key: cord-0794662-iawu2ylw
authors: Shamseldin, Mohamed M.; Zani, Ashley; Kenney, Adam; Evans, Jack; Zeng, Cong; Read, Kaitlin A.; Caution, Kyle; Hall, Jesse M.; Brown, Jessica M.; Gunsch, Gilian; Corps, Kara N.; Chaiwatpongsakorn, Supranee; Mahesh, KC; Lu, Mijia; Deora, Rajendar; Peeples, Mark E.; Li, Jianrong; Oestreich, Kenneth J.; Liu, Shan-Lu; Yount, Jacob S.; Dubey, Purnima
title: Prime-pull immunization of mice with a BcfA-adjuvanted vaccine elicits mucosal immunity and prevents SARS CoV-2 infection and pathology
date: 2022-04-07
journal: bioRxiv
DOI: 10.1101/2022.04.06.487394
sha: a49a6b59dc58c405a9682a70ee6f0caba5dd2adb
doc_id: 794662
cord_uid: iawu2ylw

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. We show that intramuscular priming of mice with an alum and BcfA-adjuvanted Spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17 polarized tissue resident CD4+ T cells, and mucosal and serum antibodies. The serum antibodies efficiently neutralized SARS-CoV-2 and its Delta variant, suggesting cross-protection against a recent variant of concern (VOC). Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 and reduced viral replication in the nose and lungs. Histopathology showed a strong leukocyte and polymorphonuclear (PMN) cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. In contrast, viral load was not reduced in the upper respiratory tract of IL-17 knockout mice immunized with the same formulation, suggesting that the Th17 polarized T cell responses are critical for protection. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, protect against SARS-CoV-2 infection without causing enhanced respiratory disease. SIGNIFICANCE There remains a need for SARS CoV-2 booster vaccines that generate mucosal immunity and prevent transmission. We show that systemic priming followed by a mucosal booster with a BcfA-adjuvanted subunit vaccine generates neutralizing antibodies and Th17 polarized systemic and tissue-resident immune responses that provide sterilizing immunity against wildtype SARS CoV-2, and a variant of concern. Importantly, in contrast to alum alone, the addition of BcfA prevents respiratory pathology. These results suggest that a BcfA-adjuvanted mucosal booster may elicit mucosal immunity in individuals previously immunized systemically with approved vaccines. This foundational study in mice sets the stage for testing our vaccine regimen in larger animal models as a booster vaccine.

COVID-19 is a respiratory and multi-organ disease caused by severe acute respiratory influenza. Thus, there is a critical need to create a vaccine that will protect against primary 55 infection and re-infection with the virus (Amanat and Krammer, 2020) . 56

The symptoms of SARS-CoV-2 infection include headache, fever, chills, and a persistent 57 dry cough. Additional symptoms may include gastrointestinal distress, diarrhea, vomiting and loss 58 of smell or taste. The main transmission route of SARS-CoV-2 is via respiratory exposure 59 although infection may also occur via fecal-oral and ocular exposure. The causes of morbidity 60

To determine whether our immunization regimens prevented virus infection in vivo, we 231 challenged naive and immunized mice i.n. with 5x10 4 PFU of mouse-adapted SARS-CoV-2 (strain 232 MA10) ( Figure 6A ). We recorded daily body weight in one cohort of infected mice until d10 p.i. to 233 assess whether immunization affected disease severity. Naive mice and mice immunized with S 234 alone lost 10-15% body weight beginning on d2 post-infection and recovered to pre-challenge 235 weight by d8 p.i. ( Figure 6B ). In contrast, mice immunized and boosted with S/A, or immunized 236 with S/A/B and boosted with S/B did not lose weight ( Figure 6B ), indicating that the vaccines 237 prevented severe disease. We quantified viral load in the nose and lungs on d2 p.i. in a second 238 cohort infected at the same time. Viral load in the nose ( Figure 6D ) and lungs ( Figure 6E naïve challenged mice. Lymphocytes were increased in this group compared to naïve challenged 275 mice ( Figure 7H ). Total cellularity, edema and hemorrhage were similar between naïve and 276 immunized mice ( Figure S4 ). We then tested the presence of SARS-CoV-2 N protein as a second 12 measure of virus presence in the lungs. IHC analysis revealed strong and widespread N 278 protein staining in epithelial cells of naïve challenged mice ( Figure 7I ) and mice immunized with 279 S alone ( Figure 7J ). Mice primed and boosted with S/A showed foci of N protein staining in 2 of 5 280 mice ( Figure 7K and Table S1 ), suggesting that protection from infection was inefficient in this 281 group. Remarkably, N protein staining was completely absent from the lungs of all mice 282 immunized with S/A/B and boosted with S/B ( Figure 7L and showed milder pathology overall compared with wild-type mice. Naïve challenged mice displayed 288 thickening of the alveolar wall with hemorrhage, type II pneumocyte hyperplasia and inflammation, 289 degeneration and sloughing in the airways with an inflammatory infiltrate and tissue necrosis 290 was significantly higher ( Figure S5F ) in immunized mice compared to naïve mice, suggesting that 296 while the immunization recruited lymphocytes to the tissues, these did not have antiviral function. 297

These data show that while IL-17 KO mice had milder respiratory disease compared to wild-type 298 C57BL/6 mice, immunized mice were not protected from respiratory pathology. Thus, Th17 299 polarized T cells generated by BcfA-adjuvanted vaccines contribute to prevention of respiratory 300 damage following SARS CoV-2 challenge. IHC did not detect N protein staining in lungs of IL-17 301 KO naïve or immunized mice (data not shown), likely due to the overall lower viral titer in the 302 respiratory tract compared with wild-type C57BL/6 mice. 303 In this study, we identified an immunization strategy and vaccine formulation that elicits 308 protective immunity against SARS-CoV-2 both systemically and at the infection site. Generation Together our data show that a subunit vaccine that elicits Th1/17 polarized systemic and mucosal 376 immunity is highly effective against SARS-CoV-2. In naïve IL-17 KO mice, although lung 377 pathology was milder than in wild-type mice, immunized mice also displayed respiratory disease. Given that global vaccination rates are on the rise, there will be an increasing need for booster 393 vaccines that extend protection provided by currently approved mRNA vaccines. Thus, it will be 394 important to test whether i.n. booster with S/B generates mucosal immunity in individuals 395 previously immunized with mRNA vaccines, and thereby increases the longevity of protection. 396

All experiments were performed in accordance with standard operating procedures at Biosafety 399 C57Bl/6J mice and IL-17 KO mice (male and female, older than 6 weeks old) were bred in-house. 409

BcfA was produced and purified as described previously (24) with stringent endotoxin removal. 411

Endotoxin level was <5 EU/mg protein (51). Stabilized S protein containing six proline 412 substitutions was produced and purified as described (Hsieh et al., 2020) . Aluminum hydroxide 413 (alum) and fetal bovine serum (FBS) was from Sigma-Aldrich (St. Louis, MO). ELISA kits were 414 from eBioscience (Thermo Fisher Scientific). Flow cytometry antibodies were from eBioscience, 415 BD Biosciences or R&D Systems (see Table 1 ). 416 

Virus neutralization by serum and lung antibodies was performed as described (Zeng et al., 2020) . 1302) for 1 hour at RT, then developed with TMB as above. 544

Lung samples from mice were processed per a standard protocol. Briefly, the tissues were fixed 546 in 10% neutral buffered formalin. Tissues were processed and embedded in paraffin. Five-547 micrometer sections (3 per tissue) were stained with hematoxylin and eosin by the Comparative 548 24 veterinary pathologist (K.N.C.) was blind to the experimental groups, and sections were scored 550 qualitatively on a scale ranging from 0 to 5 for the degree of cellularity and consolidation, the 551 thickness of the alveolar walls, degeneration and necrosis, edema, hemorrhage, infiltrating 552 alveolar/interstitial polymorphonuclear cells (PMNs), intrabronchial PMNs, perivascular and 553 peribronchial lymphocytes and plasma cells, and alveolar macrophages. The total inflammation 554 score was calculated by totaling the qualitative assessments in each category. 555

Immunohistochemistry to detect nucleocapsid protein expression in lung sections was conducted 556 by HistoWiz, Inc. Briefly, tissue sections were stained with a rabbit monoclonal anti-nucleocapsid 557 antibody (Genetex # GTX635686) using standard methodology. 558

Anesthetized (isoflourane) mice were intranasaly infected with 10 5 PFU of SARS-CoV-2 MA10 560 diluted in PBS where indicated. Clinical signs of disease (weight loss) were monitored daily. Mice 561 were euthanized by isoflurane overdose at 2 days post infection and samples for titer (caudal right 562 lung lobe and nasal septum) and histopathological analyses (left lung lobe) were collected. 563

Importantly, mice were randomized and assigned to specific harvest days before the start of the 564 experiment. Lung viral titers were determined by plaque assay. Briefly, right caudal lung lobes 565 were homogenized in 1mL PBS using glass beads and serial dilutions of the clarified lung 566

homogenates were added to a monolayer of Vero E6 cells. After three days CPE was examined 567 via staining for viral nucleoprotein (Sino Biological Cat. 40143-MM08-100). The left lung lobe was 568 stored in 10% phosphate buffered formalin for 7 days prior to removal from the BSL3 for 569 processing. After paraffin embedding, sectioning and staining histopathological scoring was 570 Gating strategy in Figure S1 . (F) The percentage and number of CD45-CD3+CD8+CD44+CD62L-CD69+

antigen-experienced CD8+ TRM and (G) IFNγ+ subset is shown. One-way ANOVA with Tukey's multiple comparisons was used to detect differences between all experimental groups. Significance is indicated above for each group ( * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001). The data are representative of 2 independent experiments. Mean and SEM are displayed. with Tukey's multiple comparisons was used to detect differences between all experimental groups.

Significance is indicated above for each group ( * p < 0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001). N= 5 animals per group, mean and SEM of the results are displayed. Dissociated lung cells stimulated with S1 and S2 peptide pools were stained and sequentially gated to identify the CD45-and CD45+ fractions, as described in Figure S1 . 

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