key: cord-0903439-ww7lekxq
authors: Stark, Felicity C.; Akache, Bassel; Deschatelets, Lise; Tran, Anh; Stuible, Matthew; Durocher, Yves; McCluskie, Michael J.; Agbayani, Gerard; Dudani, Renu; Harrison, Blair A.; Renner, Tyler M.; Makinen, Shawn R.; Bavananthasivam, Jegarubee; Duque, Diana; Gagne, Martin; Zimmermann, Joseph; Zarley, C. David; Cochrane, Terrence R.; Handfield, Martin
title: Intranasal Immunization with a Proteosome-Adjuvanted SARS-CoV2 Spike Protein-Based Vaccine is Immunogenic and Efficacious in Mice & Hamsters
date: 2022-03-02
journal: bioRxiv
DOI: 10.1101/2022.03.02.482651
sha: 0fd29d53fea3fe9b119889eb6c4d961f406ee5bb
doc_id: 903439
cord_uid: ww7lekxq

With the persistence of the SARS-CoV-2 pandemic and the emergence of novel variants, the development of novel vaccine formulations with enhanced immunogenicity profiles could help reduce disease burden in the future. Intranasally delivered vaccines offer a new modality to prevent SARS-CoV-2 infections through the induction of protective immune responses at the mucosal surface where viral entry occurs. Herein, we evaluated a novel protein subunit vaccine formulation containing a resistin-trimerized prefusion Spike antigen (SmT1v3) and a proteosome-based mucosal adjuvant (BDX301) formulated to enable intranasal immunization. In mice, the formulation induced robust antigen-specific IgG and IgA titers, in the blood and lungs, respectively. In addition, the formulations were highly efficacious in a hamster challenge model, reducing viral load and body weight loss. In both models, the serum antibodies had strong neutralizing activity, preventing the cellular binding of the viral Spike protein based on the ancestral reference strain, the Beta (B.1.351) and Delta (B.1.617.2) variants of concern. As such, this intranasal vaccine formulation warrants further development as a novel SARS-CoV-2 vaccine.

through the induction of protective immune responses at the mucosal surface where viral entry occurs. 23

Herein, we evaluated a novel protein subunit vaccine formulation containing a resistin-trimerized 24 prefusion Spike antigen (SmT1v3) and a proteosome-based mucosal adjuvant (BDX301) formulated to 25 enable intranasal immunization. In mice, the formulation induced robust antigen-specific IgG and IgA 26 titers, in the blood and lungs, respectively. In addition, the formulations were highly efficacious in a 27 hamster challenge model, reducing viral load and body weight loss. In both models, the serum antibodies 28 had strong neutralizing activity, preventing the cellular binding of the viral Spike protein based on the 29

The etiological agent of the COVID-19 pandemic, SARS-CoV-2, has proven to be highly virulent and 45 adaptable. Despite the development and deployment of multiple relatively safe and efficacious vaccines 46 worldwide, the emergence of novel viral variants with decreased sensitivity to vaccine-induced immune responses has caused the pandemic to persist and still present a major challenge to human health 1-3 . 48 In the initial response to the COVID-19 pandemic, vaccine developers were able to produce efficacious advanced through clinical trials to regulatory approval due to their strong immunogenicity/efficacy, but 54 also in part to their relatively rapid manufacturing processes 4-6 . As such, they were the first wave of 55 SARS-CoV-2 vaccines to become widely available in many parts of the world including China, North 56 America and Europe. However, questions remain regarding their efficacy and safety in the long term. 57

Although rare in frequency, severe cases of anaphylaxis, myocarditis and/or thrombocytopenia have been 58 linked specifically to the mRNA and viral vector vaccine platforms 7-9 . With the emergence of novel COVID-59 19 variants of concern capable of partially evading the protection induced by the currently approved 60 vaccines 1-3,10 , the development of novel vaccines with improved safety and efficacy profiles could reduce 61 the impact of SARS-CoV-2 and its variants in the future. 62 SARS-CoV-2 is an airborne pathogen that enters the body primarily through the upper respiratory tract 63 (URT; nose and mouth). The URT is likely the initial location of infectivity as it contains a much greater 64 concentration of ACE2 receptors than the lower respiratory tract 11Error! Reference source not found. . Immune 65 responses initiated in these mucosal sites often involve mucosa-associated lymphoid tissues (MALT), 66 of SmT1v3 alone or adjuvanted with BDX301 on Days 0 and 21. SmT1v3 based on the ancestral reference 103 strain originally identified in Wuhan was used for all vaccine formulations in this study. Controls included 104 mice injected with the vaccine vehicle, phosphate-buffered saline (PBS), or SmT1v3 adjuvanted with 105 aluminum phosphate (AdjuPhos TM ). Aluminum phosphate is a conventional vaccine adjuvant found in a 106 number of approved vaccines and is a potent inducer of antigen-specific antibody responses 26,27 . SmT1v3-107

AdjuPhos TM formulations were delivered intramuscularly 28 . To assess the ability of the vaccine 108 formulations to induce systemic immune responses following one or two vaccine doses, serum samples 109 were taken on Days 21 and 35, respectively. 110

Following a single vaccine dose, SmT1v3 adjuvanted with BDX301 or aluminum phosphate induced 111 statistically similar anti-Spike IgG Geomean Titers (GMT) of 5,288 and 7,091 (p>0.05), respectively, in the 112 sera of immunized mice (Fig. 1A) . These levels were significantly greater than those observed in animals 113 immunized with antigen alone or vehicle control (p<0.0001). Antigen-specific antibody titers were 114 increased >20-fold in the sera of mice following administration of a second vaccine dose of SmT1v3 115 adjuvanted with BDX301 or aluminum phosphate, with measured GMTs of 392,690 and 163,003, 116 respectively (p>0.05; Fig. 1B ). Again, they were significantly greater than both control groups (p<0.0001), 117 which still showed low or non-detectable antibody titers. 118

While the induction of anti-Spike IgG antibodies is currently thought to be a strong predictor of vaccine 120 efficacy against COVID-19 29 , the functionality of these antibodies as measured by their ability to prevent 121 viral Spike binding to the cells and prevent infection is especially important. Using a surrogate cell-based 122 neutralization assay previously shown to have a strong correlation to responses obtained with viral-based by antibodies induced by the reference strain 30 . SmT1v3-BDX301 vaccine formulations induced strong 128 neutralizing activity against both the ancestral and Beta SARS-CoV2 Spike proteins, which was significantly 129 higher than with antigen alone (Fig. 2; p<0 .0001). Additionally, the BDX301-adjuvanted formulation 130 induced significantly greater neutralization than that observed with the SmT1v3-aluminum phosphate 131 formulation against the Beta variant (71 vs. 35%; p<0.05), while neutralization was statistically similar 132 against the Spike from the ancestral reference strain (93 vs. 80%; p>0.05). 133

IgA antibody response in immunized mice 134

As enhancement of local IgA responses in the mucosal compartment is typically one of the main 135 advantages of mucosal proteosome-adjuvanted vaccine formulations, antigen-specific IgA titers were also 136 assessed in the bronchoalveolar lavage (BAL) fluid and serum of mice collected on Day 35. Only SmT1v3-137 BDX301 immunized mice showed detectable anti-SmT1v3 IgA titers, as titers in mice immunized with 138 antigen alone or SmT1v3-aluminum phosphate were below the assay's limit of detection (Fig. 3 ). In both 139 the BAL fluid and serum, IgA titers were significantly higher than the control groups with a GMT of 1,000 140 and 5,074 in the BAL and serum, respectively (p<0.0001). Interestingly, the anti-SmT1v3 IgG/IgA ratio was 141 ~25-fold lower in the BAL vs. serum (p<0.01; Fig. 3D ), indicating that the antigen-specific IgA detected in 142 the BAL was not simply due to cross-contamination with blood or leakage of blood through the mucosal 143 epithelium. Having established the immunogenicity of a vaccine formulation based on BDX301 and 144 SmT1v3 in mice, we next sought to evaluate its efficacy against a live SARS-CoV-2 challenge in hamsters. days post the second immunization, the GMT titers were still higher with 15 µg vs. 5 µg, but they did not 158 reach a level of statistical significance (19,492 vs. 8,510; p>0.05; Fig. 4B ). 159

As with mice, the neutralization activity of the immunized hamster serum was assessed, but in addition 161 to the ancestral reference strain and Beta VOC, the Spike protein from the Delta VOC was also included. 162

The BDX301 formulations with either 5 µg or 15 µg antigen induced strong neutralization activity to the 3 163 different Spike variants. The strongest neutralization activity was seen against the ancestral reference 164 strain, with BDX301 with 5 or 15 µg of SmT1v3 inducing an average of 55 and 71% neutralization, 165 respectively (p<0.0001 vs. vehicle or adjuvant alone groups; Fig. 5A ). While neutralization activity against 166 the Beta variant was detected in many animals immunized with BDX301-SmT1v3, it did not reach a level 167 of statistical significance when compared to the control groups ( This assay was performed exclusively within a containment level 3 facility (CL3). Whole lung from each 353 hamster was homogenized in 1 mL PBS. The samples were centrifuged and the clarified supernatants were 354 used in a plaque assay. The plaque assay, in brief, was carried out by diluting the clarified lung homogenate 355 in a 1 in 10 serial dilution in infection media (1x DMEM, high glucose media supplemented with 1x non-356 albumin). Vero E6 cells were infected for 1 h at 37 o C before the inoculum was removed and overlay media 358 was added, which consisted of infection media with 0.6% ultrapure, low-melting point agarose. The cells 359 were incubated at 37 o C/5% CO2 for 72 h. After incubation, cells were fixed with 10% formaldehyde and 360 stained with crystal violet. Plaques were enumerated and PFU was determined per gram of lung tissue. 361 362

All steps carried out for the PRNT assay was performed in a CL3 facility. Serum samples were inactivated 364

at 56 o C for 30 min and stored on ice. A 1-in-2 serial dilution was carried out using inactivated serum. 365

Diluted serum was incubated with equal volume containing 100 PFU of SARS-CoV-2 at 37 o C for 1 h, 366 followed by infection of Vero E6 cells. Adsorption of virus were carried out for 1 h at 37 o C. Inoculum was 367 removed after adsorption and overlay media as described above was added over the infected cells. The 368 assay was incubated at 37 o C/5% CO2 for 72 h. After incubation, cells were fixed with 10% formaldehyde 369 and stained with crystal violet. Controls included naïve animal serum, as well as a no serum, virus-only 370 back-titer control. PRNT80 is defined as the highest dilution of serum that results in 80% reduction of 371 plaque-forming units. Samples that did not result in an 80% reduction in PFUs were assigned the value of 372 the lowest tested dilution (i.e. 40) for analysis purposes. 373 374

Data were analyzed using GraphPad Prism® version 9 (GraphPad Software, San Diego, CA, USA). Statistical 376 significance of the difference between groups was calculated by one-way or two-way analysis of variance 377 (ANOVA) followed by post-hoc analysis using Tukey's (comparison across all groups) multiple comparison 378 test. A Student's t-test was applied when analyzing the significance of the difference between the antigen-379 specific IgG/IgA ratios in the BAL and serum. Data was log transformed (except for % neutralization, 380

IgG/IgA ratio and % body weight loss) prior to statistical analysis. For all analyses, differences were 381 considered to be not significant with p > 0.05. Significance was indicated in the graphs as follows: *p < 382 0.05, **p < 0.01, ***p<0.001 and ****: p<0.0001. 

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