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. NT90 and 485 S protein-binding titers were shown in Fig. 1J and Fig. S3B . Data are means ± SEM 486 (standard errors of means). P values were analyzed with one-way ANOVA (ns, P > 487 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). The dashed line 488 indicates the limit of detection. values were analyzed with one-way ANOVA (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001). The dashed line indicates the limit of detection.

A novel coronavirus from patients with pneumonia in China

WHO Coronavirus Disease (COVID-19) Dashboard

A novel coronavirus outbreak of global 544 health concern

Learning from the past: development of safe and effective 546 COVID-19 vaccines

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, 548 BBIBP-CorV: a randomised, double-blind, placebo-controlled, phase 1/2 trial. The 549 Lancet Infectious Diseases

Effect of an inactivated vaccine against SARS-CoV-2 on safety and 551 immunogenicity outcomes: interim analysis of 2 randomized clinical trials

Development of an inactivated vaccine candidate for SARS-CoV-2

Development of an inactivated vaccine candidate, BBIBP-CorV, with 555 potent protection against SARS-CoV-2

Randomized, double-blinded and placebo-controlled phase II trial of an 557 inactivated SARS-CoV-2 vaccine in healthy adults

Cold-adapted live attenuated SARS-Cov-2 vaccine completely 559 protects human ACE2 transgenic mice from SARS-Cov-2 infection

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces 561 protective immunity

Phase 1-2 trial of a SARS-CoV-2 recombinant spike protein nanoparticle 563 vaccine

A universal design of betacoronavirus vaccines against COVID-19, MERS, 565 and SARS

NVX-CoV2373 vaccine protects cynomolgus macaque upper 567 and lower airways against SARS-CoV-2 challenge

A COVID-19 vaccine candidate using SpyCatcher multimerization of the 569

SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising 570 antibody responses

Newcastle disease virus-like particles displaying prefusion-stabilized 572 SARS-CoV-2 spikes elicit potent neutralizing responses

Phase 1 trial of a candidate recombinant virus-like particle vaccine for 574 randomised, double-blind, placebo-controlled, phase 2 trial

Safety and immunogenicity of an rAd26 and rAd5 vector-based 583 heterologous prime-boost COVID-19 vaccine in two formulations: two open, 584 non-randomised phase 1/2 studies from Russia

Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine 586 against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised 587 controlled trial

A single-dose intranasal ChAd vaccine protects upper and lower 589 respiratory tracts against SARS-CoV-2

Replication-competent vesicular stomatitis virus vaccine vector protects 591 against SARS-CoV-2-mediated pathogenesis in mice

Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus 594 macaques

An adenovirus-vectored COVID-19 vaccine confers protection from 596 SARS-COV-2 challenge in rhesus macaques

COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell 598 responses

Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults. 600

Safety and immunogenicity of two RNA-Based Covid-19 vaccine 602 candidates

A thermostable mRNA vaccine against COVID-19

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Immunogenicity of a DNA vaccine candidate for COVID-19

ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia 610 in rhesus macaques

Evaluation of the immunogenicity of prime-boost vaccination with 612 the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19

Ad26 vaccine protects against SARS-CoV-2 severe clinical disease 615 in hamsters

Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized 617

Spike immunogen induces potent humoral and cellular immune responses. 618 NPJ Vaccines

Structural and functional basis of SARS-CoV-2 entry by using human 620 ACE2

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is 622 blocked by a clinically proven protease inhibitor

Structural basis of receptor recognition by SARS-CoV-2

Distinct conformational states of SARS-CoV-2 spike protein

A human neutralizing antibody targets the receptor-binding site of 627 SARS-CoV-2

Convergent antibody responses to SARS-CoV-2 in convalescent 629 individuals

Human neutralizing antibodies elicited by SARS-CoV-2 infection

Structures of human antibodies bound to SARS-CoV-2 spike reveal 633 common epitopes and recurrent features of antibodies

Potent neutralizing antibodies from COVID-19 patients define 635 multiple targets of vulnerability

Potent neutralizing antibodies against SARS-CoV-2 identified by 637 high-throughput single-cell sequencing of convalescent patients' B cells

Structurally resolved SARS-CoV-2 antibody shows high efficacy in severely 640 infected hamsters and provides a potent cocktail pairing strategy

Aerosol and surface distribution of severe acute respiratory syndrome 643 coronavirus 2 in hospital wards

Studies in humanized mice and convalescent humans yield a 646 SARS-CoV-2 antibody cocktail

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection 648 from disease in a small animal model

Broad neutralization of SARS-related viruses by human monoclonal 650 antibodies

A noncompeting pair of human neutralizing antibodies block COVID-19 652 virus binding to its receptor ACE2

Potently neutralizing and protective human antibodies against 654 SARS-CoV-2

Early death after feline infectious peritonitis virus challenge due to 656 recombinant vaccinia virus immunization

A study on the 658 mechanism of antibody-dependent enhancement of feline infectious peritonitis virus 659 infection in feline macrophages by monoclonal antibodies

Antibody-dependent 662 enhancement occurs upon re-infection with the identical serotype virus in feline 663 infectious peritonitis virus infection

Role of antibody-dependent 665 enhancement (ADE) in the virulence of SARS-CoV-2 and its mitigation strategies for the 666 development of vaccines and immunotherapies to counter COVID-19

Antibody-dependent infection of human macrophages by severe acute 669 respiratory syndrome coronavirus

Antibodies against trimeric S glycoprotein protect hamsters against 671

SARS-CoV challenge despite their capacity to mediate FcgammaRII-dependent entry 672 into B cells in vitro

A perspective on potential antibody-dependent enhancement of 674 SARS-CoV-2

The human immune response to Dengue virus is dominated by 676 highly cross-reactive antibodies endowed with neutralizing and enhancing activity

Zika virus infection enhances future risk of severe dengue disease

Potent neutralizing antibodies directed to multiple epitopes on SARS-CoV-2 681 spike

Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction 683 with ACE2

Immunization with the receptor-binding domain of SARS-CoV-2 elicits 685 antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent 686 enhancement

Elicitation of potent neutralizing antibody responses by designed 688 protein nanoparticle vaccines for SARS-CoV-2

Neutralizing antibody responses to enterovirus and adenovirus in healthy 690 adults in China. Emerging microbes & infections

Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman 692 primate adenovirus in the presence of immunity against human adenovirus. Human 693 gene therapy

A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, 695 and SARS

Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD 697 protein vaccine against COVID-19 in adults: pooled analysis of two randomized, 698 double-blind, placebo-controlled, phase 1 and 2 trials

Mucosal IgA responses in influenza virus 701 infections; thoughts for vaccine design

Inducing mucosal IgA: A challenge for vaccine adjuvants and delivery 703 systems

Broad and strong memory CD4(+) and CD8(+) T cells induced by 705 SARS-CoV-2 in UK convalescent individuals following COVID-19

SARS-CoV-2-derived peptides define heterologous and 708 COVID-19-induced T cell recognition

Targets of T cell responses to SARS-CoV-2 coronavirus in humans with 710 COVID-19 disease and unexposed individuals

SARS-CoV-2-reactive T cells in healthy donors and patients with 712 COVID-19

SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, 714 and uninfected controls

A SARS-CoV-2 infection model in mice demonstrates protection by 716 neutralizing antibodies

The architecture of SARS-CoV-2 transcriptome

Antibody-dependent enhancement 720 and SARS-CoV-2 vaccines and therapies

Viral targets for vaccines against COVID-19

Prospects for a safe COVID-19 vaccine

Immunization with inactivated Middle East Respiratory Syndrome 725 coronavirus vaccine leads to lung immunopathology on challenge with live virus

Effects of Toll-like receptor stimulation on eosinophilic 728 infiltration in lungs of BALB/c mice immunized with UV-inactivated severe acute 729 respiratory syndrome-related coronavirus vaccine

Prior immunization with severe acute respiratory syndrome 732 (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein causes severe 733 pneumonia in mice infected with SARS-CoV

Immunization with Low Doses of Recombinant Postfusion or 736

Prefusion Respiratory Syncytial Virus F Primes for Vaccine-Enhanced Disease in the 737

Cotton Rat Model Independently of the Presence of a Th1-Biasing

Atypical measles and enhanced respiratory syncytial virus disease (ERD) 740 made simple

Cryo-EM structure of the 2019-nCoV spike in the prefusion 742 conformation

Recombinant Chimpanzee Adenovirus Vaccine AdC7-M/E Protects against

Zika Virus Infection and Testis Damage

Establishment and validation of a pseudovirus neutralization assay for 746 SARS-CoV-2. Emerging microbes & infections 9

SARS-CoV-2 infection protects against rechallenge in rhesus 748 macaques

Virological assessment of hospitalized patients with COVID-2019