Raw materials/formulation ingredients

GMP-grade E6020 was obtained from Eisai Inc. (Cambridge, MA). Highly purified QS21 was obtained from Desert King (San Diego, CA). Dioleoylphosphatidylcholine (>99% pure synthetic DOPC) and Cholesterol (>99% pure plant-derived cholesterol) were obtained from Avanti Polar Lipids (Alabaster, AL). Monophosphoryl lipid A from Salmonella minnesota R595 (MPLA) was obtained from InVivogen (MPLA-SM VacciGrade™; cat # vac-mpla) and from Sigma (cat # L6895). Pseudomonas aeruginosa lipopolysaccharide (LPS) and a synthetic TLR7/8 agonist (R848) used as an assay control in the MIMIC® PTE module were obtained respectively from Sigma (cat # L8643) and InVivogen (cat # TLRL-R848). AS01B was retrieved from commercial packages of Shingrix® (GSK) and comprised 100 µg/mL of MPL®, 100 µg/mL of QS21, 2 mg/mL of DOPC and 0.5 mg/mL of cholesterol in phosphate-buffered saline (PBS), pH 6.1.

Model vaccine antigens

Recombinant CMV-gB and CMV-PC were expressed in Chinese hamster ovary (CHO) cells. The CMV-gB (Towne strain) was expressed as a glycoprotein deleted from its transmembrane domain to enable secretion from the CHO cells. The CMV-PC, i.e., gH/gL/pUL128/pUL130/pUL131 (BE/28/2011 strain), was obtained as a secreted glycoprotein complex by transfection of CHO cells with 5 plasmids, each plasmid coding for one of the 5 proteins constituting the CMV PC. Glycoprotein H (gH) was deleted from its transmembrane domain to enable secretion of the PC58. CMV-gB and CMV-PC were purified to ≥ 99% as assessed by SDS PAGE, by using affinity chromatography and size exclusion chromatography, respectively. Endotoxin content in the recombinant antigens assessed by using the limulus amoebocyte assay (BioWhittaker Inc., Walkersville, MD) was less than 0.05 EU of LPS/mg of protein.

Determination of E6020 and MPLA solubility in ethanol

The solubility of E6020 and MPLA (Sigma) in ethanol was determined by using nephelometry to follow opalescence due to insolubility. The products were resuspended at 0.5, 1.0, 2.0 and 10 mg/mL in absolute ethanol (Carlo Erba). One mL of each suspension was stirred for 3 h at room temperature. Nephelometry was performed on a BMG-Labtech Nephelostar with 0.200 mL samples on a UV 96-well microplate (Thermo UV Flat Bottom 96; Ref. 8404) with an absolute ethanol blank.

Preparation and characterization of liposomal adjuvants

SPA14-20, SPA14-8, and the QS21-Liposomes were prepared by an ethanol injection technique as follows. Typically, a stock solution of QS21 at 1.0 mg/mL in CBS (10 mM citrate buffer, 140 mM NaCl, pH 6.3) was prepared by resuspending 3.0 mg of QS21 powder into 3.0 mL of CBS and sterile filtration on a 25 mm Pall Acrodisc® 0.2 µm membrane. A stock solution of E6020 at 2.0 mg/mL in ethanol was prepared by dissolving 2.0 mg of E6020 powder in 0.998 mL of ethanol. A fourfold concentrated stock solution of liposomal lipids in ethanol at 40 mg/mL of DOPC, 10 mg/mL of cholesterol and 0.2 mg/mL of E6020 was prepared by dissolving 40 mg of DOPC and 10 mg of cholesterol in 0.850 mL of ethanol and adding 0.100 mL of the E6020 stock solution. For the preparation of E6020-Liposomes, 1.0 mL of the liposomal lipid solution was injected into 3.0 mL of CBS under stirring at 1000 rpm, at room temperature, by using a Hamilton syringe with a 22-gauge needle and a syringe pump at 0.1 mL/min. E6020-Liposomes were dialyzed using Slide-A-Lyzer™ 10 K MWCO dialysis cassettes (Thermo Fischer Scientific, Courtaboeuf, France) three times (half a day, overnight and one full day) against CBS pH 6.3 to remove ethanol, sterile filtered on 33 mm Millex-GV PVDF 0.22 µm filters (Merck-Millipore), and stored at +4 °C under nitrogen until used. At this stage, E6020-Liposome components concentration was estimated according to the dilution factor of dialysis. For a typical 1.6 dilution factor, the liposomes composition was 6.25 mg/mL of DOPC, 1.56 mg/mL of Chol, and 0.031 mg/mL of E6020. SPA14 was prepared in sterile conditions by addition of 1.563 mL of the QS21 stock solution and 1.250 mL of CBS pH 6.3 to 5.0 mL of E6020-Liposomes. Under these conditions, the final concentration of SPA14 components was 4 mg/mL DOPC, 1 mg/mL Chol, 0.02 mg/mL E6020, and 0.2 mg/mL QS21. The mixture was vortexed for 10 s and stored at +4 °C under nitrogen. This preparation was termed SPA14-20 (20 referring to the E6020 content in µg/mL). SPA14-8 with a lower E6020 content (8 µg/mL) was prepared by following the same procedure but starting with a 0.8 mg/mL stock solution of E6020 in ethanol. QS21-Liposomes were prepared as described for SPA14 without including E6020 in the starting lipid solution in ethanol.

Liposome characterizationSize measurements

Liposome size (z-Average) was determined by dynamic light scattering (DLS) using the Malvern Zetasizer NanoZS (Malvern, Worcestershire, UK).

Electron microscopy of SPA14

SPA14 was visualized by cryo-transmission electron microscopy (cryo-TEM). A 4 μL sample was deposited on Quantifoil R2/2 cupper 200mesh grids (Quantifoil Instruments GmbH, Germany) after 90 s of glow discharge on an ELMO ionizer (Cordouan, France). Grids were blotted and frozen using a Vitrobot MARK IV (Thermo Scientific) and transferred for observation onto a TEM Tecnai-G20 (Thermo Scientific) operated at 200 kV using a 910 cryo-holder (Gatan Inc., USA). Images were recorded at 3 μm defocus and at low-dose mode (electron doses ≤ 10 e-/Å2) using an ssCCD Ultrascan 4000 (Gatan Inc., USA). Pixel size of recorded images was estimated to 0.221 nm after TEM calibration using a cross-line grid (EMS, USA) with pitch spacing of 500 nm and 2000 lines/mm.

HPLC determination of E6020 and QS21 content and integrity in SPA14

E6020 was determined and analyzed by HILIC HPLC/MS/MS as previously described22 after solubilization of SPA14 in isopropanol/hexane/methanol 64:16:20 (v/v/v). QS21 was determined and analyzed by RP-HPLC (Agilent 1200 series) according to Brunner et al59. In brief, the separation and analysis of QS21 was performed by using an Hypersil Gold C4 column, 5 µm, 4.6 ×250 mm (Thermo Fisher Scientific; Ref. 25505-254630) maintained at 30 °C and eluted with a discontinuous acetonitrile gradient at a flow rate of 1 mL/min. The mobile phase was degassed by ultrasonic bath and consisted of 0.1% TFA in water in line A and 0.1% TFA in acetonitrile in line B. The gradient was as follows: T0: 30% B, T20 min: 55% B, T21 min: 30% B, T25 min: 30% B. The UV detector was set at 214 nm. Chromelon® 7.2 (Thermo Fisher Scientific) was used to process the data.

Hemolysis assay

The assay was performed in round bottom 96-well plates (Corning, cat#353077) using sheep red blood cells (RBCs) obtained from Rockland (Pennsylvania, PA; Ref. R405-0050). Before use, the RBCs (5 mL in a 15 mL Falcon tube) were washed twice with cold PBS by centrifugation at 4 °C for 10 min at 700 g and resuspended in 5 mL of PBS. Next, 100 µL of PBS, 100 µL of test item (QS21 in twofold serial dilutions from 200 to 1.6 µM in CBS pH 6.3) and 25 µL of the RBC suspension were subsequently added into the microwells of the 96-well plate. After 30 min of incubation at 37 °C, the plates were centrifuged for 5 min at 700 g at room temperature and 80 µL of well supernatants were transferred into a flat bottom 96-well plate (Corning, cat#3599) for absorbance reading at 540 nm using a plate reader (VersaMax – Molecular Devices). The percentage of cell hemolysis was calculated according to the formula: 100 x [(sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance)]. Negative and positive controls were obtained by replacing the test item with plain CBS or distilled water, respectively.

Immunological studiesStatements of ethics

Animal study protocols were reviewed by Sanofi Ethics Committee (Marcy L’Etoile, France). The macaque study was included in the project number APAFIS#16801–2018092011168040 v2 and the mouse studies in the project AFAPIS#16508–2018081313297024 v1, both approved by the French Ministry of Higher Education, Research and Innovation (MHERI). All experiments were conducted in accordance with the European Directive 2010/63/UE as published in the French Official Journal of February 7th, 2013. Mice and monkeys were housed in animal care facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Profiling the adjuvants by using the PTE module of the MIMIC® system PBMC preparation

Apheresis blood products were collected from donors at OneBlood (Orlando, FL). The study protocol and donor program were reviewed and approved by Chesapeake Research Review, Inc. (Columbia, MD). At the time of collection, peripheral blood mononuclear cells (PBMCs) from healthy donors were enriched by Ficoll density gradient separation and cryopreserved in DMSO-containing freezing media.

MIMIC® PTE assay

The MIMIC® PTE construct was assembled on a robotic line using published methods27,33,34. Briefly, endothelial cells were grown to confluence atop a collagen matrix (Advanced Biomatrix, San Diego, CA). Thereafter, donor PBMCs prepared from frozen stocks were applied to the assay wells. After an incubation of 90 min, non-migrated cells were removed by washing and treatments were added to the cultures at different dilutions. A mixture of 100 ng/mL LPS and 10 µg/mL R848 was used as a positive control in these assays. This study, evaluating the ability of SPA14 and AS01B to stimulate human immune cells, was designed to test two concentrations of E6020 in SPA14, i.e., 8 and 20 µg/mL, keeping constant all other SPA14 ingredients. Test items were diluted 1:20 to 1:160 in a 2-fold dose curve. To understand the contribution of the QS21-liposomes to the innate immune signature induced by SPA14 or AS01B, QS21-liposomes (minus any TLR4 agonist) were also examined at the 1:40 dilution. E6020, the TLR4 agonist in SPA14, was also dosed alone in the assay at a concentration of 200 ng/mL (corresponding to the 1:40 dilution of the SPA14 dose range) and can be compared to MPLA (InvivoGen) at 5 µg/mL (corresponding to the 1:20 dilution of the AS01B dose range). Cells and culture supernatants were harvested after a 48-hr treatment period. The culture supernatants were analyzed for cytokines/chemokines using the Milliplex® human 12-plex multi-cytokine detection system (Millipore) based on the manufacturer protocol. The kit included IFN-α2, IFNγ, IL-1β, IL-6, IL-8, IL-10, IL-12p40, IP-10, MCP-1, MIP-1β, RANTES, and TNFα. PGE2 secretion was evaluated by ELISA following the manufacturer’s protocol (Arbor assay, MI, cat # K051-H5, MI). The cells were phenotyped using various markers by flow cytometry. Data were exported to GraphPad Prism (GraphPad Software, San Diego, CA, USA) for graph preparation.

Cytokine secretion assay

Analyte concentrations were calculated based on relevant standard curves using the Bio-Plex® manager software. For run acceptance criteria, LLOQ and ULOQ for each analyte were established based on the percent recovery (Observed/Expected*100) of each point against a 5-parameter logistic (5PL) curve fit of the standard values. A recovery percentage of 80 - 120% was considered acceptable, such that values falling within this range were used to set the lower and upper bounds of the standard curve. The raw data file was reviewed for bead counts; a data point was considered valid when a minimum of 35 beads per region was counted.

Cellular phenotype by flow cytometry

MIMIC® PTE-derived cells were washed with PBS and labeled with Live-Dead Aqua (Thermo Fisher Scientific, USA) for 20 min on ice. After washing and performing an IgG-Fc block (Normal mouse serum; Cat # 015-000-120, Jackson Immuno Research Laboratories), the cells were incubated with a cocktail of fluorochrome-labeled monoclonal antibodies, such as anti-CD14, anti-HLA-DR, anti-CD11c, anti-CD86, anti-CD25, anti-CD3 and anti-CD19, that are specific for non-myeloid lineage cells and immune ligands (BD Biosciences, San Jose, CA). Thereafter, the cells were washed with buffered media and acquired on a BD Fortessa flow cytometer equipped with BD FACS Diva software (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR). For flow gating, doublets were first excluded from the live-cell population and then lymphocytes (CD3 + , CD19 + ) were removed from the analysis using a dump-channel approach. Next, HLA-DR+ cells were gated into CD11c+ monocytic DCs. Thereafter, mDC subpopulations were analyzed for their expression of HLA-DR and maturation/activation markers (CD14, CD86 and CD83) by Mean Fluorescent Intensity (MFI) and % of positive cells.

Preparation of experimental vaccine formulations for animal studies

The experimental vaccine formulations comprising CMV gB + PC were prepared by diluting and mixing concentrated antigen stock solutions, followed by the addition of the adjuvant. Specifically, for AS01B, 50 µl of an antigen mixture at 400 µg/mL of CMV-gB and 400 µg/mL of CMV-PC in PBS were added into 500 µL of AS01B (as retrieved from the Shingrix package). Mice and NHPs received 55 µL and 550 µL of vaccine formulation, respectively. The adjuvanted vaccine formulations were used within 3 h of preparation. In this way, monkeys in the AS01B group received 20 µg CMV-gB + 20 µg CMV-PC + 50 µg QS21 + 50 µg MPL®.

For SPA14-20 and SPA14-8, 250 µl of an antigen mixture at 80 µg/mL of CMV-gB and 80 µg/mL of CMV-PC in PBS were added to 250 µL of SPA14 (or to 250 µl of plain CBS in case of antigen alone group). Mice were immunized within 3 h with 50 µl and monkeys with 500 µl of the preparations. In this way, monkeys in the SPA14 groups received 20 µg CMV-gB + 20 µg CMV-PC + 50 µg QS21 + 5 µg or 2 µg E6020.

Immunization of mice

C57BL/6 J female mice (16 per group), aged 6–8 weeks (body weight 18–20 g) at the time of first immunization were obtained from Charles River Laboratories (Saint Germain sur l’Arbresle, France). Mice were immunized twice, 3 weeks apart (D0, D21), by intramuscular injection into the quadriceps. Immunization was performed under gaseous anesthesia with 5% isoflurane using a TEM SEGA gas anesthesia table for rodents. Blood samples were collected at different time points following immunization for the analysis of antibody responses (D20, D35, D91, D179) by ELISA and/or virus plaque reduction assays. Intermediate blood samplings were performed under 5% isoflurane gas anesthesia. Terminal blood samplings were performed under deep chemical anesthesia. Mice were injected by the intra-peritoneal route with 0.1 mL/10 g body weight of a Ketamine plus Xylazine mixture (80 mg/kg of Imalgène® 1000 + 16 mg/kg Rompun® 2%). After anesthesia, terminal blood sampling was performed by carotid section. After spleen collection, mice were euthanized by cervical dislocation.

Eight mice per group were euthanized at D35 and the remaining mice were euthanized at D179. Individual spleens were collected in a sterile 50 mL Falcon tube containing RPMI medium and mechanically dissociated using GentleMACS (Miltenyi Biotec). The tubes were centrifuged at 500 g for 10 min at 4 °C and the supernatants discarded. Spleen cell pellets were resuspended with 1 mL of red blood cell lysis buffer (Sigma; cat # R 7757) and gently mixed for 1 min. The lysis reaction was stopped by placing the tubes in crushed ice followed by an addition of 20 mL of cold RPMI medium. The tubes were then centrifuged at 500 g for 7 min and the supernatants discarded. Spleen cell pellets were resuspended in RPMI containing 10% fetal calf serum (FCS) and prepared for cell counting before use in B and T cell ELISPOT assays using Fluorospot kits described below.

Immunization of NHPs (macaques)

Groups of 5 male cynomolgus macaques (2.4–3.5 kg body weight) obtained from Noveprim (Camarney, Spain) were housed in collective cages based on social affinity under controlled humidity, temperature, and light (12-hour light/12-hour dark cycles) and allowed to acclimatize for 4 weeks before study procedures. The macaques were monitored and fed 1–2-times daily with commercial monkey chow and fruits, and water ad libitum. Environmental enrichment provided included toys, novel foodstuffs, and music. They were immunized twice 8 weeks apart (D0, D56) and boosted 6 months later (D189) with 500 µL of the vaccine formulations by intramuscular injection into the upper arm deltoid muscle. Blood samples were collected pre-immunization (D-8) and at different time points following immunization (D28, D55, D84, D118, D168, D181, D217, D237, D273, D300, D331, D357 and then every 2-3 months over a 1.5-year follow-up period). For experimental procedures such as animal handling, weighing injection point observation, animals were trained during the acclimatization period and these procedures were conducted with conscious animals. Immunizations and blood sampling were conducted after mild sedation using ketamine hydrochloride (Imalgene 1000, Boehringer, 10 mg/kg, intramuscular route). Blood and serum samples were used for the analysis of virus-neutralizing antibody responses by plaque reduction assay, and specific T cell responses (D197, 8 days post-boosting) and memory B cell responses (D217, four weeks postboosting) by ELISPOT. For ELISPOT assays, blood was collected in Sodium-Heparin tubes (Becton-Dickinson Vacutainer®; Ref. 267876) for PBMC isolation. PBMCs were stored frozen in liquid nitrogen until analysis. Clinical observations, including temperature and injection site observation, were performed over three days after each injection. Animals body weight was also monitored all along the study.

Determination of binding antibody titers in mouse serum

Serum antibodies directed against CMV gB or PC were titrated by ELISA. In brief, Dynex 96-well microplates were coated overnight at 4 °C with 100 µL of CMV-gB (1 µg/mL) or CMV-PC (2 µg/mL), in 0.05 M carbonate/ bicarbonate buffer, pH 9.6 (Sigma). Next, plates were blocked for at least 1 h at 37 °C with 150 μL/well of PBS-Tween-milk (PBS pH 7.1, 0.05% Tween 20, 1% (w/v) powdered skim milk (DIFCO). All subsequent incubations were carried out in a final volume of 100 μL, each followed by 3 washes with PBS-Tween. Two-fold serial dilutions of serum samples were performed in PBS-Tween-milk (starting from 1/100, 1/1000 or 1/10000) and were added to the wells. Plates were incubated for 90 min at 37 °C. After washing, goat anti-mouse IgG1- or IgG2c-HRP (Southern Biotech, cat# 1070-05 and 1079-05, respectively) diluted in PBS-Tween-milk at 1/2000 were added to the wells. Plates were incubated for 90 min at 37 °C, washed and further incubated in the dark for 30 min at room temperature with 100 μL/well of a ready-to-use Tetra Methyl Benzidine (TMB) substrate solution (TEBU, cat# TMBW1000-01). The reaction was stopped with 100 μL/well of HCl 1 M (Prolabo, cat# 30024290). Optical density (OD) was measured at 450 nm-650 nm with a plate reader (VersaMax – Molecular Devices). Specific antibody titers were determined using the CodUnit software, for an OD value range of 0.2 to 3.0 from the titration curve (reference mouse hyperimmune serum put on each plate). The titer of the references, expressed in arbitrary ELISA units (EU), was previously calculated as the reciprocal dilution giving an OD of 1.0. The threshold of antibody detection was 10 EU (1.0 log10).

Determination of neutralizing antibody titers

Neutralizing antibodies in sera from immunized mice and macaques were titrated using a plaque reduction seroneutralization assay on ARPE-19 epithelial cells and on MRC-5 fibroblasts as described previously60.

Specific CMV-gB and PC memory B cell frequencies in mice and NHPs

For the NHP study, PBMCs were isolated from whole blood collected into Vacutainer tubes with sodium heparin (Becton Dickenson) at regular time points. Whole blood was diluted to 1:3 in PBS and 30 ml of diluted whole blood layered over 15 mL of Lymphoprep 95% (Stemcell). After centrifugation, the buffy coat was harvested and washed with RPMI + 10% FBS. Splenocytes (for mouse studies) or PBMCs (for NHP studies) were counted using a Muse cell counter. Fresh PBMCs or mouse splenocytes were plated for 3 days in 6-well culture plates in RPMI medium plus R848 (1 µg/mL) and IL-2 (10 ng/mL).

For NHP studies, CMV-gB and CMV-PC specific memory B cell ELISPOT were performed using a commercial kit from Mabtech (Human IgG/IgM Fluorospot kit, cat # FS-05R17G-10). Multiscreen™ 96-well IPFL plates (Mabtech; Ref. 3654-FL-10) were pre-wetted with 25 µL of 35% ethanol for 1 min, washed with PBS and coated overnight at 5 °C with 100 µL/well of either CMV-gB, CMV-PC (10 µg/mL) or anti-IgG or -IgM antibodies from Mabtech (15 µg/mL). Next, the plates were washed with PBS and blocked for 2 h at 37 °C with RPMI containing 10% FBS, 200 mM L-Glutamine, 100 U/mL penicillin, and 10 μg/mL streptomycin. After washing with PBS, 4x105 R848 plus IL-2 treated PBMCs from immunized NHPs were plated per well and incubated for 5 h at 37 °C in a 5% CO2 incubator. After 6 washes in PBS-0.5% BSA, the plates were incubated for 2 h at 37 °C with 100 μL/well of anti-human IgG-Cy3 conjugate (Mabtech; cat # MT78/145), and anti-human IgM-FITC conjugate (Mabtech; cat # MT22). After extensive washing with PBS, fluorescent spots were enumerated with an automated spot reader equipped with filters for Cy3 and FITC (Microvision Instruments, Evry, France). The same protocol was used for mouse studies, with anti-mouse antibodies: goat anti-mouse total IgG (KPL, cat#01-18-02), goat anti-mouse IgG1-PE conjugate (Southern biotech; cat # 1070-09), goat anti-mouse IgG2c-FITC conjugate (Southern biotech; cat # 1079-02), goat anti-mouse IgG-PE conjugate (Southern biotech; cat # 1032-09).

Determination of IFN-γ / IL-5 (mouse) or IFN-γ / IL-4 (NHP) secreting T cells specific to CMV-gB and CMV-PC

Mouse and NHP Fluorospot kits were purchased from Mabtech (cat # FS-4143-10 for mouse and Fluorospot flex for NHP). Multiscreen™ 96-well IPFL plates were prepared as described above for the B cell assay and coated overnight at 5 °C with 100 µL/well of rat anti-mouse IFN-γ or rat anti-mouse IL-5 mAb (15 μg/mL). Next, the plates were washed with PBS and blocked for 2 h at 37 °C with RPMI containing 10% FBS, 200 mM L-Glutamine, 100 U/mL penicillin and 10 μg/mL streptomycin. After washing with PBS, 5×105 freshly isolated spleen cells (mice) or PBMCs (NHPs) were added per well and incubated for 24 h with CMV-gB (0.1 μg/mL), CMV-PC (0.1 μg/mL), medium alone as negative control or concanavalin A (2.5 μg/mL) as a positive control. After 6 washes with 0.1% bovine serum albumin (BSA) in PBS (200 µL per well), anti-mouse or NHP IFN-γ BAM-conjugated or biotinylated anti-mouse IL-5 or anti-NHP IL-4 antibodies at 2 µg/ml in PBS-0.1% BSA were added (100 μL per well) and incubated for 2 h at room temperature in the dark. After 6 washes in PBS-0.1% BSA, 100 µL of streptavidin-Cy3 conjugate or FITC conjugated anti-BAM antibody (1/200 dilution) in PBS-0.1% BSA were added per well and incubated for 2 h in the dark. After 6 washes with PBS-0.1% BSA, fluorescent spots were enumerated with an automated spot reader equipped with a filter for PE fluorescence (Microvision). Results were expressed as number of specific IFN-γ-, IL-5- or IL-4-secreting cells per 106 cells after subtracting fluorospot counts from negative control wells.

Statistical analyses

For the MIMIC® PTE study, statistical analysis was performed by unpaired, two-tailed t-test for relevant conditions using GraphPad Prism version 10.1.2. For the cytokine heatmap analysis, geometric mean values were calculated for all donors, treatment, and analytes. The heatmap was made in R studio Version 4.1.2 with heatmap package [https://cran.r-project.org/package=pheatmap] using log2 (fold change) values relative to geometric mean values. The heatmap package was downloaded from the Comprehensive R Archive Network (CRAN) repository. For certain comparisons, one-way ANOVA with Tukey post-tests (GraphPad Prism) were performed, with p < 0.05 being considered statistically significant. For mouse and NHP studies, group comparisons were performed using one- or two-way or repeated ANOVA as a function of the readout on SAS v9.2 (SAS Institute, Cary, NC). For data with several timepoints (i.e., antibody responses over time) an ANOVA from a mixed model with group, time and their interaction was applied. The type of covariance structure used in the model was decided based on the lowest AIC criterion obtained. To account for multiple testing, a Dunnett or Tukey adjustment was applied for group comparisons.

Computer code and algorithm

The heatmap was made in R studio Version 4.1.2 with heatmap package [https://cran.r-project.org/package=pheatmap].