface markers followed by membrane permeabilization (eBioscience™ Foxp3/Transcription Factor Staining Buffer Set [catalog number 00-5523-00], Invitrogen™/Thermo Fisher) to stain for intracellular markers. Samples were collected on BD LSRFortessa, and data were analyzed using FlowJo™ v10.7.1 (BD, Ashton, OR, USA) by the gating strategy outlined in Supplementary Fig. 11.

Env IgG enzyme-linked immunoassay

Detection of IgG antibodies to Env was conducted through enzyme-linked immunoassay. Thermo Scientific Nunc™ MaxiSorp 384 well assay plates (Thermo Fisher) were coated overnight at 4 °C with SIV gp120 recombinant proteins (2 μg/mL). Plates were washed three times with phosphate-buffered saline (PBS) with Tween pH 7.4 containing 0.05% Tween 20 (PBST) and blocked with Dulbecco’s PBS pH 7.4 containing 5% skim milk and 1% goat serum for 1 h at room temperature. The blocking reagent was then aspirated, and 25 μL of an eight-point, threefold serial dilution (1:15 starting dilution, followed by 1:3 dilution) of heat-inactivated sera prepared in diluent (Dulbecco’s modified Eagle media containing 2% fetal bovine serum [FBS]) was added to the plates. Pooled sera from three naïve NHPs was diluted similarly to the test sera and used as a negative control. Plates were incubated for 1 h at 4 °C, followed by three washes with PBST. Immediately after washing, 25 μL of goat anti-monkey IgG (H + L)-HRP secondary antibody diluted 1:100,000 in Dulbecco’s PBS pH 7.4 containing 1% bovine serum albumin were added to each well of the plate and incubated for 30 min at room temperature. Plates were then washed three times with PBST, 25 μL of tetramethylbenzidine substrate was added, incubated at room temperature for 20 min, and the reaction was quenched with 25 μL of 0.16 M sulfuric acid. The absorbance of the plates was read at 450 nm on an EnVision XCite multimode plate reader (PerkinElmer, Waltham, MA, USA). Duplicate A450 negative control values (naïve NHP sera) were averaged. Endpoint titers were reported as the highest dilution of serum samples with average A450 values that were three standard deviations above the negative controls.

ADCC assay

Target CEM-NKR-CCR5-Luc T cells were activated with diethylaminoethyl (DEAE)-dextran 50 µg/mL for 10 min, followed by infection with SIVMAC251 (1.26 ng virus/million target cells). After 3–4 days at 37 °C and 5% CO2, infected and uninfected cells were plated in 96-well white flat-bottom plates at 20,000 cells/well. Serially diluted serum samples or media controls were added to the plate and incubated at 37 °C for 30–60 min. Effector natural killer cells (KHYG1-MmCD16) were then added at 100,000 cells/well and incubated overnight at 37 °C. Cell viability was determined using ONE-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) per the manufacturer’s instructions and luminescence read on the Envision plate reader.

SIV neutralizing antibodies

Four-fold serially diluted NHP sera in complete Roswell Park Memorial Institute (RPMI) media starting at 1:60 was added to a solid white 384-well plate in a seven-series dilution. SIVSME543, SIVSME660, SIVMAC239 pseudoviruses, and SIVMAC251 virus were diluted in media for a final dilution of 1/32 and added to the sera in 384-well plates, followed by a 1-min spin at 50 g. Samples were incubated at 37 °C for 1 h. CEM.NKR cells at 1.5 × 106 cells/mL were activated with 100 μg/mL DEAE-dextran. After incubation, 30,000 activated CEM.NKR cells were added to each well-containing sera and virus (final DEAE dextran concentration, 50 μg/mL) and incubated at 37 °C for 72 h. Cell viability was determined using ONE-Glo per the manufacturer’s instructions and luminescence read on the Envision plate reader. Values obtained from CEM.NKR cells infected in triplicate with the pseudovirus/virus in the absence of NHP sera were averaged and set to 100% infection (no virus inhibition). Triplicate samples for each dilution were averaged to calculate percent inhibition. Percent inhibition was plotted (y-axis) against log-transformed serum dilution (x-axis) for each of the tested viruses and fit a four-parameter dose response using GraphPad Prism (GraphPad Software Boston, MA, USA). Half-maximal effective concentration (EC50) values were interpolated from the fit curve. The EC50 was reported as the serum dilution (titer) that inhibited 50% luciferase signals versus control (cells and virus only) after subtraction of the negative control. Sera for which no EC50 was obtained were assigned an arbitrary titer of 1.

LCMV neutralization assay

ARPE-19 cells (ATCC CRL-2302) were seeded in Dulbecco’s modified Eagle media with 2% FBS in 384-well plates overnight before infection. Test sera were preincubated at 56 °C for 1 h and eight-point serial threefold dilutions were made. An equal volume of LCMV-green fluorescent protein (GFP) vector was added to the diluted sera samples to achieve a final multiplicity of infection of 10,000 and incubated for 90 min at 37 °C in 5% CO2. Cells were infected by transferring 40 µL of the sera/LCMV-GFP vector mixture and incubated for 24 h at 37 °C in 5% CO2. The next day, after the media was removed, the cell monolayer was washed with PBS, fixed with 4% paraformaldehyde, and stained with 4′,6-diamidino-2-phenylindole (1:1000) for 30 min at room temperature. Assay plates were washed three times with PBS, followed by fluorescent imaging (CellInsight CX7 Pro HCS Platform, Thermo Fisher). The reduction in GFP signal was reported as the ID50 titers for the sera evaluated in duplicates. The ID50 values were calculated from the dose-response curves fit to a four-parameter equation. All ID50 values represent geometric mean values of a minimum of two determinations. For serum samples with no detectable LCMV neutralization antibodies, ID50 titer of the starting sera dilution of 60 was reported.

PICV neutralization assay

Baby hamster kidney fibroblast cells (BHK-21) were plated at 10,000 cells/well in tissue culture-treated flat-bottom 96-well plates and cultured overnight at 37 °C in RPMI media supplemented with 10% FBS. Seven 4-fold serial dilutions of sera from NHPs were made in RPMI media starting at 1:40 in clear 96-well U-bottom plates. Replicating PICV-based vector encoding luciferase (PICV-Nanoluc) was added at a final concentration of 3 × 103 RCV focus forming units in 50 µL to each well. Serum mixed with PICV-Nanoluc was incubated at 37 °C for 2 h in 5% CO2. Serum dilutions and PICV-Nanoluc were mixed and added to cells (100 µL/well). Plates were incubated overnight at 37 °C with 5% CO2. Cell viability was determined using the Nano-Glo® Dual-Luciferase® Reporter Assay System NanoGlo (Promega) following manufacturer’s instructions and a luminescence signal was acquired on the Envision plate reader. Values of six replicates of uninfected BHK-21 cells were averaged and subtracted as background from values obtained from individual test sera samples. Values obtained from BHK-21 cells in quadruplicate infected in the absence of sera were averaged and set to 100% infection (no virus inhibition). The luminescence signal from duplicate samples for each serum dilution was averaged to calculate percent inhibition and plotted against log-transformed serum dilution (x-axis) and fit to a four-parameter dose response using GraphPad Prism. The EC50 values were interpolated and reported as the serum dilution (titer) that inhibited 50% luciferase signal compared with control after background subtraction. Sera for which no EC50 was determined were given a titer of 40 based on the starting sera dilution. Titers were reported for each sample as a geometric mean from two independent experiments.

SIVMAC251 challenge

All animals in the study were challenged 4 weeks after the last vaccine dose (week 32) with a single intravenous inoculation of a heterologous SIV virus swarm (SIVMAC251: 8.19 TCID50), as in Liu et al.23. The estimated animal ID50 of the SIVMAC251 challenge stock was 0.29 TCID50 via the intravenous route. After the SIV challenge, all animals were monitored for clinical and laboratory progression, as well as viral load, to determine peak viral load (calculated 2 weeks after the challenge) and setpoint viral load (calculated over weeks 10–40 after the challenge). Clinical illness after SIVMAC251 infection was characterized by a decrease in CD4 T cell counts and body weight, non-resolving diarrhea, anemia, mild edema, loss of appetite, jaundice, lymph node swelling along with an increase in viral load and was closely monitored by the veterinarians at Bioqual.

Plasma viral load quantification

For the assessment of viremia, viral RNA was extracted from 0.2 mL of cell-free plasma using the QIAamp MinElute Virus Spin Kit (QIAGEN, Hilden, Germany). Quantitative polymerase chain reaction (Applied Biosystems™ 7500 Real-Time PCR System, Thermo Fisher) was performed by using the Taqman RT PCR kit (Thermo Fisher) in triplicate using the following primer and probe combination: Forward: 5′-GTCTGCGTCATCTGGTGCATTC-3′, Reverse 5′-CACTAGGTGTCTCTGCACTATCTGTTTTG-3′, Probe: 6-FAM-5′-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-3′-Idaho Black. The amplification conditions were 48 °C for 30 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 1 min at 60 °C.

SIV sequencing

Illumina short-read data for SIV were generated using the Nextera-transposon-based method as previously described38 at the Wisconsin National Primate Research Center (University of Wisconsin–Madison). Sequencing reads were trimmed using Trimmomatic v0.3639 for low quality (sliding window 4 bp, average phred 15), and read lengths <50 base pairs were filtered out. Reads were then aligned to reference sequence M33262 using SMALT v0.7.6 aligner (Wellcome Sanger Institute, Cambridgeshire, UK). Each aligned read was then parsed using codons and evaluated for amino-acid sequence changes and in-frame indels. Amino acid changes in Gag, Pol, Env, and Nef were reported per gene position at frequency ≥15%, excluding any changes overlapping with amplification primers, average phred score <30, and read depth <50. The amino acid changes in each treated sample were then compared with changes found in SIVMAC251 viral stock to obtain the number of developed amino acid changes.

Statistical analysis

Immunologic and virologic data from the study were analyzed using GraphPad Prism 8.1.2. To compare groups, a two-sided Wilcoxon matched-pairs signed-rank test, Friedman’s test with Dunn’s post-test for multiple comparisons, or two-way ANOVA with Dunnett’s post-test was utilized as appropriate. A two-sided Spearman rank test was performed for correlation analyses and corrected for multiple comparison testing by the Benjamini–Hochberg method where indicated. Sample sizes were predetermined by statistical methods. The experiments were not randomized, and investigators were not blinded during analyses.

Reporting summary

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