Immunogenicity and efficacy of XBB.1.5 rS vaccine against EG.5.1 variant of SARS-CoV-2 in Syrian hamsters

The continued emergence of SARS-CoV-2 variants necessitates updating COVID-19 vaccines to match circulating strains. The immunogenicity and efficacy of these vaccines must be tested in pre-clinical animal models. In Syrian hamsters, we measured the humoral and cellular immune response after immunization with the nanoparticle recombinant Spike (S) protein-based COVID-19 vaccine (Novavax, Inc.). We also compared the efficacy of the updated monovalent XBB.1.5 variant vaccine to previous COVID-19 vaccines for the induction of XBB.1.5 and EG.5.1 neutralizing antibodies and protection against a challenge with the EG.5.1 variant of SARS-CoV-2. Immunization induced high levels of spike-specific serum IgG and IgA antibodies, S-specific IgG and IgA antibody secreting cells, and antigen specific CD4 + T-cells. The XBB.1.5 and XBB.1.16 vaccines, but not the Prototype vaccine, induced high levels of neutralizing antibodies against XBB.1.5 and EG.5.1 variants of SARS-CoV-2. Upon challenge with the Omicron EG.5.1 variant, the XBB.1.5 and XBB.1.16 vaccines reduced the virus load in the lungs, nasal turbinates, trachea and nasal washes. The bivalent vaccine continued to offer protection in the trachea and lungs, but protection was reduced in the upper airways. In contrast, the monovalent Prototype vaccine no longer offered good protection, and breakthrough infections were observed in all animals and tissues. Thus, the protein-based XBB.1.5 vaccine is immunogenic and can protect against the Omicron EG.5.1 variant in the Syrian hamster model.


INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused hundreds of millions of infections worldwide and over 7 million deaths.Vaccines targeting the SARS-CoV-2 spike protein were developed within one year of the start of the pandemic, and they were remarkably effective in protecting against severe coronavirus disease 2019 (COVID- 19), with e cacy rates ranging from 75 to 95% depending on the vaccine, the circulating strain, and age of the individual [1][2][3] .In November of 2021, the Omicron variant of SARS-CoV-2 emerged, quickly spreading globally and replacing previous variants of concern (VOC) of SARS-CoV-2.Omicron variants harbor more than 30 amino acid substitutions in the spike (S) protein, which results in evasion of humoral immune responses and escape from protection of the original vaccines 4 .The Omicron lineage of SARS-CoV-2 has continued to evolve away from neutralizing antibodies generated by previous infection or vaccination with ancestral vaccines, a process referred to as antigenic drift.Because of this drift, in 2022, global regulatory agencies recommended updating the COVID-19 vaccine to include the BA.5 variant of SARS-CoV-2.In late 2022, XBB-lineage Omicron variants of SARS-CoV-2 emerged and became successful 5 .The XBB variants were resistant to antibodies induced by the BA.5 vaccine, prompting another update of the COVID-19 vaccine; the monovalent XBB.1.5vaccine 6 .Due to the continued evolution and drift of the SARS-CoV-2 virus, the ability of the updated vaccines to generate cross-protective immunity against future viral variants is crucial, and must be evaluated in preclinical animal models.
Novavax Inc. developed a SARS-CoV-2 recombinant S protein nanoparticle vaccine comprised of fulllength prefusion S trimers co-formulated with a saponin-based adjuvant, Matrix-M™ (Prototype rS).In preclinical studies in mice and non-human primates, this vaccine was effective against a homologous challenge with SARS-CoV-2 7,8 .Similarly, in mice, a Beta (B.1.351rS) version of this vaccine was effective against heterologous challenge with the Omicron BA.1 variant of SARS-CoV-2 9 .In Syrian hamsters, we showed that a boost with the BA.5 rS vaccine offered robust protection against a BA.5 virus challenge 10 .In humans, immunization with the monovalent Prototype vaccine was effective against mild, moderate, or severe COVID-19 in clinical trials [11][12][13] .Several trials reported the vaccine e cacy against symptomatic infection of 96% for the ancestral strain of SARS-CoV-2 and 86% for the alpha (B.1.1.7)variant 13,14 .Boosting with a third or fourth dose of NVX-CoV2373 reduced the antigenic distance between the ancestral and Omicron BA.4/5 variants of SARS-CoV-2 15,16 , suggesting that repeated exposure to a subunit vaccine containing ancestral S protein induces a cross-reactive and crossneutralizing antibody response.
Here, we evaluated the immunogenicity and e cacy of protein-based nanoparticle vaccines containing recombinant S proteins from Wuhan-1, BA.5, XBB.1.5,and XBB.1.16variants of SARS-CoV-2 in hamsters.These vaccines induced robust S-speci c cellular immune responses, S-speci c IgG and IgA serum antibodies, and virus neutralizing antibodies in this pre-clinical animal model.Furthermore, the monovalent XBB.1.5 and XBB.1.16vaccines provided protection against a heterologous challenge with the EG.5.1 variant of SARS-CoV-2.

RESULTS
Nanoparticle protein-based COVID-19 vaccine induces S protein speci c IgG and IgA antibody secreting cells in Syrian hamsters.Groups of 5-6 week-old male Syrian hamsters (n = 7) were immunized intramuscularly twice at four-week intervals with 1 µg of the nanoparticle protein-based vaccine containing Prototype rS (Wuhan-1) and serum was collected 21 days later.Sixteen weeks later, the animals received a third dose of Prototype rS, and S-speci c cellular responses were quanti ed 7 days later by B-cell and T-cell ELISpot.Compared to unvaccinated control animals, ~ 2,300 and ~ 3,200 Wuhan-1 (WT) S-speci c IgG antibody secreting cells (ASC)/million were detected in the spleen and draining inguinal lymph nodes (DLN), respectively (Fig. 1A-C).In the same samples, we also detected WT Sspeci c IgA ASC, albeit the frequency per million cells was signi cantly reduced in the spleen (~ 1,150 ASC/million; P < 0.001) and DLN (~ 1,110 ASC/million; P < 0.001) compared to the frequency of IgG ASC (Fig. 1A-C).The ratio of IgG to IgA S-speci c ASC was ~ 2.5:1 in both tissues 7 days after immunization.To support the presence of S-speci c IgG and IgA ASC, an ELISA was performed on sera collected 21 days after the second immunization.Relatively high levels of S-speci c IgG and IgA antibodies were detected in the serum of these animals (Fig. 1D-E).
To evaluate the impact of antigenic variation in the S protein on the B-cell cellular response, we compared the frequency of IgG and IgA ASC speci c for WT and EG.5.1 S protein.A 1.8-2-fold reduction in the frequency of EG.5.1 S-speci c IgG and IgA ASC was observed in the spleen (P < 0.001 and P < 0.05 for IgG and IgA respectively) and DLN (P < 0.05 and P < 0.001 for IgG and IgA respectively) of these animals (Fig. 1F-I).
Immunization with a nanoparticle protein-based COVID-19 vaccine induces predominantly CD4 + T-cell response in Syrian hamsters.We also determined the CD4 + and CD8 + T-cell response in the spleen and DLN of hamsters immunized three times with the Prototype rS vaccine by ow cytometry and interferongamma (IFN-γ) ELISpot assay.Flow cytometry analysis found that the average number of cells collected from the spleen and DLN was 50 and 36 million, respectively (Fig S1).Within the spleen, ~ 22% of the cells were CD4 + B220-cells and ~ 10% were CD8 + B220-cells.In the DLN, these frequencies doubled to ~ 40% and ~ 22% for the CD4 + and CD8 + T-cell population, respectively (Fig S1).Next, we measured the number of IFN-γ secreting cells/million following re-stimulation with pools of overlapping 15-mer peptides corresponding to the S1 and S2 subunit of Wuhan-1 SARS-CoV-2 Spike protein (Fig. 2A).Compared to our unstimulated negative control wells, re-stimulation with S1 or S2 subunit peptide pools induced IFN-γ secretion (purple spots in Fig. 2A).On average, we detected ~ 160 and ~ 80 IFN-γ secreting cells/million in the spleen and DLN of these hamsters, respectively (Fig. 2B-C).In all three hamsters, we detected more S1 speci c cells compared to S2 speci c IFN-γ secreting cells.To assess if the IFN-γ secretion was predominantly CD4 + or CD8 + cell mediated, we depleted the CD4 + cells ex vivo, con rmed depletion by ow cytometry (Fig. 2D-F) and quanti ed the number of S1 and S2 speci c IFN-γ secreting cells in this CD4-depleted cell population.Depletion of CD4 + cells greatly reduced the number IFN-γ secreting cells detected by ELISpot assay (Fig. 2A and D).Overall, a 5-fold reduction (P < 0.05) in the number of IFN-γ secreting cells was detected in the spleen and DLN of these three hamsters (Fig. 2D).These data suggest that the nanoparticle protein-based vaccine induced a predominantly CD4-mediated T-cell response in hamsters.
Syrian hamsters immunized twice with the bivalent Prototype + BA.5 rS vaccine also demonstrated signi cantly reduced amounts of viral RNA and infectious titers in the lungs, nasal wash and trachea with no discernible differences compared to the XBB.1.5rS and XBB.1.16rS immunized animals (Fig. 4).However, in the nasal turbinate, the amount infectious virus was signi cantly lower (P < 0.05) in the XBB.1.5rS (~ 24-fold) and XBB.1.16rS (~ 18-fold) immunized animals compared to hamsters that received the bivalent vaccine.Similarly, the amount of viral RNA was also lower (~ 3-fold), but this did not reach statistical signi cance.Finally, immunization with the Prototype rS vaccine signi cantly reduced the amount of viral RNA in nasal turbinates (~ 7-fold, P < 0.001), trachea (~ 9-fold, P < 0.0001), and lungs (~ 22-fold, P < 0.0001) compared to unvaccinated controls.It also reduced the amount of infectious virus in the nasal wash (~ 13-fold, P < 0.001), nasal turbinates (~ 15-fold, P < 0.05), trachea (~ 49-fold, P < 0.0001) and lungs (~ 97-fold, P < 0.0001) of these same animals.However, breakthrough infections were detected in all tissues tested in 100% of the animals.Compared to the Prototype + BA.5 rS, XBB.1.5rS and XBB.1.16rS immunized animals, protection from EG.5.1 challenge was greatly reduced in all four respiratory tissues of Prototype rS immunized animals (Fig. 4A-H).Combined, these data demonstrate the e cacy of the XBB.The XBB.1.5 and XBB.1.16rS vaccines induced robust XBB.1.5 and EG.5.1 speci c antibodies capable of neutralizing both Omicron variants of SARS-CoV-2.Importantly, we did not observe a signi cant decrease in neutralization between XBB.1.5 and EG.5.1 despite the two amino-acid differences between the two strains (Phe456Lue and Gln52His).This observation is in line with previous studies in mice and nonhuman primates immunized twice with the XBB.1.5rS or XBB.1.16rS vaccine, or boosted once in preimmune animals 18 .In humans, the EG.5.1 variant was more resistant to neutralization compared to the XBB.1.16virus in a cohort of individuals with a XBB breakthrough infection, albeit the different was less than 2-fold 19 .However, a second study using convalescent sera from Prototype immunized and XBB variant infected individuals, did not detect any difference in neutralization between the XBB.1.5 and EG.5.1 virus 20 .The high levels of neutralizing antibodies against EG.5.1 Omicron variant of SARS-CoV-2 were associated with a complete protection of the lower airways upon EG.5.1 challenge and a signi cant reduction in virus load in the upper airways.This is the rst evidence in vivo that the XBB.1.5vaccine can protect against EG.5.1 virus.
While the XBB.1.5 and XBB.1.16rS vaccine induced signi cantly higher neutralizing antibody titers compared to the previous bivalent vaccines, immunization with the bivalent (Prototype + BA rS) vaccine did induce cross-neutralizing antibodies against XBB.1.5 and EG.5.1 Omicron variant SARS-CoV-2.The fold reduction in neutralization of the XBB.1.5variant (~ 20-fold compared to BA.5 virus) was similar to that observed with a intranasal Chimpanzee adenovirus vectored bivalent vaccine in Syrian hamsters 21 , suggesting that both vaccine platforms induce similarly broadly protective antibodies in this pre-clinical animal model.This highlights the need to periodically update the COVID-19 vaccine to better match contemporary and emerging variants of SARS-CoV-2.This data also demonstrates the power of the preclinical hamster model to be able to differentiate the vaccine e cacy between current and prior COVID-19 vaccines.
Despite the increasing utilization of hamsters in vaccine research, there is still a shortage of immunological tools speci cally designed to evaluate immune responses in this model 22 .Previous studies have highlighted the signi cance of T-and B-cells on SARS-CoV-2 infection and clearance in Syrian hamsters [23][24][25] .In this study, we have developed the T-and B-cell ELISpot assay to provide the most complete immunogenicity analysis of a COVID-19 vaccine in Syrian hamsters to date.We show robust induction of S-speci c IFN-γ secreting cells in the spleen and DLN of hamsters immunized with a protein-based vaccine, and discovered that the S-speci c T-cell response was dominated by CD4 + cells in this setting.The frequency of IFN-γ secreting cells was on par with what was observed in mice and nonhuman primates that received the same vaccine 18 .This study also showed for the rst time, the induction of IgG and IgA ASC in the spleen and DLN following immunization with the Prototype rS vaccine.Importantly, we detected a ~ 2-fold reduction in the number of EG.5.1 variant speci c ASC compared to the Wuhan-1 prototype S protein.This reduction in ASC coincided with a reduction in binding antibodies and a complete lack of neutralizing antibodies against the EG.5.1 variant of SARS-CoV-2.It also associated with a partial but signi cant loss of protection from a challenge with EG.5.1 variant of SARS-CoV-2.
Limitations of the study.We note several limitations of our study.(a) The vaccines were not tested in the context of pre-existing infection-or vaccine-induced immunity.While this would be valuable to investigate, we expect that all XBB.1.5boosted animals will be fully protected against a challenge with the EG.5.1 variant of SARS-CoV-2 as was previously demonstrated by our group in a study that demonstrated that boosting mRNA vaccine-immunized hamsters with the bivalent Prototype + BA.5 rS vaccine conferred complete protection against the BA.5 variant of SARS-CoV-2 10 .(b) We did not evaluate the e cacy of each vaccine booster in male and female hamsters.Due to the number of variables (vaccines and time after vaccination), testing male and female animals in each experiment was not feasible.(c) B-and T-cell responses after immunization with variant vaccines like XBB.1.5rS or XBB.1.16rS were not measured.We expect that the T-cell response will be similar between Prototype rS and XBB.1.5rS vaccine due to the limited variability of the S protein outside of the receptor binding domain.Similarly, and based on the serum antibody responses to XBB.1.5 and EG.5.1 in the XBB.1.5rS immunized hamsters, we expect the XBB.1.5rS vaccine to induce both IgG and IgA ASC and that they cross-react with the EG.5.1 variant.(d) Studies with more recent emerging variants (e.g., BA.2.86) are warranted.(e) The impact on virus transmission was not evaluated.While EG.5.1 can transmit between naïve hamsters, airborne transmission is not as effective as was observed for pre-Omicron variants of SARS-CoV-2 26 .
Overall, our studies demonstrate that nanoparticle protein-based vaccines are immunogenic and that the XBB.1.5rS vaccine is effective against newer variants of SARS-CoV-2 in Syrian hamsters.

RESOURCE AVAILABILITY
Lead contact.Further information and requests for resources and reagents should be directed to the Lead Contact, Adrianus C.M. Boon (jboon@wustl.edu).Materials availability.All requests for resources and reagents should be directed to the Lead Contact author.This includes viruses, vaccines, and primer-probe sets.All reagents will be made available on request after completion of a Materials Transfer Agreement.
Data and code availability.All data supporting the ndings of this study are available within the paper and are available from the corresponding author upon request.This paper does not include original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Hamster experiments.Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381-01).
Immunogenicity analysis.Seven ve-week old male hamsters were obtained from Charles River Laboratories and housed at Washington University.Five days after arrival, the animals were immunized via intramuscular injection in the posterior thigh muscles with 1 µg of the protein nanoparticle Prototype rS in 100 µL (50 µL per leg), and 21 days later they were boosted with 1 µg of the same vaccine.Serum was collected 21 days later for the detection of S-speci c IgG and IgA by ELISA.After 112 days, the animals received a third dose of the Prototype rS vaccine and 7 days later, the animals were euthanized, and the spleen and draining inguinal lymph nodes (DLN) were collected into 15 mL tubes containing 5 mL of ice-cold RPMI-1640 media with 2% FBS (R2).To generate a single cell suspension from the spleen or lymph nodes, the tissues were mashed using the plunger of a 1 mL syringe and ltered through a sterile 70 µm cell strainer.The cells were spin down at 300 x g for 5 min at 4 °C and red blood cells were lysed with 500 μL RBC lysis buffer (BioLegend) for 1 minute at room temperature.Next, 10 mL of R2 media was added, the cells were spin down, and resuspended in 1 mL ice-cold RPMI-1640 / 10% FBS (R10).Live and dead cells were counted using Acridine orange (AO) and propidium iodide (PI) (Sigma) using a cell counter (Nexcelom Bioscience), and the cells were diluted in R10 to a concentration of 10 7 cells/mL and used for ow cytometry analysis, and T-and B-cell ELISpot analysis.
Vaccine e cacy analysis.Five-week old male hamsters were obtained from Charles River Laboratories and housed at Washington University.Five days after arrival, the animals were immunized via intramuscular injection with 1 µg of the protein nanoparticle Prototype rS, Prototype + BA.5 rS (bivalent), XBB.1.5rS, or XBB.1.16rS vaccine.Control animals received PBS alone.Serum samples were obtained 21 days later and one week later the animals were immunized with a second dose of the same vaccine, and serum was collected 21 days later.Approximately two months later (day 59), the animals were randomly divided into two groups and one group was transferred to the enhanced Biosafety level 3 laboratory and challenged via intranasal route with 1 × 10 4 PFU of Omicron EG.5.1 variant.The second group followed a week later and was also challenged with 1 × 10 4 PFU of the EG.5.1 variant.Animal weights were measured daily for the duration of the experiment.Three days after challenge, the animals were necropsied, and their lungs, trachea, and nasal turbinates were collected for virological analysis.These tissues were homogenized in 1 mL of DMEM, clari ed by centrifugation (1,000 × g for 5 min) and used for viral titer analysis by quantitative RT-PCR (RT-qPCR) using primers and probes targeting the N gene, and by plaque assay.A nasal wash was also collected, by inoculating 1 mL of PBS with 0.1% bovine serum albumin into one nostril and collecting the wash from the other nostril.The nasal wash was clari ed by centrifugation (2,000 × g for 10 min) and used for viral titer analysis by RT-qPCR using primers and probes targeting the N gene, and by plaque assay.
Homogenates of lungs, trachea and nasal turbinates, and nasal washes were diluted serially by 10-fold, starting at 1:10, in cell infection medium (DMEM + 2% FBS + 100 U/mL of penicillin-streptomycin).Two hundred and fty microliters of the diluted virus were added to a single well per dilution per sample.After 1 h at 37°C, the inoculum was aspirated, the cells were washed with PBS, and a 1% methylcellulose overlay in MEM supplemented with 2% FBS was added.Ninety-six hours after virus inoculation, the cells were xed with 10% formalin, and the monolayer was stained with crystal violet (0.5% w/v in 25% methanol in water) for 30 min at 20°C.The number of plaques were counted and used to calculate the plaque forming units/mL (PFU/mL).
To quantify viral load in lung tissue homogenates and nasal washes, RNA was extracted from 100 µL samples using the MagMax Viral Pathogen Kit (ThermoFisher) on the KingFisher Flex Puri cation System following the manufacturer's protocol and eluted with 50 µL of water.Four microliters RNA was used for real-time RT-qPCR to detect and quantify N gene of SARS-CoV-2 using TaqMan™ RNA-to-CT 1-Step Kit (Thermo Fisher Scienti c) as described 31 using the following primers and probes: Forward: GACCCCAAAATCAGCGAAAT; Reverse: TCTGGTTACTGCCAGTTGAATCTG; Probe: ACCCCGCATTACGTTTGGTGGACC; 5'Dye/3'Quencher: 6-FAM/ZEN/IBFQ.Viral RNA was expressed as N gene copy numbers per mg for lung tissue homogenates or mL for nasal washes, nasal turbinates, and trachea based on a standard included in the assay, which was created via in vitro transcription of a synthetic DNA molecule containing the target region of the N gene.
ELISA.Ninety-six-well microtiter plates (Nunc MaxiSorp; ThermoFisher Scienti c) were coated with 100 µL of recombinant SARS-CoV-2 S protein (Wuhan-1 strain, BA.2, BA.5, or XBB.1.5,generated by Novavax as described above) at a concentration of 1 µg/mL in PBS (Gibco) at 4°C overnight; negative control wells were coated with 1 µg/mL of BSA (Sigma).Plates were blocked for 1.5 h at room temperature with 280 µL of blocking solution (PBS supplemented with 0.05% Tween-20 (Sigma) and 10% FBS (Corning)).The sera were diluted serially in blocking solution, starting at 1:100 dilution and incubated for 1.5 h at room temperature.The plates were washed three times with T-PBS (1X PBS supplemented with 0.05% Tween-20), and 100 µL of HRP-conjugated anti-hamster IgG(H+L) antibody (Southern Biotech Cat. #6061-05) diluted 1:1000 in blocking solution, was added to all wells and incubated for 1 h at room temperature.Alternatively, plates were incubated with biotinylated anti-hamster IgA antibody (Brookwood Biomedical, Cat.# sab3002a) diluted 1:1000 in blocking solution for 1 hours, followed by three washes with T-PBS and 1:5000 diluted HRP-conjugated streptavidin (Zymed).Plates were washed 3 times with T-PBS and 3 times with 1X PBS, and 100 µL of 1-step Ultra TMB-ELISA substrate solution (Thermo Fisher Scienti c) was added to all wells.The reaction was stopped after 10 min using 100 µL of 1N H 2 SO 4 , and the plates were analyzed at a wavelength of 450 nm using a microtiter plate reader (BioTek).B-cell ELISpot assay.Enzyme-linked immune absorbent spot (ELISpot) assays were performed to determine the number of S-speci c IgG and IgA ASC ELISpot Multiscreen Filter Plates (Millipore) were coated overnight at 4°C with 1 μg/mL of rS from the Wuhan-Hu-1 or EG.5.1 strains of SARS-CoV-2.
Control plates were either coated with anti-Syrian hamster IgG (1:100, Jackson ImmunoResearch) or left uncoated.The next day, the plates were blocked for 60 min at 37°C with RPMI 1640 supplemented with 10% FBS.Single cell suspensions of freshly isolated spleen or DLN cells (500,000 cells/well) were added in duplicate to the rst row followed by 3-fold serial dilution of the cells.After 6 hours at 37°C, the cells were washed off, and secreted hamster IgG or IgA were detected with a biotinylated anti-Syrian hamster IgG (1:1000, Jackson ImmunoResearch) or anti-Syrian hamster IgA (1:1000, Brookwood Biomedical) detection antibody respectively.Following overnight incubation at 4°C, the plates were washed 3x with T-PBS and streptavidin-conjugated horseradish peroxidase (HRP, Invitrogen) diluted 1:5000 in PBS was added for 1.5 hours at RT.Following another three washes with T-PBS and 1 wash PBS, the plates were developed, and spots were formed through an enzymatic reaction in the presence of 3-Amino-9-Ethyl Carbazole (AEC) and H 2 O 2 (Sigma).ELISpot plates were analyzed using an ELISpot counter (Cellular Technology Limited).Each spot represents an individual ASC and the number of spots indicates the frequency of B cells in the original sample that produces antibodies against the target antigen.
T-cell ELISpot assay.Interferon-gamma (IFN-γ) ELISpot was done according to ELISpot Flex: Hamster IFNγ kit (MABTECH) speci cations.Brie y, the Polyvinylidene di uoride (PVDF)-lined microplates (Millipore)   were coated overnight at 4°C with an IFN-γ capture antibody diluted in PBS (15 μg/ ml).Prior to the addition of cells, the wells were washed 5 times with PBS.A total of 500,000 cells in R10, were incubated peptide pools (10 µg/mL) of 15-mer overlapping peptides (BEI-Resources) corresponding to the S1 (1-668) and S2 (659-1273) subunit of S, PMA (phorbol myristate acetate, 0.5 μg/mL) plus ionomycin (1μg/mL) as a positive control, or 1% DMSO as a negative control.After 24 hours, the cells were washed off with PBS and the plates were incubated with 1 μg/mL of biotinylated IFN-γ-speci c detection antibody in PBS-0.5% FBS for 2 hours at room temperature.Following another washing step 5 times with PBS the plates were incubated for 1 hours with streptavidin-conjugated alkaline phosphatase (ALP, 1:1000) in PBS-0.5% FBS.After washing 5x with PBS, BCIP/NBT substrate was added until the spots appeared.The color development was stopped by washing the plates extensively with water.ELISpot plates were analyzed using an ELISpot counter (Cellular Technology Limited).
CD4+ depletion.CD4+ cell depletion was performed on cells collected from the spleen or draining lymph nodes using Dynabeads TM Biotin Binder kit (Invitrogen) containing magnetic beads.In short, the beads were washed twice with 2% FBS in PBS (P2).As per manufacturer, 50 μL of pre-washed beads were incubated with 10 µg/mL of biotinylated anti-CD4 (GK1.5, BioLegend) for 45 minutes at room temperature.The beads were washed 5 times with P2 and added to one million cells from the spleen or draining lymph node.The mixture was incubated for 30 min on ice with occasional shaking.Using the magnetic stand, the CD4+ cells were removed from the cell population and used for Flow cytometry and ELISpot assay.Flow cytometry.Staining was performed on the supernatant of CD4-depleted cells or 1x106 of nondepleted cells from the spleen or lymph node.The cells were stained for 30 min on ice with CD4-PE (GK1.5, 1:100, BioLegend), CD8b-BB700 (341, 1:100, BD Biosciences), B220-PE/Cyanine7 (RA3-6B2, 1:100, BioLegend) and Zombie Aqua (1:200, BioLegend) prepared in P2.Then, the cells were xed with 2% paraformaldehyde and re-suspended in P2.Sample acquisition was done on an Aurora using SpectroFlo v2.2 (Cytek).Flow cytometry data were analyzed using FlowJo v10 (BD Biosciences).CD4 cells and CD8 were selected as live, singlet, and B220-cells.

QUANTIFICATION AND STATISTICAL ANALYSES
Statistical signi cance was assigned when P values were < 0.05 using GraphPad Prism version 9.
1.5 rS and XBB.1.16rS vaccines and highlight the need for updating COVID-19 vaccines with contemporary variants of SARS-CoV-2 to more closely match newly emerging variants of SARS-CoV-2.DISCUSSION In this study, we evaluated the immunogenicity of a nanoparticle protein-based COVID-19 vaccine in Syrian hamsters and compared the e cacy of the XBB.1.5 and XBB.1.16variant vaccines to the original and bivalent COVID-19 vaccines, for protection against a challenge with the EG.5.1 variant of SARS-CoV-2.The nanoparticle protein-based subunit vaccine is highly immunogenic in Syrian hamsters and induced robust B-and T-cell responses against the Spike protein of SARS-CoV-2.Importantly, immunization with the XBB.1.5 or XBB.1.16rS vaccine induced strong serum neutralizing antibody responses against XBB.1.5 and EG.5.1 variant of SARS-CoV-2.The antibody responses were associated with reduced viral burden after intranasal challenge with the EG.5.1 variant of SARS-CoV-2.Overall, these data demonstrate the e cacy of the XBB.1.5vaccine against the novel EG.5.1 Omicron variant of SARS-CoV-2 in the preclinical hamster model of COVID-19.
3. Tests, number of animals, median and geometric mean values, and statistical comparison groups are indicated in the Figure legends.Analysis of weight change was determined by two-way ANOVA.Changes in infectious virus titer, viral RNA levels, or serum antibody responses were compared between all conditions, and were analyzed by one-way ANOVA with multiple comparisons correction on lntransformed data.Pairwise comparisons were done using a pairwise t-test.Declarations DECLARATION OF INTERESTS The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences Inc., and Nano targeting & Therapy Biopharma Inc.The Boon laboratory has received funding support from AbbVie Inc., for the commercial development of SARS-CoV-2 mAb.Novavax authors are current employees of Novavax, Inc., a for-pro t organization, who own stock or hold stock options.The Ellebedy laboratory has received funding under sponsored research agreements from Moderna, Emergent BioSolutions, and AbbVie.A.H.E. has received consulting and speaking fees from InBios International, Inc, Fimbrion Therapeutics, RGAX, Mubadala Investment Company, AstraZeneca, Moderna, P zer, GSK, Danaher, Third Rock Ventures, Goldman Sachs, and Morgan Stanley.A.H.E. is the founder of ImmuneBio Consulting and a recipient of royalties from licensing agreements with Abbvie and Leyden Laboratories B.V. AUTHOR CONTRIBUTIONS N.S. performed all the T-and B-cell analysis.N.S. and K.S. performed ELISA assays.N.P., G.S., M.G.X., and A.C.M.B. designed the hamster study.T.L.B. performed hamster experiments and quanti ed virus titers in collected tissues.T.L.D. performed hamster experiments, virus neutralization assays, and RT-qPCR assays.A.C.M.B. had unrestricted access to all the data, analyzed the data, and performed the statistical analysis.M.S., M.E.D.D., N.P., G.S., M.G.X.provided key reagents.A.C.M.B. supervised experiments and acquired funding.A.C.M.B. wrote the rst draft of the manuscript and all authors reviewed and edited the nal version.All authors agreed to submit the manuscript, read and approved the nal draft, and take full responsibility for its content.

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