key: cord-0963437-h1h6ypzk
authors: Adam, Awadalkareem; Shi, Qing; Wang, Binbin; Zou, Jing; Mai, Junhua; Osman, Samantha R; Wu, Wenzhe; Xie, Xuping; Aguilar, Patricia V; Bao, Xiaoyong; Shi, Pei-Yong; Shen, Haifa; Wang, Tian
title: A modified porous silicon microparticle promotes mucosal delivery of SARS-CoV-2 antigen and induction of potent and durable systemic and mucosal T helper 1 skewed protective immunity
date: 2021-11-24
journal: bioRxiv
DOI: 10.1101/2021.11.22.469576
sha: 83488a33ca24474bc4d8e818075e49fcb0cf93d2
doc_id: 963437
cord_uid: h1h6ypzk

Development of optimal SARS-CoV-2 vaccines to induce potent, long-lasting immunity and provide cross-reactive protection against emerging variants remains a high priority. Here, we report that a modified porous silicon microparticle (mPSM)-adjuvanted SARS-CoV-2 receptor-binding domain (RBD) vaccine activated dendritic cells and generated more potent and durable SARS-CoV-2-specific systemic humoral and type 1 helper T (Th) cell-mediated immune responses than alum-formulated RBD following parenteral vaccination, and protected mice from SARS-CoV-2 and Beta variant infection. mPSM facilitated the uptake of SARS-CoV-2 RBD antigens by nasal and airway epithelial cells. Parenteral and intranasal prime and boost vaccinations with mPSM-RBD elicited potent systemic and lung resident memory T and B cells and SARS-CoV-2 specific IgA responses, and markedly diminished viral loads and inflammation in the lung following SARS-CoV-2 Delta variant infection. Our results suggest that mPSM can serve as potent adjuvant for SARS-CoV-2 subunit vaccine which is effective for systemic and mucosal vaccination.

challenged with 2 x 10 4 PFU of the mouse-adapted SARS-CoV-2 strain CMA4 at 4.5 months post 161 vaccination. While mice in both mock and alum-RBD groups exhibited 10 2 to 10 3 PFU/ml viral loads in 162 the lung tissues; no detectable viral titers were measured in the mPSM-RBD group at day 4 post infection 163 (Fig 4A-B ). In addition, lung inflammation was assessed by measurement of proinflammatory cytokines 164 (IL-1, IL-6) and chemokines (CCL2, CCL7, CXCL10) levels (Fig 4C-H) . The mPSM-RBD-vaccinated 165 mice had significantly reduced levels of inflammation compared to the mock and the alum-RBD group.

166 Furthermore, to assess protective efficacy from a single dose vaccination, 6-8-week-old K18 hACE2 mice 167 were treated i.p. with PBS (mock), alum-RBD (25 µg), or mPSM-RBD (25 µg). Mice were challenged 168 i.n. with 4x10 3 PFU of SARS-CoV-2 Beta variant 1 month post vaccination. While both alum-RBD and 169 mPSM-RBD groups showed reduced viral loads in the lung compared to the mock group, mice in the 170 mPSM-RBD group had 40% lower viral load in the lungs than those in the alum-RBD group (Fig 4I, J) .

In summary, these data showed that the mPSM-RBD vaccine triggered more durable and stronger RPMI2650 cells, with a higher binding affinity to SAECs based on the average number of particles in 186 each cell type (Fig 5A) . mPSM-RBD co-localized with early endosome (EEA1 + , green) as soon as 0.5 h 187 after incubation. After 2 h and 6 h incubation, mPSM-RBD vaccine was gradually released from the 188 particles and reached the surrounding area inside the cells. These results suggest that mPSM can 189 effectively deliver RBD antigen and promote its uptake by upper respiratory epithelial cells. Next, we 190 assessed SARS-CoV-2-specific immune responses in BALB/c mice following primed i.p. with PBS

(mock), RBD alone, m-PSM-RBD or alum-RBD (5 µg) on day 0 and boosted i.n. with the same dose on 192 day 21 (Fig 5B) . Blood, bronchoalveolar lavage fluids (BAL), lung and spleen tissues were collected on 193 day 35. In the lung, there were stronger SARS-CoV-2-specific Th1 responses in mPSM-RBD group than 194 the alum-RBD group, and both CD4 + and CD8 + T cells produced more IFNthan the alum-RBD group 195 (Fig 5C-E) . While both alum-RBD and mPSM-RBD vaccinations triggered more RBD-specific IgA + B 196 cells in the lung compared to that of the mock group, the mPSM-RBD group produced at least 2-fold as 197 many RBD-specific IgA + B cells as those in the alum-RBD group (Fig 5F) . In the spleen, the mPSM-i.n. challenged with 1 x 10 4 PFU of SARS-CoV-2 Delta variant at day 35. On day 4 post infection, plaque 208 and Q-PCR assays showed that mPSM-RBD group had about 685-fold and 50-fold decrease in lung viral 209 loads compared to the mock and alum-RBD groups, respectively (Fig 6A, B) . In addition, the mPSM-210 RBD-vaccinated mice also showed significantly diminished levels of inflammatory cytokines in the lung 211 compared to those in the mock group; in comparison, no difference was detected between the alum-RBD 212 and mock groups (Fig 6C-E) .

In conclusion, these studies demonstrated that i.n. boost with mPSM-RBD 

The S protein, including RBD, can elicit highly potent and persistent NAbs and contain many T cell 227 epitopes 3 . Therefore, adjuvanted S or RBD protein subunit vaccines likely represent some of the most 

Furthermore, there is a potential concern of "vaccine-induced disease enhancement", which was reported 283 for certain SARS-CoV vaccine candidates 12 and inactivated RSV vaccines 48 . The potential risk of ADE 284 mediated by Fc-receptor could be increased due to waning immunity after vaccination and possibly 285 mutations in the SARS-CoV-2 S protein 49 . Due to the above concerns, the optimal COVID-19 vaccines 286 will need to exhibit long-lasting immunity, be effective for various populations globally, and provide 

Neutralizing Activity of BNT162b2-Elicited Serum

BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants

Respiratory syncytial virus disease in infants despite prior administration of 567 antigenic inactivated vaccine

Unique 570 Challenges of the Virus and Vaccines

A familial cluster of pneumonia associated with the 2019 novel coronavirus 573 indicating person-to-person transmission: a study of a family cluster

Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal 576 encephalitis

Dysregulation of Toll-Like Receptor 7 Compromises Innate and Adaptive T Cell

CoV-2 r-RBD measured in serum collected at various time points after vaccination

PRNT80 622 titers are shown, n = 4 or 6. F-H. SARS-CoV-2 specific memory B cell (MBC) responses by ELISPOT 623 analysis at 7 months post vaccination. F. Images of wells from MBC culture

RBD (H) specific ASCs per 10 6 input cells in MBC cultures from the subject. I-J. ELISPOT 625 quantification of vaccine-specific splenic T cells at 7 months post vaccination. Mouse splenocytes were 626 ex vivo stimulated with overlapping peptide pools spanning SARS-CoV-2 S protein, -CD3, or blank for 627 20 h. I. Images of wells from T cell culture

Data are shown as # of SFC per 10 6 splenocytes. n= 4. ** P < 0.01 compared to the mock 629 group. ## P < 0.01 compared to alum-RBD group. were collected at 24 h post-vaccination for analysis using Biochemistry Panel Plus analyzer discs

Abaxis) or proinflammatory cytokine levels by Q-PCR (F-H). Data are presented as the fold increase 682 compared to naïve mice (means ± SEM)

Supplementary Figure 2. mPSM-RBD induces SARS-CoV-2 specific immune responses in

C57BL/6 mice one month post parenteral vaccination. A-B. Endpoint IgG subtype titers against 685

SARS-CoV-2 rRBD measured in serum collected from vaccinated mice. n= 5. C-D. SARS-CoV-2 686 specific memory B cell (MBC) responses by ELISPOT analysis. C. Images of wells from MBC culture

2 and seeded onto ELISPOT plates 688 coated with Ig capture Ab or SARS-CoV-2 RBD. Images of total ASCs, RBD specific MBCs, and 689 negative control (NC) wells are shown. D. Frequencies of SARS-CoV-2 RBD specific ASCs per 10 6 690 input cells in MBC cultures from the subject

Six-to eight-week-old BALB/c mice (n =5) 704 were prime-boost immunized with mock (PBS), alum-RBD, or mPSM-RBD. One month post 705 vaccination, all mice were i.n. challenged with 2 x10 4 PFU mouse-adapted SARS-CoV-2 CMA4. At day 706 2 post infection (pi), lung tissues were collected. A. Study design and vaccination timeline

CoV-2 viral titers in lung tissues were measured by Q-PCR assay. C-E. Measurement of chemokine 708 levels in lung tissues by Q-PCR assays at day 2 post infection. Data are presented as the fold increase 709 compared to naïve mice (means ± SEM). ** P < 0

Supplementary Figure 5. Parenteral and mucosal prime-boost vaccination induced strong 711

At day 31 post 713 vaccination, blood and spleen tissues were collected for immunogenicity studies. A. ELISPOT 714 quantification of vaccine-specific splenic T cells at 1 month post vaccination