key: cord-1026371-rcjpwkk6
authors: Luo, Shengxue; Zhang, Panli; Zou, Peng; Wang, Cong; Liu, Bochao; Wu, Cuiling; Li, Tingting; Zhang, Ling; Zhang, Yuming; Li, Chengyao
title: A Self-Biomineralized Novel Adenovirus Vectored COVID-19 Vaccine for Boosting Immunization of Mice
date: 2021-09-28
journal: Virol Sin
DOI: 10.1007/s12250-021-00434-3
sha: e2ae4b747d2f781e051943ce88b30468a0adc3f5
doc_id: 1026371
cord_uid: rcjpwkk6

SARS-CoV-2 has caused more than 3.8 million deaths worldwide, and several types of COVID-19 vaccines are urgently approved for use, including adenovirus vectored vaccines. However, the thermal instability and pre-existing immunity have limited its wide applications. To circumvent these obstacles, we constructed a self-biomineralized adenovirus vectored COVID-19 vaccine (Sad23L-nCoV-S-CaP) by generating a calcium phosphate mineral exterior (CaP) based on Sad23L vector carrying the full-length gene of SARS-CoV-2 spike protein (S) under physiological condition. This Sad23L-nCoV-S-CaP vaccine was examined for its characteristics of structure, thermostability, immunogenicity and avoiding the problem of preexisting immunity. In thermostability test, Sad23L-nCoV-S-CaP could be stored at 4 °C for over 45 days, 26 °C for more than 8 days and 37 °C for approximately 2 days. Furthermore, Sad23L-nCoV-S-CaP induced higher level of S-specific antibody and T cell responses, and was not affected by the pre-existing anti-Sad23L immunity, suggesting it could be used as boosting immunization on Sad23L-nCoV-S priming vaccination. The boosting with Sad23L-nCoV-S-CaP vaccine induced high titers of 10(5.01) anti-S1, 10(4.77) anti-S2 binding antibody, 10(3.04) pseudovirus neutralizing antibody (IC(50)), and robust T-cell response of IFN-γ (1466.16 SFCs/10(6) cells) to S peptides, respectively. In summary, the self-biomineralization of the COVID-19 vaccine Sad23L-nCoV-S-CaP improved vaccine efficacy, which could be used in prime-boost regimen for prevention of SARS-CoV-2 infection in humans. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12250-021-00434-3.

The coronavirus disease 2019 (COVID-19) usually presents as severe acute respiratory syndrome caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Wang et al. 2020a) , which has Shengxue Luo and Panli Zhang authors contributed equally to this work.

The online version contains supplementary material available at https://doi.org/10.1007/s12250-021-00434-3. become worldwide pandemic and more than 3.8 million people died worldwide (14 June 2021, WHO COVID-19 report; https://covid19.who.int/). Currently the development of safe, effective and easily preserved vaccines to prevent SARS-CoV-2 infection is urgently needed. SARS-CoV-2 genome encodes four major structural proteins, including spike (S), envelope (E), membrane (M) and nucleocapsid (N) (Srinivasan et al. 2020; Walls et al. 2020) . The S protein is a glycoprotein which contains S1 and S2 subunits, and is a major protective antigen that may elicit potent neutralizing antibody (NAb) and cellular immunity. The receptor-binding domain (RBD) within S1 binds to the human angiotensin-converting enzyme 2 (hACE2) for viral entry into human cell (Srinivasan et al. 2020; Walls et al. 2020) . Therefore, S antigen has been used as the primary antigen to develop candidate vaccines.

According to the World Health Organization (WHO) report of a draft landscape of COVID-19 candidate vaccines (15 June 2021, WHO COVID-19 report), some vaccines have been approved and registered for emerging use including adenovirus vector vaccine. Recombinant adenovirus vectors can induce strong immune responses and present good safety, and are widely used for research and development of vaccines (Abbink et al. 2015) . According to previously published data of Ad5, ChAdOx1 and Ad26 COVID-19 vaccines (Doremalen et al. 2020; Folegatti et al. 2020; Graham et al. 2020; Mercado et al. 2020; Tostanoski et al. 2020; Zhu et al. 2020a, b) , the relatively weaker immune response was found in humans with preexisting immunity or induced by a single-shot vaccine in comparison with prime-boost immunizations by two or three doses of inactivated virus or mRNA vaccines (Corbett et al. 2020; Gao et al. 2020; Jackson et al. 2020; Mulligan et al. 2020; Wang et al. 2020b ). Therefore, the major limitations for wide application of adenovirus vectors are the preexisting anti-vector immunity in prime and boost immunizations, as well as the thermal instability that leads to incomplete immunization and loss of efficacy during storage and delivery of vaccines.

The novel adenoviral vector Sad23L was constructed by simian adenovirus type 23 (SAdV23), which was lowseroprevalence (\ 10%) comparing with high pre-existing anti-Ad5 antibody (75.2%) in humans (Luo et al. 2019 (Luo et al. , 2021 . In addition, the calcium phosphate (CaP), as the major component of bones and teeth, is the excellent bioinorganic material and was widely used for biological applications, in particular for virus surface engineering because of its non-toxicity, biocompatibility and biodegradability (Walters and Welsh 1999; Shen et al. 2004; Lin et al. 2019 ). More recently, some studies showed CaP could serve as an excellent mineral shell candidate to stabilize virus and escape from preexisting anti-vector immunity (Wang et al. 2012 (Wang et al. , 2013 (Wang et al. , 2016 , as well as adjuvant with immunostimulatory features to enhance systemic immune responses in mice (Jiang et al. 2004; Dorozhkin 2013; Lin et al. 2017; Amanat and Krammer 2020) .

In this study, the novel adenovirus vector Sad23L carried the full-length S gene of SARS-CoV-2, which was designated as Sad23L-nCoV-S vaccine in a separate study (Luo et al. 2021) . By using CaP, Sad23L-nCoV-S was selfbiomineralized, which designated as Sad23L-nCoV-S-CaP vaccine and characterized for thermostability, immunogenicity and escaping preexisting anti-Sad23L immunity in mice.

HEK-293A, HEK-293T and HEK293T-hACE2 cells were maintained in complete Dulbecco's modified Eagle's medium (DMEM, Gibco) and incubated at 37°C in 5% CO 2 . Female BALB/c mice were obtained from the Animal Experimental Centre of Southern Medical University, Guangdong, China.

Sad23L-nCoV-S vaccine strain was previously constructed with a simian adenovirus type 23 based vector Sad23L, carrying the full-length spike protein (S) gene of SARS-CoV-2 (GenBank: MN908947.3) (Luo et al. 2019 (Luo et al. , 2021 . Sad23L-nCoV-S vaccine was propagated from HEK-293A cells, and purified by cesium chloride density gradient centrifugation as previously described (Luo et al. 2019 ).

The purified 10 9 PFU Sad23L-nCoV-S vaccine was incubated with 10 mmol/L calcium chloride (CaCl 2 ) at 4°C for 4 h, and then dropwise titration of sodium hydrogen phosphate under a consistent pH (pH = 7.6) at room temperature. The Sad23L-nCoV-S-CaP vaccine was recollected by centrifugation at 8000 9g as described (Wang et al. 2012 ).

The morphological observation of Sad23L-nCoV-S-CaP nanoparticles was conducted by Transmission Electron Microscope (TEM, JEM-1400, JEOL, Japan) directly without any stain. Briefly, Sad23L-nCoV-S-CaP particles Virologica Sinica were drop-cast onto TEM grids and blown away to keep dried without stain. In comparison, Sad23L-nCoV-S particles were stained with phosphotungstic acid. Energydispersive X-ray spectroscopy (EDS) analysis of Sad23L-nCoV-S-CaP and Sad23L-nCoV-S was conducted using Scanning Electron Microscope (SEM, S-3000 N, Hitachi, Japan).

Titers of purified viruses were calculated in PFU/mL by standard plaque-forming assay on HEK-293A, in which the cells are infected with serially diluted adenovirus stock and then over laid with agar, and a plaque will form as the result of a single infectious event. Sad23L-nCoV-S and Sad23L-nCoV-S-CaP were incubated at 4°C, 26°C and 37°C, respectively, and samples were collected periodically. The infectivity of the remainings was detected by standard plaque-forming assay on HEK-293A.

HEK-293A cells were infected with Sad23L-nCoV-S or Sad23L-nCoV-S-CaP strains, respectively, and Sad23L-GFP or Sad23L-GFP-CaP was used as controls. The expression of SARS-CoV-2 S protein was analyzed by Western blotting with rabbit polyclonal antibody to SARS-CoV-2 RBD (Sino Biological, China). The nitrocellulose (NC) membrane was developed by Supersignal West Pico Plus chemiluminescent substrate (Thermo Scientific, USA).

For immunological detection of Sad23L coat proteins, the CaP, Sad23L-nCoV-S and Sad23L-nCoV-S-CaP (native and denatured) were spotted onto a nitrocellulose filter (NC) membrane, then air-dried and blocked using 5% skim milk in PBST. The NC membrane was incubated with a polyclonal antibody anti-Sad23L, followed by an alkaline phosphatase-conjugated goat anti-mouse antibody. Signals were generated by the addition of 5-bromo-4-chloro-3-isdolyl phosphate/nitro blue tetrazolium (BCIP/NBT).

Mouse plasma samples were collected and detected for the neutralizing antibody (NAb) titer to Sad23L-GFP or Sad23L-GFP-CaP viruses in HEK-293A cells by green fluorescent activity assay as previously described. NAb titers were defined as the maximum serum dilution that neutralized 50% of green activity (Luo et al. 2019; Wang et al. 2019 ).

Female BALB/c mice (5-6 weeks, n = 5/group) were individually inoculated intramuscularly (i.m.) with a dose of 10 7 PFU Sad23L-nCoV-S or Sad23L-nCoV-S-CaP vaccine, respectively. A dose of 10 7 PFU Sad23L-GFP virus and an equivalent volume of CaP were used as sham controls (Supplementary Table S1 ). Female BALB/c mice (5-6 weeks, n = 5 each group) were primed by intramuscular inoculation with PBS or 10 7 PFU Sad23L-GFP, and then were boosted with a dose of 10 7 PFU Sad23L-nCoV-S or Sad23L-nCoV-S-CaP at 4-week interval (Supplementary Table S2 ). Female BALB/c mice (5-6 weeks, n = 5/group) were intramuscularly primed with 10 7 PFU Sad23L-nCoV-S, and then were boosted with 10 7 PFU Sad23L-nCoV-S or Sad23L-nCoV-S-CaP at 4-week interval, respectively (Supplementary Table S3 ).

The plates were coated overnight with 1 lg/mL of recombinant S1 or S2 protein of SARS-CoV-2 (Sino Biological, China). Sera were threefold serially diluted and specific binding antibodies (BAb) were detected by ELISA. Endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance [ 0.2, and a ratio of signal than cutoff (S/CO) [ 1. Log10 end point titers were reported ).

SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated for measuring of specific neutralizing antibody (NAb) titer as previously described . Briefly, the plasmids psPAX2 (Addgene), pLenti-CMV Puro-Luc (Addgene) and pcDNA3.1-SDCT were co-transfected into HEK-293T cells. The supernatants were collected 48 h post-transfection and purified by filtration through 0.45 lm filter. The two-fold serial dilutions of heat-inactivated serum samples mixed with 50 lL of pseudovirus incubated for 1 h at 37°C, the mixture was added to HEK293T-hACE2 cells. After 48 h, cells were lysed with Steady-Glo Luciferase Assay (Promega). The pVNT NAb titer to SARS-CoV-2 was defined as the reciprocal serum dilution at which a 50% inhibition rate was obtained. Inhibition rate (%) = (1 -sample (Relative light units, RLU)/virus control RLU) 9 100. Log10 pVNT (IC 50 ) titer was reported.

Mouse splenocytes (5 9 10 5 cells/well) were stimulated with S peptides (3 lg/mL) in triplicates, and detected SARS-CoV-2 S antigen-specific T lymphocyte response by Mice IFN-c ELISpot kits (MabTech). SARS-CoV-2 S peptides were predicted (http://www.iedb.org/) and synthesized by Guangzhou IGE Biotechnology LTD (Supplementary Table S4 ). Spots were counted with a CTL Immunospot Reader (Cellular Technology Ltd). The results were expressed as spot forming cells (SFCs) per million cells.

Mouse splenocytes (2 9 10 6 cells/well) were isolated and stimulated with S peptide pools (3 lg/mL each peptide), or medium as negative control. After 4 h, the cells were incubated with Golgi Plug (BD) for 12 h at 37°C. Cells were collected and stained with anti-mouse CD3, CD4 and CD8 surface marker antibodies, then fixed with IC fixation buffer, permeabilized with permeabilization buffer and stained with anti-mouse interferon-c (IFN-c), interleukin-2 (IL-2) and tumor necrosis factor a (TNF-a) (eBioscience) antibodies. All samples were tested with BD FACS Canton flow cytometer (BD).

Data are analyzed with unpaired two-tailed t test, one-way ANOVA, Mann-Whitney test. Statistically significant differences are indicated with asterisks (*P \ 0.05; **P \ 0.01 and ***P \ 0.001). All graphs are generated with GraphPad Prism 7 software.

The novel adenovirus vector COVID-19 vaccine (Sad23L-nCoV-S) was produced by HEK-293A cell culture (Supplementary Fig. S1 ). In a calcium-rich culture medium under physiological condition, the surface of Sad23L-nCoV-S virus was coated with calcium phosphate (CaP) to form a biomineral virus nanoparticle as Sad23L-nCoV-S-CaP vaccine (Fig. 1A) . The Sad23L-nCoV-S viruses were presented as typical adenovirus particles observed under transmission electron microscopy (TEM) with phosphotungstic acid negative staining (Fig. 1B) . The CaP-coated layer on Sad23L-nCoV-S-CaP nanoparticles was seen by Scanning Electron Microscope (SEM) (Fig. 1C) , and the mineral phase from Ca, P or O ions of Sad23L-nCoV-S-CaP vaccine was measured by the energy dispersive X-ray spectroscopy (EDS) (Fig. 1D ). The biomineral structure was identified by TEM without any staining, which presented a superficial CaP circle on Sad23L-nCoV-S-CaP viral particle (Fig. 1E) , while generally, a native virus could not be observed by TEM without staining. By using anti-Sad23L serum in a dot blot assay, the viral surface protein was detected on both native and denatured Sad23L-nCoV-S, but not from native Sad23L-nCoV-S-CaP (Fig. 1F) , indicating that CaP blocked the binding of antibody to surface protein of Sad23L-nCoV-S virus. Expression of SARS-CoV-2 S protein was identified in Sad23L-nCoV-S or Sad23L-nCoV-S-CaP vaccines infected HEK-293A cells by Western blot with rabbit polyclonal antibodies specific to RBD, but not in the vector controls of Sad23L-GFP or Sad23L-GFP-CaP viruses infected cells (Fig. 1G) . The infectivity of Sad23L-nCoV-S-CaP vaccine to HEK-293A cells was also tested. Cytopathic effect was observed after 48 h post infection, which is consistent with Sad23L-nCoV-S (Fig. 1H ).

The thermostability of Sad23L-nCoV-S-CaP and Sad23L-nCoV-S vaccines was examined at 4°C (cold chain), 26°C (room temperature) and 37°C (high temperature) by measuring the infectious titers of vaccine with plaque assays (Fig. 2) . The infectious titers of Sad23L-nCoV-S-CaP and Sad23L-nCoV-S vaccines were appeared different at three temperatures with prolonging incubation, in which the initial titer of Sad23L-nCoV-S-CaP vaccine (10 7.98 PFU) was slowly decreased to 10 7.03 PFU in 20 d at 4°C (Fig. 2A) , to 10 4.63 PFU in 14 d at 26°C (Fig. 2B) or 10 4.58 PFU in 144 h at 37°C (Fig. 2C) , while the initial titer of Sad23L-nCoV-S vaccine (10 8.23 PFU) was quickly decreased to 10 5.93 PFU in 20 d at 4°C, 10 1.69 PFU in 14 d at 26°C or 10 1.27 PFU in 144 h at 37°C, respectively. At 2-log 10 reduction of the initial titer after 17.2 d, 3.2 d and 26.5 h of Sad23L-nCoV-S at 4°C, 26°C and 37°C, respectively (Fig. 2) , but after 45.8 d, 8.4 d and 68.6 h of Sad23L-nCoV-S-CaP at 4°C, 26°C and 37°C. Therefore, Sad23L-nCoV-S-CaP exhibited a significantly slower inactivation rate (0.0408 d -1 , 0.2372 d -1 , 0.02256 h -1 ) and its storage could be prolonged to more than 45 d at 4°C, 8 d at 26°C and 68 h at 37°C, respectively (Fig. 2) . These results suggested that CaP biomineralization obviously improved Sad23L-nCoV-S-CaP vaccine thermostability, especially its infectious titer retained over 10 6.07 PFU for 8 days at room temperature.

To evaluate the immunogenicity, BALB/c mice were intramuscularly injected with a single dose of 10 7 PFU

Sad23L-nCoV-S-CaP vaccine in comparing with Sad23L-nCoV-S vaccine, Sad23L-GFP and an equal volume of CaP controls ( Fig. 3A; Supplementary Table S1 ). After 4 weeks post-immunization, the specific serum binding antibody (BAb) to S1 or S2 protein (S1-BAb or S2-BAb) was measured by ELISA. Sad23L-nCoV-S-CaP vaccine raised BAb titers of 10 4.63 to S1 and 10 4.08 to S2, respectively, which were the same with Sad23L-nCoV-S vaccine (10 4.55 S1-BAb and 10 3.93 S2-BAb) but significantly higher than the control groups (P \ 0.001, Fig. 3B, 3C) . The neutralizing antibody (NAb) to SARS-CoV-2 were quantified by pseudovirus-based NAb test (pVNT) at 50% inhibition concentration (IC 50 ) (Fig. 3D) . Similarly, Sad23L-nCoV-S-CaP vaccine induced NAb titer of 10 2.59 pVNT (IC 50 ) equal to Sad23L-nCoV-S vaccine induced NAb titer (10 2.53 pVNT at IC 50 ) (Fig. 3E) .

Specific T-cell response of splenocytes to S peptides was detected by ELISpot ( Fig. 3F; Supplementary Fig. S2 ). The IFN-c secreting T cell response was 1075.36 SFCs/ million cells in Sad23L-nCoV-S-CaP vaccine injected mice while the similar level of 923.21 SFCs/million cells was in Sad23L-nCoV-S vaccine group (Fig. 3F ). Both vaccines elicited T cell response significantly higher than sham controls (P \ 0.001).

Overall, the self-biomineralized vaccine can develop high level of specific humoral and T cell responses to SARS-CoV-2 S antigen in mice, suggesting Sad23L-nCoV-S-CaP vaccine carries strong immunogenicity. A BALB/c mice (n = 5/group) were immunized with 10 7 PFU Sad23L-nCoV-S or Sad23L-nCoV-S-CaP. Sera and splenocytes were collected for the detection of antibody and T cell response 4-week post-immunization. B S1-BAb or C S2-BAb titers were measured by ELISA. D Inhibition of SARS-CoV-2 pseudovirus by anti-sera from vaccinated or control mice. E NAb titers were measured by pseudovirus neutralization test. F IFN-c secreting T cell response (SFCs/million cells) of splenocytes to S peptides was measured by ELISpot. Data is shown as mean ± SEM. P values are analyzed by one-way ANOVA with twofold Bonferroni adjustment. Statistically significant differences are shown with asterisks (***, P \ 0.001; ns, P [ 0.05 or no significant difference).

Sad23L-GFP Anti-Sad23L Serum Fig. 4 The self-biomineralized Sad23L virus escaped pre-existing immunity in vitro and in vivo. A Neutralization of Sad23L-GFP and Sad23L-GFP-CaP in HEK-293A cells with anti-Sad23L serum. Scale bar = 1 mm. B Immunization regimen in naïve and Sad23L preexposed mice. C S1-BAb or D S2-BAb titers were detected by ELISA. E NAb titers were obtained by pVNT. F Splenocytes was stimulated by S peptides and T cell response and measured by ELISpot. Frequency of intracellular IFN-c, TNF-a or IL-2 expressing in CD4 ? (G) or CD8 ? (H) T cell response to S peptides was determined by ICS, respectively. Data is shown as mean ± SEM. P values are analyzed by one-way ANOVA and unpaired t test. Statistically significant differences are shown with asterisks (*, P \ 0.05; **, P \ 0.01; ***, P \ 0.001 and ns, P [ 0.05 or no significant difference).

The Self-Biomineralized Virus Avoided Impact of Preexisting Immunity on Boosting Immunization In Vitro and In Vivo

The influence of preexisting immunity of adenovirus vector Sad23L was compared between Sad23L-GFP-CaP and Sad23L-GFP virus. Mouse anti-Sad23L serum inhibited Sad23L-GFP virus infection in HEK-293A cells, of which the GFP fluorescent signal was remarkably reduced depending on the dilution of antiserum (Fig. 4A, upper  panel) , while the anti-Sad23L serum did not neutralize Sad23L-GFP-CaP infection in cells (Fig. 4A, lower panel) .

The results suggested that CaP-coated Sad23L-GFP virus (Sad23L-GFP-CaP) could avoid being neutralized by anti-Sad23L antibody in vitro.

In order to explore whether Sad23L-nCoV-S-CaP vaccine could evade pre-existing immunity in vivo, three groups of naïve mice were prime immunized with 10 7 PFU Sad23L-GFP virus, and the neutralizing antibody (NAb) to Sad23L-GFP was determined as 1:906.67 (mean) in week 4 ( Supplementary Fig. S3 ). These Sad23L pre-exposed mice were secondarily injected with 10 7 PFU Sad23L-GFP-CaP (GFP), Sad23L-nCoV-S (G3) or Sad23L-nCoV-S-CaP (G4) virus in the boost immunization, respectively. In comparison, Sad23L non-exposed (PBS injected) mice were inoculated with CaP (CaP), Sad23L-nCoV-S (G1) or Sad23L-nCoV-S-CaP (G2) viruses ( Fig. 4B ; Supplementary Table S2 ). Serum S1-BAb or S2-BAb titers were detected by ELISA after four weeks post boost-immunization. The mean endpoint S1-BAb titers were 10 4.45 in G1, 10 4.57 in G2, 10 1.87 in G3 or 10 4.21 in G4 group (Fig. 4C) , and corresponding S2-BAb titers were 10 3.89 , 10 3.91 , 10 1.79 or 10 3.59 in G1 to G4 group (Fig. 4D) , respectively, but no S1-BAb or S2-BAb were detected in CaP and GFP control mice (P \ 0.001). NAb (pVNT) titers to SARS-CoV-2 were tested for 10 2.65 (G1), 10 2.74 (G2), 10 1.18 (G3) and 10 2.46 (G4), respectively ( Fig. 4E ; Supplementary Fig. S4 ). Sad23L-nCoV-S-CaP vaccine group (G4) exhibited significantly higher S1-BAb, S2-BAb and pVNT titers than Sad23L-nCoV-S vaccine group (G3) based on Sad23L pre-exposed mice ( Fig. 4C-4E , P \ 0.001).

Specific T cell response to S peptides was measured by ELISpot after 4 weeks post boost-immunization of mice. The IFN-c secretion T cell response of Sad23L-nCoV-S-CaP (G4, 744.8 SFCs/10 6 cells) in Sad23L pre-exposed mice paralleled with G1 (827.4 SFCs/10 6 cells) and G2 (899.6 SFCs/10 6 cells) in Sad23L non-exposed mice (Fig. 4F , P [ 0.05), which was significantly higher than Sad23L-nCoV-S (G3, 251.4 SFCs/10 6 cells) in Sad23L pre-exposed mice, CaP and GFP control groups (Fig. 4F , P \ 0.001). Frequency of intracellular IFN-c, TNF-a and IL-2 expressing CD4 ? or CD8 ? T cell response to S peptides was measured by ICS (Fig. 4G, 4H ; Supplementary  Fig. S5 ), in which G4, G1 and G2 induced similar IFN-c or TNF-a expressing CD4 ? /CD8 ? T cell response, but significantly higher than Sad23L-nCoV-S (G3) in Sad23L preexposed mice (P \ 0.05). Four groups (G1 to G4) varied insignificantly in intracellular IL-2 expressing CD4 ? /CD8 ? T cell responses (Fig. 4G, 4H; Supplementary Fig. S5 ).

The prime-boost regimen was applied for inoculating BALB/c mice (n = 5/group) by priming with 10 7 PFU Sad23L-nCoV-S and boosting with 10 7 PFU Sad23L-nCoV-S (V1) or Sad23L-nCoV-S-CaP (V2) at 4-week interval in comparison with PBS/CaP (CaP) and Sad23L-GFP/Sad23L-GFP-CaP (GFP) control groups ( Fig. 5A ; Supplementary Table S3 ). The booster V2 induced significantly higher S1-BAb titer (10 5.01 ) than the booster V1 (10 4.83 ) (P = 0.038, Fig. 5B ), while the booster V2 or V1 raised similar S2-BAb (10 4.71 vs 10 4.77 , P [ 0.05, Fig. 5C ) and pVNT NAb titers (10 3.04 vs 10 2.78 , P [ 0.05, Fig. 5D ), respectively.

Regarding to specific T cell response to S peptides (Fig. 5E ), the booster Sad23L-nCoV-S-CaP induced the higher level of specific IFN-c secreting T cell response (1466.16 SFCs/10 6 cells) in V2 than Sad23L-nCoV-S in V1 (1170 SFCs/10 6 cells, P = 0.0016). In contrast, the frequency of IFN-c, TNF-a and IL-2 of CD4 ? and CD8 ? T cell responses were not statistically different between V1 and V2 (P [ 0.05) (Fig. 5F-5I) , excepting for that the higher frequency of TNF-a producing CD8 ? T cell response in V2 (0.81%) than V1 (0.53%) (P = 0.0409, Fig. 5I ).

Taken together, the results suggest that priming immunization with Sad23L-nCoV-S vaccine and boosting with self-biomineralized Sad23L-nCoV-S-CaP vaccine could elicit higher immune response to SARS-CoV-2 antigen than a single shot of Sad23L-nCoV-S or Sad23L-nCoV-S-CaP vaccine (Supplementary Fig. S6 ).

Although 102 COVID-19 candidate vaccines are in clinical trials (15 June 2021, WHO report), and some of them have been approved or registered for emerging use, the development of safe, thermostable, effective and easily produced COVID-19 vaccines is still a big challenge, especially for the vaccines used in the poorest countries. Our study may provide an alternative vaccine candidate.

In this study, we evaluated a novel adenovirus vector vaccine Sad23L-nCoV-S and its self-biomineralized form Sad23L-nCoV-S-CaP vaccine in prime-boost vaccination regimen. The self-biomineralization could improve the thermostability and immunogenicity of the vaccine, and overcome the preexisting anti-vector immunity from priming immunization with Sad23L-nCoV-S when it was used as a booster in vaccination. In comparison with other COVID-19 vaccines, the immunization regimen by priming with Sad23L-nCoV-S and boosting with Sad23L-nCoV-S-CaP vaccines presented several advantages, which are highlighted below as four attractive aspects.

Firstly, the novel adenovirus vector Sad23L is lowseroprevalence in humans. Comparing with over 75% prevalence of Ad5 in Chinese population, NAb prevalence of Sad23L is less than 10%, suggesting that Sad23L can be a better vector for vaccine development and avoid the preexisting immunity to common human adenoviruses (Luo et al. 2019 (Luo et al. , 2020 (Luo et al. , 2021 .

Secondly, CaP-masked vaccine (Sad23L-nCoV-S-CaP) could be considered as the vaccine that had a shell to protect the vaccine from preexisting antibody binding and improve its storage stability at room temperature. Therefore, this Sad23L-nCoV-S-CaP vaccine is particularly suitable for application in developing countries, where the vaccine delivery can be less dependent on cold-chain.

Thirdly, the self-biomineralized Sad23L-nCoV-S-CaP retained high immunogenicity and could induce stronger humoral and cellular immune response in mice than Sad23L-nCoV-S (Fig. 3) .

Fourthly, the self-biomineralized vaccine could overcome preexisting anti-Sad23L immunity in vitro and in vivo. Furthermore, Sad23L-nCoV-S-CaP induced significantly higher S1-BAb, S2-BAb, NAb, and specific IFN-c secreting Fig. 5 Specific humoral and T cell response of mice immunized with Sad23L-nCoV-S and Sad23L-nCoV-S-CaP vaccine by prime-boost regimen. A Immunization regimen. (B) S1-BAb or (C) S2-BAb titers were tested by ELISA. D NAb titers were measured by pVNT (IC 50 ). E IFN-c secreting level to S peptides was measured by ELISpot. (F-I) Frequency of intracellular IFN-c, TNF-a and IL-2 expressing CD4 ? or CD8 ? T cell response to S peptides was determined by intracellular cytokine staining, respectively. Data are shown as mean ± SEM. P values are calculated with two-tailed t test. Statistically significant differences are shown with asterisks (*, P \ 0.05; **, P \ 0.01; ***, P \ 0.001; ns, P [ 0.05 or no significant difference).

T cell response, and IFN-c or TNF-a expressing CD4 ? / CD8 ? T cell responses than Sad23L-nCoV-S in Sad23L preexposed mice ( Fig. 4; Supplementary Fig. S6 ). Therefore, two inoculations of vaccines for prime-boost vaccination become possible, in which Sad23L-nCoV-S and Sad23L-nCoV-S-CaP are used as priming and boosting immunization, respectively. Compared with homologous boosting of ChAdOx1 nCoV-19 or Ad5-S vaccines Logunov et al. 2020) , the prime-boost vaccination regimen with Sad23L-nCoV-S and Sad23L-nCoV-S-CaP vaccines has the advantage of avoiding vector's immunity from priming immunization (Luo et al. 2019 (Luo et al. , 2020 (Luo et al. , 2021 . In this study, a self-biomineralized Sad23L-nCoV-S-CaP vaccine was evaluated in comparison with Sad23L-nCoV-S vaccine, which had better thermostability and was able to induce robust humoral and cellular immune responses in mice. Sad23L-nCoV-S-CaP vaccine could be used as a booster in combination with priming vaccine Sad23L-nCoV-S for prime-boost vaccinations against SARS-CoV-2 infection in humans.

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Acknowledgements The authors thank professors Kwok-Yung Yuen from The University of Hong Kong for his assistance for purchasing simian adenoviruses type 23; Dongming Zhou from The Institut Pasteur in Shanghai for his technical help for construction of adenovirus vectors. This work was supported by the grants from the National Natural Science Foundation of China (No. 32070929, 81871655 and 31770185), the Special Funding for COVID-19 Prevention and Control of China (2020M670013ZX), the China Postdoctoral Science Foundation (2021M691474) and Guangzhou Bai Rui Kang (BRK) Biological Science and Technology Limited Company, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.