Carlos Ortiz

A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates

DISCUSSION

The recent success in the rapid development of safe and efficacious SARS-CoV-2 vaccines has been tempered by the emergence of virus variants to which vaccine-induced immunity has shown diminished potency or efficacy (3033). Thus, there remains a need for next-generation vaccines that target the broadening antigenic diversity of SARS-CoV-2 and related coronaviruses. The major vaccines that have progressed to human efficacy trials and have been granted either approval or emergency use authorization all present the SARS-CoV-2 spike protein that is based on the genetic sequence of the Wuhan-Hu-1 isolate. All of these vaccines have demonstrated protective efficacy in NHPs against respiratory mucosal challenge with the closely matched USA-WA1/2020 (25, 3438). These earlier animal studies, however, did not evaluate the neutralization capacity of serum against other coronavirus species. In the current study we demonstrate that an adjuvanted recombinant nanoparticle vaccine, SpFN, elicited high titers of that antibodies neutralized SARS-CoV-2 and rapidly protected against SARS-CoV-2 infection. We also found that, compared to wild type virus, SpFN elicited serum virus neutralizing activity that was either higher or equivalent against four major VOCs (B.1.1.7, B.1.351, P.1, B.1.617.2) in an authentic virus neutralization assay and equivalent or mildly diminished against two VOCs in a pseudovirus neutralization assay (B.1.1.7, B.1.351). Finally, SpFN induced robust neutralizing activity against SARS-CoV-1, a separate species that has 26% and 36% sequence divergence in the spike protein and S1 subunit, respectively (39), which is important for protection in animal models (40, 41).SARS-CoV-2 vaccine efficacy studies in NHPs generally compare elicited antibody responses to those from patients who have recovered from COVID-19. We found neutralizing activity in the two-dose 50 μg SpFN group to be ten-fold higher than that in recovering patients. The lack of standardization across convalescent serum panels and absence of head-to-head comparisons of candidate vaccines makes it difficult to compare immunogenicity profiles. Direct comparisons come with a qualification that immunogenicity output assays have been in the process of harmonization. Bearing this caveat in mind, we found that SARS-CoV-2 antibody responses in animals vaccinated with high-dose SpFN were robust when set in the context of the entire vaccine landscape, to include genetic vaccines (34, 38), recombinant virus vector vaccines (25, 37), and adjuvanted protein subunit vaccines (35). Although direct quantitative comparisons of NHP vaccine studies can be difficult to interpret, we have made attempts to mitigate differences in assay outputs by analyzing specimens with orthogonal assays harmonized to consensus platforms. Specifically, the pseudovirus neutralization assay we developed for our immunogenicity assessments demonstrated equivalence to platforms used for the assessment of other major vaccines through participation in the SARS-CoV-2 Neutralizing Assay Concordance Survey 1 coordinated by the External Quality Assurance Program Oversight Laboratory/Virology Quality Assurance Program at the Duke Human Vaccine Institute (42).Potent neutralizing antibody responses may offer advantages for both vaccine efficacy and durability. Thus far, neutralizing activity has been predictive of efficacy in human trials, as vaccines that generate lower antibody titers have diminished efficacy (43). An open question remains, however, regarding the length of immunity conferred by SARS-CoV-2 vaccines. Among viral infections for which neutralizing antibodies are the primary correlate of protection, peak titers have been shown to be predictive of durability and may serve as one of several indicators of length of vaccine-elicited protective immunity (4446). As such, SpFN may offer some measure of a durable immune response; though this will require empirical confirmation.Overall, SpFN vaccination induced serum cross-neutralizing antibody responses. Additionally, we found serum binding to mutated SARS-CoV-2 RBD was either unaffected or mildly reduced. We also found serum binding to mutated SARS-CoV-2 RBD was either unaffected or mildly diminished. Other reports of nanoparticle vaccine approaches presenting RBD also have shown a breadth of neutralizing antibody responses against multiple sarbecoviruses (4749). Similar to other studies, SpFN vaccination induces comprehensive binding and neutralizing antibody responses and a balanced cellular immune response against SARS-CoV-1. Although background neutralizing activity was high in one assay, neutralizing potency against SARS-CoV-1 was confirmed in an orthogonal virus neutralization assay. Still, we plan to test purified IgG from vaccine serum in both assays to control for the background activity at baseline and in controls.We hypothesize that the breadth of immune response elicited by the SpFN vaccine may be the result of several factors. First, the quantity of the polyclonal antibody response may surpass a threshold that overcomes resistance to neutralization of antigenically distinct virus variants. Second, repetitive, ordered display of antigen on a self-assembling nanoparticle has been shown to drive an expanded germinal center reaction with resultant increases in B cell receptor mutation, affinity maturation and plasma cell differentiation (57). Lastly, the adjuvant, ALFQ, may drive some of the breadth through CD4 T cell activation (50, 51), especially given the high Th1 response elicited by the co-formulation. ALFQ, as compared to aluminum hydroxide (Alhydrogel), previously has demonstrated superior immunogenicity when administered with SpFN to C57BL/6 and BALB/c mice (20). As NHPs are a more predictive model with respect to adjuvant performance in humans, we are now conducting a follow-on adjuvant comparison study in NHPs to evaluate the impact of ALFQ on immunogenicity potency and breadth. Additionally, as NHPs do not exhibit the same degree of fidelity as Syrian golden hamsters in terms of developing severe COVID-19 disease (21), we have also found SpFN to protect against VOCs in challenge experiments in the latter model (52).The interpretation of the results from this study is limited by several factors. First, we were able evaluate efficacy our vaccine candidate only against challenge with the Wuhan-Hu-1 strain of SARS-CoV-2. This is because VOCs were only beginning to emerge and circulate when this study was conceived and initiated. Our assessment of breadth of the immune response was, instead, evaluated by serum neutralizing activity against a panel of SARS-CoV-2 VOCs as well as SARS-CoV-1. Second, we cannot define, from the design and outputs of this study, the relative contributions of the immunogen and adjuvant of our vaccine formulation to the potency and breadth of immune response observed. Finally, we have yet to understand the role and repertoire of the humoral and cellular immune responses elicited by this SpFN. We are carrying out follow on studies examine the mechanisms of immunity conferred by our vaccine candidate.

The collective immune response elicited by SpFN translated into a robust and rapid reduction in replicating virus in the upper and lower airways of animals and resultant prevention of pulmonary pathology. It is notable that SpFN protected against a potent viral challenge, as replicating virus concentrations detected in the upper and lower airways of unvaccinated controls reached a mean of 106 to 107 copies/ml. Despite this higher challenge, SpFN still protected lower airway viral burden and disease as early as within one day of virus inoculation. The rapid elimination of replicating virus in the upper airways also lends evidence for potential near-sterilizing immunity, which again may have implications for preventing viral transmission. Altogether, these findings support the further development of SpFN, which has now moved to human evaluation in a phase 1 clinical trial (53).

MATERIALS AND METHODS

Study design. Thirty-two male and female specific-pathogen-free, research-naïve Chinese-origin rhesus macaques (age 3 to 7 years) were distributed—on the basis of age, weight, and sex—into 4 cohorts of 8 animals (table S1). Sample sizes were set based animal availability and prior experience with other nonhuman primate vaccination/challenge studies. Animals were assigned to groups without a specific randomization scheme; however, no outliers were skewed to any one group (table S1). Animals were vaccinated intramuscularly with either 50 or 5 μg of SpFN, formulated with ALFQ, or 1 ml of PBS in the anterior proximal quadriceps muscle, on alternating sides with each dose in the series. Immunizations were administered twice, 4 weeks apart, or once, 4 weeks prior to challenge. Animals were challenged with virus stock obtained through Biodefense and Emerging Infections Research Resources Repository Resources (BEI Resources), National Institute of Allergy and Infectious Diseases, National Institutes of Health: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-53780 (Lot# 70038893). Virus was stored at -80°C prior to use, thawed by hand and placed immediately on wet ice. Stock was diluted to 5×105 TCID50 per ml in PBS and vortexed gently for 5 s prior to inoculation by combined intratracheal (1 mL) and intranasal routes (0.5 mL per nostril).

All procedures were carried out in accordance with institutional, local, state and national guidelines and laws governing animal research included in the Animal Welfare Act. Animal protocols and procedures were reviewed and approved by the Animal Care and Use Committee of both the US Army Medical Research and Development Command (USAMRDC, protocol 11355007.03) Animal Care and Use Review Office as well as the Institutional Animal Care and Use Committee of Bioqual, Inc. (protocol number 20-092), where nonhuman primates were housed for the duration of the study. USAMRDC and Bioqual, Inc. are both accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and are in compliance with the Animal Welfare Act and Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Vaccine and adjuvant design and production. The spike ferritin nanoparticle (SpFN) vaccine immunogen was produced by linking Helicobacter pylori ferritin to the C-terminal region of the pre-fusion stabilized ectodomain (residues 12-1158) of the SARS-CoV-2 spike protein in a genetic fusion. The immunogen was expressed, purified, and characterized as previously described (20). Briefly, SpFN was derived from the Wuhan-Hu-1 strain genome sequence (GenBank MN9089473), synthesized by Genscript Inc, and subcloned into a modified cytomegalovirus promoter (CMVR) expression vector. Expi293F cells (Thermo Fisher Scientific) were transiently transfected with SpFN plasmid DNA using ExpiFectamine 293 transfection reagent (Thermo Fisher Scientific). Cells were grown in polycarbonate baffled shaker flasks at 34°C and 8% CO2 at 120 revolutions per minute (rpm). Cells were harvested 5 days post-transfection by centrifugation at 3,500 × g for 30 min. Culture supernatants were filtered with a 0.22-μm filter and stored at 4°C prior to purification using GNA lectin affinity chromatography. Briefly, 25 mL GNA-lectin resin (VectorLabs) was used to purify SpFN from 1L of expression supernatant. GNA resin was equilibrated with 10 column volumes (CV) of PBS (pH 7.4) followed by supernatant loading twice at 4°C. Unbound protein was removed by washing with 20 CV of PBS buffer. Bound protein was eluted with 250mM methyl-α-D mannopyranoside in PBS buffer (pH 7.4). SpFN was further purified by size-exclusion chromatography using a 16/60 Superdex-200 purification column. Purification purity for all the proteins was assessed by SDS-PAGE. Endotoxin concentrations for SpFN was assessed (Endosafe nexgen-PTS, Charles River Laboratories) and 5% v/v glycerol was added prior to filter-sterilization with a 0.22-μm filter, flash-freezing in liquid nitrogen and storage at -80°C. SpFN antigenicity was assessed by Octet Biolayer Interferometry with a set of antibodies that included CR3022 (20) and SR1-SR5 (potent neutralizing antibodies targeting the ACE2 binding site of the RBD; kindly provided by S. Rajan, P.M. McTamney, and M.T. Esser of Astra Zeneca). Size and consistency of the SpFN immunogen was determined by negative-stain electron microscopy. These measurements ensured lot-to-lot consistency of the SpFN immunogen.The adjuvant, Army Liposomal Formulation with QS21 (ALFQ), was prepared as previously described (54). Briefly, ALFQ is a unilamellar liposome that contains saturated phospholipids, cholesterol, monophosphoryl lipid A and the saponin, QS-21. It is comprised of dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol (Chol), and synthetic monophosphoryl lipid A (3D-PHAD) (Avanti Polar Lipids) and QS-21 (Desert King). DMPC and Chol were dissolved in chloroform and DMPG and 3D-PHAD were dissolved in chloroform:methanol at a ratio of 9:1. The lipids were mixed in a molar ratio of 9:1:12.2:0.114 (DMPC:DMPG:Chol:3D-PHAD) and the solvent was removed by rotary evaporation. 3D-PHAD and QS-21 doses were 200 and 100 μg, respectively. The lipids were suspended in Sorenson’s PBS, pH 6.2, microfluidized to form small unilamellar vesicles (SUV) and filtered. QS-21 was solubilized in Sorenson’s PBS, pH 6.2, filtered and added to the SUV to form ALFQ. The final lipid ratio was 9:1:12.2:0.114:0.044 (DMPC:DMPG:Chol:3D-PHAD:QS-21).

Immunogen was diluted in Dulbecco’s PBS (Lot#723188, Quality Biological) to 0.1 mg/ml or 0.01 mg/ml and mixed 1:1 with 2X ALFQ liposomes on a tilted slow-speed roller at room temperature for 10 min, followed by incubation at 4°C for 50 min. All reagents were equilibrated to room temperature before use and immunizations were performed within 4 hours of vaccine formulation. Each vaccine comprised a 1.0 ml solution of SpFN formulated with ALFQ.

Convalescent Plasma Samples. A panel of 41 human convalescent-phase plasma samples were obtained from BEI Resources Repository (n=30), StemExpress (n=7) and a Walter Reed Army Institute of Research institutional review board-approved leukapheresis protocol (#1386H) (n=4) for which written informed consent was provided by participants. Samples were collected from males (n=20) and females (n=21) ranging in age from 31 to 71 years. Individuals donated plasma specimens approximately four-to-eight weeks after laboratory-confirmed SARS-CoV-2 infection and had histories of asymptomatic-to-mild-to-moderate clinical presentation.

Binding antibody measurements. SARS-CoV-2-specific binding IgG antibody responses were measured using MULTI-SPOT 96-well plates from Meso Scale Discovery (MSD). Multiplex wells were coated with spike (S), RBD and nucleocapsid (N) antigens at a concentration of 200-400 ng/ml and bovine serum antigen, which served as a negative control. Four-plex MULTISPOT plates were blocked with MSD Blocker A buffer for 1 hour at room temperature, shaking at 700 rpm. Plates were washed with buffer before the addition of reference standard and calibrator controls. Serum samples were diluted at 1:1,000 to 1:100,000 in diluent 100 buffer (provided with manufacturer kit), then added to each of four wells. Plates were incubated with shaking at 700 rpm for two hours at room temperature and then washed. MSD SULFO-TAG anti-IgG antibody was added to each well. Plates were incubated for 1 hour at room temperature with shaking at 700 rpm and washed, then MSD GOLD Read buffer B was added to each well. Plates were read by the MESO SECTOR SQ 120 Reader. IgG concentration was calculated using DISCOVERY WORKBENCH MSD Software and reported as arbitrary units (AU)/ml.

The ability of SARS-CoV-2 spike-specific binding antibodies to inhibit spike protein or RBD binding to the ACE2 receptor was also measured using MULTI-SPOT 96-well plates (MSD). Antigen-coated plates were blocked and washed as described above. Assay calibrator and samples were diluted at 1:25 to 1:1,000 in MSD Diluent buffer, then added to the wells. Plates were incubated for 1 hour at room temperature, shaking at 700 rpm. ACE2 protein conjugated with MSD SULFO-TAG was added and plates were incubated for 1 hour at room temperature, shaking at 700rpm. Plates were washed and read as described above. AU/ml concentration of inhibitory antibodies was calculated with DISCOVERY WORKBENCH MSD Software.

Binding antibody measurements by octet biolayer interferometry were made on biosensors of the Octet FortéBio Red96 instrument (Sartorius) that were hydrated in PBS prior to use. All assay steps were performed at 30°C with agitation set at 1,000 rpm. Baseline equilibration of the anti-His-tag biosensors (HIS1K biosensors with a conjugated Penta-His antibody (Sartorius) was carried out with PBS for 15 s, prior to SARS-CoV2-RBD (30 μg/ml diluted in PBS) loading for 120 s. After dipping in assay buffer (15 s in PBS), biosensors were dipped in the serum samples (100-fold dilution) for 180 s. Binding response (nm) at 180 s was recorded for each sample.

SARS-CoV-1 and SARS-CoV-2 Pseudovirus Neutralization. The spike protein expression plasmid sequence for SARS-CoV-2 was codon optimized and modified to remove an 18 amino acid endoplasmic reticulum retention signal in the cytoplasmic tail to improve spike protein incorporation into pseudovirions (PSV) and thereby enhance infectivity. SARS-CoV-2 PSV were produced by co-transfection of HEK293T/17 cells with a SARS-CoV-2 spike protein-expressing plasmid (pcDNA3.4), derived from the Wuhan-Hu-1 genome sequence (GenBank accession number: MN908947.3) and an HIV-1 (pNL4-3.Luc.R-E-, NIH HIV Reagent Program, Catalog number 3418). Infectivity and neutralization titers were determined using ACE2-expressing HEK293 target cells (Integral Molecular) in a semi-automated assay format using robotic liquid handling (Biomek NXp Beckman Coulter). Virions pseudotyped with the vesicular stomatitis virus (VSV) G protein were used as a non-specific control. Serum samples were diluted 1:40 in growth medium (10% fetal bovine serum, 2.5% HEPES, 0.5% Gentamicin, 0.1% Puromycin in Dulbecco’s Modified Eagle Medium); then 25 μl/well was added, in triplicate, to a white 96-well plate. An equal volume of diluted SARS-CoV-2 PSV was added to each well and plates were incubated for 1 hour at 37°C. Target cells were added to each well (40,000 cells/ well) and plates were incubated for an additional 48 hours. Relative light units (RLU) were measured with the EnVision Multimode Plate Reader (Perkin Elmer) using the Bright-Glo Luciferase Assay System (Promega). Neutralization dose–response curves were fitted by nonlinear regression using the LabKey Server, as previously described (55). Final titers are reported as the reciprocal of the dilution of serum necessary to achieve 50% (ID50) and 90% neutralization (ID90). Assay equivalency was established by participation in the SARS-CoV-2 Neutralizing Assay Concordance Survey (SNACS) run by the Virology Quality Assurance Program and External Quality Assurance Program Oversite Laboratory (EQAPOL) at the Duke Human Vaccine Institute, sponsored through programs supported by the National Institute of Allergy and Infectious Diseases, Division of AIDS.

Authentic SARS-CoV-2 wild-type neutralization assay. Authentic virus neutralization was measured using SARS-CoV-2 (2019-nCoV/USA_WA1/2020) that was obtained from the Centers for Disease Control and Prevention and passaged once in Vero CCL81 cells (American Type Culture Collection (ATCC)). Rhesus serum samples were serially diluted and incubated with 100 focus-forming units (FFU) of SARS-CoV-2 for 1 hour at 37°C. Serum-virus mixtures were added to Vero E6 cells in 96-well plates and incubated for 1 hour at 37°C. Cells were overlaid with 1% (w/v) methylcellulose in Minimal Essential Media (Sigma-Aldrich). After 30 hours, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature then washed and stained overnight at 4°C with 1 μg/ml of antibody CR3022 in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. Cells were subsequently stained with horseradish peroxidase (HRP)-conjugated goat anti-human IgG for 2 hours at room temperature. SARS-CoV-2-infected cell foci were visualized with TrueBlue peroxidase substrate (KPL) and quantified using ImmunoSpot microanalyzer (Cellular Technologies). Neutralization curves were generated with Prism software (GraphPad Prism 8.0).

Authentic SARS-CoV-2 variant and SARS-CoV-1 neutralization assay. The SARS-CoV-2 viruses USA-WA1/2020 (WA1), USA/CA_CDC_5574/2020 (B1.1.7), hCoV-19/South Africa/KRISP-EC-K005321/2020 (B.1.351), hCoV-19/Japan/TY7-503/2021, hCoV-19/USA/PHC658/2021 (B.1.617.2) were obtained from BEI Resources (NIAID, NIH) and propagated for one passage using Vero clone E6 cells. Virus infectious titer was determined by an end-point dilution and cytopathic effect (CPE) assay on Vero-E6 cells as described previously (56, 57). An end-point dilution microplate neutralization assay was performed to measure the neutralization activity of NHP serum samples. In brief, serum samples were heat inactivated and subjected to successive three-fold dilutions starting from 1:50. Triplicates of each dilution were incubated with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 in Eagle’s Minimal Essential Medium (EMEM) with 7.5% inactivated fetal calf serum (FCS) for 1 hour at 37°C. Post incubation, the virus-antibody mixture was transferred onto a monolayer of Vero-E6 cells grown overnight. The cells were incubated with the mixture for about 70 hours. CPE of viral infection was visually scored for each well in a blinded fashion by two independent observers. The results were then reported as percentage of neutralization at a given sample dilution. A SARS-CoV-1 authentic plaque reduction virus neutralization assay was performed similarly to previously described (58), with the following modifications. The starting dilution of serum was 1:5 and about 100 plaque-forming units of virus were used for virus and serum incubation. The overlay used after virus adsorption was Dulbecco’s Modified Eagle Medium (Gibco) containing 2% FBS and 20% methylcellulose. Plates were then incubated for 5 days, and post crystal violet staining the washing step utilized water.​ Plaques were graded as follows: approximately 25 plaques/25% monolayer damage (MD; -/+); approximately 50 plaques/50% MD) (+); approximately 75 plaques/75% MD (++); approximately 100 plaques/100% MD (+++). All negative control wells were solid monolayers.

Antibody-Dependent Neutrophil Phagocytosis (ADNP). Biotinylated SARS-CoV-2 prefusion stabilized S trimer was incubated with yellow-green streptavidin-fluorescent beads (Molecular Probes) for 2 hours at 37°C. Ten μl of a 100-fold dilution of beads and protein was incubated for 2 hours at 37°C with 100μl of 8,100-fold diluted plasma samples before addition of effector cells (50,000 cells/well). Fresh human peripheral blood mononuclear cells were used as effector cells after red blood cell lysis with ACK lysing buffer (Thermo Fisher Scientific). After 1 hour, incubation at 37°C, the cells were washed, surface stained, fixed with 4% formaldehyde solution (Tousimis) and fluorescence was evaluated on an LSRII flow cytometer (BD Biosciences). Antibodies used for flow cytometry included anti-human CD3 Alexa Fluor 700 (clone UCHT1) and anti-human CD14 Allophycocyanin Cynanine Dye 7 (APC-Cy7) (clone MϕP9) from BD Biosciences, as well as anti-human CD66b Pacific Blue (clone G10F5) from BioLegend. A phagocytic score was calculated by multiplying the percentage of bead-positive neutrophils (side scatter high, CD3-CD14-CD66+) by the geometric mean of the fluorescence intensity of bead-positive cells; and dividing by 10,000.

Antibody-Dependent Cellular Phagocytosis (ADCP). ADCP was measured as previously described. Briefly, biotinylated SARS-CoV-1 or SARS-CoV-2 prefusion-stabilized spike protein trimer was incubated with red streptavidin-fluorescent beads (Molecular Probes) for 2 hour at 37°C. Ten μl of a 100-fold dilution of beads–protein was incubated for 2 hours at 37°C with 100μl of 8,100-fold (SARS-CoV-2) or 900-fold (SARS-CoV-1) diluted plasma samples before addition of THP-1 cells (20,000 cells per well; Millipore Sigma). After a 19 hour incubation at 37°C, the cells were fixed with 2% formaldehyde solution (Tousimis) and fluorescence was evaluated on an LSRII flow cytometer (BD Biosciences). The phagocytic score was calculated by multiplying the percentage of bead-positive cells by the geometric mean of the fluorescence intensity of bead-positive cells, and dividing by 10,000.

Antibody-Dependent Complement Deposition (ADCD). SARS-CoV-2 spike protein-expressing expi293F cells were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 spike protein matching the amino acid sequence of the IL-CDC-IL1/2020 isolate (GenBank ACC# MN988713). Stable transfectants were single-cell sorted and selected to obtain a high spike surface-expressing clone (293F-spike-S2A). ADCD was adapted from methods previously described (59). Briefly, SARS-CoV-2 spike protein-expressing expi293F cells were incubated with 10-fold diluted, heat-inactivated (56°C for 30 min) plasma samples for 30 min at 37°C. Cells were washed twice and resuspended in R10 media. During this time, lyophilized guinea pig complement (CL4051, Cedarlane) was reconstituted in 1 ml cold water and centrifuged for 5 min at 4°C to remove aggregates. Cells were washed with PBS and resuspended in 200 μl of guinea pig complement, which was prepared at a 1:50 dilution in Gelatin Veronal Buffer with Ca2+ and Mg2+ (IBB-300x, Boston BioProducts). After incubation at 37°C for 20 min, cells were washed in PBS 15mM EDTA (Thermo Fisher Scientific) and stained with an anti-Guinea Pig Complement C3 FITC (polyclonal, Thermo Fisher Scientific). Cells were then fixed with 4% formaldehyde solution and fluorescence was evaluated on a LSRII flow cytometer (BD Biosciences).

Opsonization. SARS-CoV-2 spike protein-expressing expi293F cells were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 spike protein matching the amino acid sequence of the IL-CDC-IL1/2020 isolate (GenBank ACC# MN988713). Stable transfectants were single-cell sorted and selected to obtain a high Spike surface-expressing clone (293F-Spike-S2A). SARS-CoV-2 spike protein-expressing cells were incubated with 200-fold diluted plasma samples for 30 min at 37°C. Cells were washed twice and stained with anti-human IgG phycoerythrin (PE), anti-human IgM Alexa Fluor 647, and anti-human IgA FITC (Southern Biotech). Cells were then fixed with 4% formaldehyde solution and fluorescence was evaluated on an LSRII flow cytometer (BD Biosciences).

Trogocytosis. Trogocytosis was measured using a previously described assay (27). Briefly, SARS-CoV-2 spike expressing expi293F cells were stained with PKH26 (Sigma-Aldrich). Cells were then washed with and resuspended in R10 media. Cells were then incubated with 200-fold diluted plasma samples for 30 min at 37°C. Effector peripheral blood mononuclear cells were next added to the R10 media at an effector to target (E:T) cell ratio of 50:1 and then incubated for 5 hours at 37°C. After the incubation, cells were washed, stained with live/dead aqua fixable cell stain (Life Technologies) and CD14 APC-Cy7 (clone MϕP9) for 15 min at room temperature, washed again, and fixed with 4% formaldehyde (Tousimis) for 15 min at room temperature. Fluorescence was evaluated on a LSRII flow cytometer (BD Biosciences). Trogocytosis was evaluated by measuring the PKH26 mean fluorescence intensity of the live CD14+ cells.

Intracellular Cytokine Staining. Cryopreserved peripheral blood mononuclear cells were thawed and rested for 6 hours in R10 with 50 U/mL Benzonase Nuclease (Sigma Aldrich). They were then stimulated for 12 hours with two pools of peptides spanning the spike protein of either SARS-CoV-2 or SARS-CoV-1 (1 μg/mL, JPT, PM-WCPV-S and PM-CVHSA-S respectively) in the presence of Brefeldin A (0.65 μL/mL, GolgiPlug, BD Biosciences Cytofix/Cytoperm Kit, Cat. 555028), co-stimulatory antibodies anti-CD28 (BD Biosciences Cat. 555725 1 μg/mL) and anti-CD49d (BD Biosciences Cat. 555501; 1 μg/mL), and CD107a (H4A3, BD Biosciences Cat. 561348, Lot 9143920 and 253441). Following stimulation, cells were stained serially with LIVE/DEAD Fixable Blue Dead Cell Stain in PBS for 20 min at room temperature (Thermo Fisher Scientific #L23105) and a cocktail of fluorescently-labeled antibodies (BD Biosciences unless otherwise indicated) to cell surface markers CD4-PE-Cy5.5 (S3.5, Thermo Fisher #MHCD0418, Lot 2118390, 1:80), CD8 BV570 (RPA-T8, BioLegend #301038, Lot B281322, 1:160), CD45RA BUV395 (5H9, #552888, Lot 154382 and 259854, 1:160), CD28 BUV737 (CD28.2, #612815, Lot 0113886, 1:20), CCR7 BV650 (GO43H7, # 353234, Lot B297645, 1:20) and HLA-DR BV480 (G46-6, # 566113, Lot 0055314, 1:640) in 4% fetal bovine serum in PBS for 20 min at room temperature. Intracellular cytokine staining was performed following fixation and permeabilization per manufacturer’s instructions (BD Cytofix/Cytoperm, BD Biosciences) with CD3 APC-Cy7 (SP34-2, #557757, Lot 6140803, 1:1,282), CD154 PE-Cy7 (24-31, BioLegend # 310842, Lot B264810 and B313191, 1:40), IFN-γ Alexa Fluor 700 (B27, # 506516, Lot B187646 and B290145, 1:1,282), TNF-α FITC (MAb11, # 554512, Lot 15360, 1:160), IL-2 BV750 (MQ1-17H12, BioLegend #566361, Lot 0042313, 1:160), IL-4 BB700 (MP4-25D2, Lot 0133487 and 0308726, 1:320), MIP-1b PE (D21-1351, # 550078, Lot 9298609, 1:160), CD69 Electron coupled dye (ECD, phycoerythrin-Texas Red conjugate) (TP1.55.3, Beckman Coulter Life Sciences # 6607110, Lot 7620070 and 7620076, 1:80), IL-21 Alexa Fluor 647 (3A3-N2.1, # 560493, Lot 9199272, 1:20), IL-13 BV421 (JES10-5A2, # 563580, Lot 9322765, 210147 and 169570, 1:20) and IL-17A BV605 (BL168, BioLegend #512326, B289357, 1:20). Sample staining was measured on a FACSymphony A5 SORP (Becton Dickenson) and data analyzed using FlowJo v.9.9 software (Tree Star, Inc.). The gating strategy is shown in fig. S10. CD4 and CD8 T cell subsets were pre-gated on memory markers prior to assessing cytokine expression as follows: single-positive or double-negative for CD45RA and CD28. Boolean combinations of cells expressing one or more cytokines were used to assess the total spike protein-specific response of memory CD4 or CD8 T cells. Responses from the two-peptide pools spanning SARS-CoV-2 spike protein or SARS-CoV-1 were summed. Display of multicomponent distributions were performed with SPICE v6.0 (NIH).

Total and Sub-Genomic Messenger (sgm) RNA Quantification. Real time quantitative polymerase chain reaction (RT-qPCR) was carried out for subgenomic messenger RNA (sgmRNA) and viral load RNA quantification from NP swab, BAL fluid and saliva samples. Primers targeted the envelope (E) gene of SARS-CoV-2 (table S2). RNA was extracted from 200 μl of NP swab media or BAL specimens using the EZ1 DSP Virus kit (Qiagen) on the EZ1 Advanced XL instrument (Qiagen). Briefly, samples were lysed in 200 μl of ATL buffer (Qiagen) and transferred to the Qiagen EZ1 for extraction. Bacteriophage MS2 (ATCC) was added to the RNA carrier and used as an extraction control to monitor efficiency of RNA extraction and amplification (60). Purified RNA was eluted in 90 μl elution buffer (AVE). The RT-qPCR amplification reactions were performed in separate wells of a 96-well Fast plate for the 3 targets: sgmRNA, RNA viral load, and MS2 RNA using 10 μl of extracted material 0.72uM of primer and 0.2uM of probe and 1x TaqPath 1-Step RT-qPCR (A15299: Life Technologies, Thermo Fisher Scientific). Amplification cycling conditions were: 2 min at 25°C, 15 min at 50°C, 2 min at 95°C and 45 cycles of 3 s at 94°C and 30 s at 55°C with fluorescence read at 55°C. An RNA transcript for the SARS-CoV-2 E gene was used as a calibration standard. RNA copy values were extrapolated from the standard curve and multiplied by 45 to obtain RNA copies/ml. A negative control (PBS) and two positive controls (heat inactivated SARS-CoV-2, ATCC, VR-1986HK at 106 and 103 copies/ml), were extracted and used to assess performance of both assays.

Histopathology. Formalin fixed sections of lung tissue were evaluated by light microscopy and immunohistochemistry. Lungs were perfused with 10% neutral-buffered formalin. Lung sections were processed routinely into paraffin wax, then sectioned at 5 μm, and resulting slides were stained with hematoxylin and eosin. Immunohistochemistry (IHC) was performed using the Dako Envision system (Dako Agilent Pathology Solutions). Briefly, after deparaffinization, peroxidase blocking, and antigen retrieval, sections were covered with a mouse monoclonal anti-SARS-CoV nucleocapsid protein antibody (#40143-MM05, Sino Biological) at a dilution of 1:4000 and incubated at room temperature for forty-five minutes. They were rinsed, and the peroxidase-labeled polymer (secondary antibody) was applied for thirty minutes. Slides were rinsed and a brown chromogenic substrate 3,3′ Diaminobenzidine (DAB) solution (Dako Agilent Pathology Solutions) was applied for eight minutes. The substrate-chromogen solution was rinsed off the slides, and slides were counterstained with hematoxylin and rinsed. The sections were dehydrated, cleared with Xyless, and then cover slipped. Tissue section slides were evaluated by a board-certified veterinary anatomic pathologist who was blinded to study group allocations.

Statistical analysis. Raw, individual level data can be found in data file S1. Primary immunogenicity outputs of binding and neutralizing antibody titers as well as T cell responses were compared across vaccination groups using Kruskal-Wallis test. Non-parametric pairwise comparisons between groups were made using the post-hoc Dunn’s test. The same hierarchical analysis was applied to comparisons of sgmRNA concentrations in the NP swabs and BAL fluids of vaccinated versus control groups. Statistical significance was preset at an alpha level of 0.05. The correlation between dependent T cell responses was assessed by non-parametric Spearman correlation (r).

Acknowledgments

ACKNOWLEDGMENTS

We thank J. Lay, E. Zografos, J. Lynch, L. Mendez-Rivera, N. Jackson, B. Slike, U. Tran, S. Peters, J. Bolton, T. Robinson, E. Duncan, H. Siegfried, R.J. O’Connell, Z. Beck and C. Alving for technical support, assistance, and advice.

Funding: We acknowledge support from the U.S. Department of Defense, Defense Health Agency (Restoral FY20 to KM). This work was partially executed through a cooperative agreement between the U.S. Department of Defense and the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (W81XWH-18-2-0040). The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense.

Author contributions: K.M. and M.G.J designed the study. I.E.N, A.A., K.K.P., C.M.C., C.S., R.E.C, J.B.C., P.V.T., W-H.C., R.S.S., A.H., E.J.M., C.E.P., W.C.C., M.C., C.S., P.J.L., A.A., K.M.W., M.S.N., H.M.S., N.dV., M.D., I.S., J.R.C., K.G.L, V.D., S.M., K.A., R.C., S.J.K. D.P.P., N.K., V.R.P., Y.H., L.L.J., G.D.G. performed immunologic and virologic assays. H.A.E, A.C., M.G.L. led the clinical care of the animals. S.P.D., X.Z., E.K.D performed histopathology. K.M., M.G.J., P.V.T., W-H.C., R.S.S., A.H., E.J.M., C.E.P., W.C.C., and M.C. designed the immunogens. M.R., G.R.M., and A.A. designed and provided the adjuvant. M.G.J., H.A.D.K. J.A.H., S.P.D., M.F.A., S.V., P.T.S., D.D.H., M.S.D., M.G.L., M.R., G.D.G., S.A.P., N.L.M. D.L.B. and K.M. analyzed and interpreted the data. K.M. wrote the paper with assistance from all coauthors.

Competing interests: K.M. and M.G.J. are primary co-inventors on related vaccine patents (U.S. Provisional Application 62/986,522 filed March 6, 2020, and to U.S. Provisional Application 63/038,600 filed June 12, 2020). M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, and Carnival Corporation. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. M.S.D. is on the Scientific Advisory Boards of Moderna and Immunome. The other authors declare no competing interests.

Data and materials availability: All data are available in the manuscript or the supplementary materials. The vaccine and adjuvant used in this study will be made available to the scientific community by contacting Kayvon Modjarrad and completion of a materials transfer agreement.

This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using this material.

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