key: cord-0786754-59v1mfen authors: Xu, Kun; An, Yaling; Li, Qunlong; Huang, Weijin; Han, Yuxuan; Zheng, Tianyi; Fang, Fang; Liu, Hui; Liu, Chuanyu; Gao, Ping; Xu, Senyu; Liu, William J.; Bi, Yuhai; Wang, Youchun; Zhou, Dongming; Wang, Qinghan; Hou, Wenli; Xia, Qianfeng; Gao, George F.; Dai, Lianpan title: Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat RBD of SARS-CoV-2 spike protein protects mice against COVID-19 date: 2021-02-06 journal: bioRxiv DOI: 10.1101/2021.02.05.429860 sha: c3f23586d333ba9124b31c80a9d5049e2c2b3e78 doc_id: 786754 cord_uid: 59v1mfen A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus vectored vaccines expressing spike (S) protein have advanced into phase 3 trials, with three approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD) or dimeric tandem-repeat RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular (Th1-based) immune responses. AdC7-RBD-tr2 showed higher antibody responses compared with both AdC7-S and AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate. As of 1 February, 2021, the pandemic of COVID-19 has accounted for more than 52 one hundred million laboratory-confirmed cases and two million deaths (1-3). The ADE (64, 65) . Therefore, we sought to develop COVID-19 87 vaccines based on both full-length S and RBD. 88 Here, we developed virus vectored vaccines based on chimpanzee adenovirus type 89 7 (AdC7), a rare serotype in the human population with the advantage of low level of 90 pre-existing immunity (66, 67). We used a design of tandem repeat RBD-dimer 91 (RBD-tr2) as antigen to increase the immunogenicity. This design has been used in 92 our protein subunit COVID-19 vaccine that has advanced in phase 3 clinical trials (68, 93 69). Our adenovirus-based vaccines are aiming to further enhance the T cell responses. 94 AdC7 expressing full-length or monomeric RBD were generated for comparison. 95 Vaccination via intramuscular and intranasal routes were evaluated to dissect the 96 systemic and mucosal immune responses, with the latter are believed to be beneficial 97 for protection in respiratory system (70, 71). These results will provide crucial 98 guidance for the further clinical trials. 99 100 We constructed three replication-incompetent AdC7 vaccines expressing 102 SARS-CoV-2 full-length S, RBD and RBD-tr2, respectively (Fig. 1A) . The 103 expression cassettes were inserted into the E1 region of the AdC7 vector with E1 and 104 E3 deletion. Western blot was conducted to confirm antigen expression in HEK 293T 105 cells infected with the recombinant adenovirus. All these antigens were detected in the 106 cell lysates (fig. S1A). Both monomeric RBD and RBD-tr2 were secreted in the 107 culture supernatants (fig. S1B). Particularly, the antigen expression of AdC7-RBD-tr2 108 was much higher than AdC7-S and AdC7-RBD (fig. S1, A and B). 109 To evaluate the immunogenicity of the recombinant AdC7 vaccines, groups of 110 BALB/c mice (n = 5) were immunized on day 0 and 28 with 1 x 10 11 vp of AdC7 111 vaccines via intramuscular (i.m.) injection (Fig. 1B) . AdC7 without any transgene 112 (AdC7-empty) was injected as the sham control. Blood samples were collected on day 113 21 and 42 (Fig. 1B) . The serological RBD-binding IgG and IgA titers was measured. 114 RBD-specific IgG were robustly induced by AdC7-S, AdC7-RBD and AdC7-RBD-tr2 115 (Fig. 1C) , while RBD-specific IgA antibodies were only moderately induced in the 116 mice vaccinated with AdC7-RBD-tr2 ( fig. S2A ). As neutralizing antibodies play a 117 crucial role in protection, pseudovirus displaying the SARS-CoV-2 S protein was used 118 to detect the neutralizating antibody titers in the sera of immunized mice. We found 119 90% neutralization titer (NT90) of the mice sera were increased post boost vaccination 120 for each AdC7 vaccine (Fig.1D) . Two doses of AdC7-S, AdC7-RBD and 121 AdC7-RBD-tr2 elicited average NT90 titers of 52, 72 and 416, respectively (Fig. 1D) . 122 AdC7-RBD-tr2 showed significantly higher neutralizing titers compared to the other 123 constructs (Fig. 1D) . and AdC7-RBD-tr2, respectively ( Fig 1M) . AdC7-RBD-tr2 group showed higher 153 mucosal pseudovirus NT90 compared to either AdC7-S or AdC7 RBD. In addition, we 154 analyzed the antibody quality in mice sera, and demonstrated RBD and 155 RBD-tr2-based vaccines exhibited higher neutralizing : binding ratio than fell-length 156 S-based vaccine (Fig 1N and S3B ). In summary, both systemic and mucosal immunity 157 were induced by recombinant vaccines through i.n. vaccination. Besides, 158 AdC7-RBD-tr2 showed the advantage of immunofocusing to induce neutralizing 159 antibodies blocking receptor-binding. 160 Aside from humoral immunity, cellular immunity also play an important role in AdC7-RBD-tr2 via both i.m. and i.n. routes (Fig. 2, A and B) . Additionally, the flow 169 cytometric analyses showed that all these recombinant AdC7 vaccines induced robust 170 induction of cytotoxic T lymphocytes (CTL), detected as IFNγ and IL-2 production, 171 via both i.m. and i.n. routes. AdC7-RBD-tr2 performed the best ( Fig. 2C and 2E ). 172 Besides, the CD4+ T cell responses cytokines were moderately induced for all these 173 three constructs ( Fig.2D and 2F ). In contrast, no substantial Th2 cytokines (IL-4 and 174 IL10) production was detected for all these three constructs ( Fig.2D and 2F ). These 175 results demonstrated Th1-biased immune responses were induced in mice received 176 AdC7-S, AdC7-RBD or AdC7-RBD-tr2 vaccines by both i.m. and i.n. routes. 177 Given the fact that AdC7-RBD-tr2 elicited the most robust RBD-binding IgG, IgA, Antibody quality of serum samples from two-dose immunized mice via the i.n. route. 484 The ratio is the pseudovirus neutralization titer : S protein-binding IgG titer. 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