Prime-boost vaccination of mice and Rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates

  1. Shengxue Luo
  2. Panli Zhang
  3. Bochao Liu
  4. Chan Yang
  5. Chaolan Liang
  6. Qi Wang
  7. Ling Zhang
  8. Xi Tang
  9. Jinfeng Li
  10. Shuiping Hou
  11. Jinfeng Zeng
  12. Yongshui Fu
  13. Jean-Pierre Allain
  14. Tingting Li
  15. Yuming Zhang
  16. Chengyao Li

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Abstract

COVID-19 vaccines are being developed urgently worldwide, among which single-shot adenovirus vectored vaccines represent a major approach. Here, we constructed two novel adenovirus vectored COVID-19 vaccine candidates on simian adenovirus serotype 23 (Sad23L) and human adenovirus serotype 49 vectors (Ad49L) carrying the full-length gene of SARS-CoV-2 spike protein (S), designated Sad23L-nCoV-S and Ad49L-nCoV-S vaccines, respectively. The immunogenicity elicited by these two vaccine strains was individually evaluated in mice. Specific humoral and cellular immune responses were proportionally observed in a dose-dependent manner, and stronger response was obtained by boosting. Furthermore, five rhesus macaques were intramuscularly injected with a dose of 5×10 9 PFU Sad23L-nCoV-S vaccine for prime vaccination, followed by boosting with 5×10 9 PFU of Ad49L-nCoV-S vaccine at 4-week interval. Three macaques were injected with Sad23L-GFP and Ad49L-GFP vectorial viruses as negative controls. Both mice and macaques tolerated well the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost vaccination regimen induced high titers of 10 3.16 S-binding antibody (S-BAb), 10 2.75 cell receptor binding domain (RBD)-BAb and 10 2.38 neutralizing antibody (NAb) to pseudovirus a week after boosting injection, followed by sustained high levels over 10 weeks of observation. Robust IFN-γ secreting T-cell response (712.6 SFCs/10 6 cells), IL-2 secreting T-cell response (334 SFCs/10 6 cells) and intracellular IFN-γ expressing CD4 + /CD8 + T cell response (0.39%/0.55%) to S peptides were detected in the vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.28.311480: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationRhesus macaques and ethics statement: Eight healthy outbred male rhesus macaques (Macaca mulatta) aged 11-14 years were randomly allocated to this study (Table S1).
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableRhesus macaques and ethics statement: Eight healthy outbred male rhesus macaques (Macaca mulatta) aged 11-14 years were randomly allocated to this study (Table S1).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-human IgG-Alexa Fluor 594 antibody (Thermo Scientific, USA) in 1% BSA-PBST was added to the cells for 30 min at 37°C.
    Anti-human IgG-Alexa Fluor 594
    suggested: None
    Secondary antibodies were goat anti-mouse IgG-HRP (Beijing Bersee Science and Technology, Co. LTd, China)
    anti-mouse IgG-HRP
    suggested: None
    Cells were collected and stained with anti-mouse or anti-monkey CD3, CD4 and CD8 surface marker antibodies (BD).
    anti-mouse
    suggested: None
    anti-monkey CD3
    suggested: None
    CD4
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Western blotting: HEK-293A cells were infected with Sad23L-nCoV-S and Ad49L-nCoV-S strains, respectively, and Sad23L-GFP and Ad49L-GFP vectorial viruses were used as mock control.
    HEK-293A
    suggested: None
    Briefly, the packaging construct psPAX2 (Addgene), plasmid pLenti-CMV Puro-Luc (Addgene), and pcDNA3.1-SARS-CoV-2 SΔCT (deletion of the cytoplasmic tail) were co-transfected into HEK-293T cells.
    HEK-293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    The pVNT titers were measured with HEK293T-hACE2 cells in 96-well tissue culture plates.
    HEK293T-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Female C57BL/6 and BALB/c mice were obtained from the Animal Experimental Centre of Southern Medical University, Guangdong, China.
    C57BL/6
    suggested: None
    Female C57BL/6 and BALB/c mice (5-6 weeks, n=5 each group) were prime inoculated intramuscularly with a dose of 109 PFU Sad23L-nCoV-S vaccine, and then at 4 week interval were boosted with a dose of 109 PFU Ad49L-nCoV-S vaccine.
    BALB/c
    suggested: None
    Software and Algorithms
    SentencesResources
    All graphs are generated with GraphPad Prism 7 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Beyond general comments on the disadvantages of major types of COVID-19 vaccines (24, 25), the limitations of inactivated SARS-CoV-2 vaccines are crucial biosafety concerns regarding large amounts of infectious virus being processed above biological safety level-3 (BSL-3) condition and production capacity; for LNP-mRNA vaccines instability and lack of evidence for efficacy are puzzling; for adenovirus vectored vaccines, pre-existing immunity to the carrier virus and the relative weakness of single-shot immunization appear limiting factors. In this study, we generated two novel adenovirus vectored COVID-19 vaccines encoding the full-length S gene of SARS-CoV-2. The intact S glycoprotein rather than the shorter S or RBD proteins was shown to be the most effective antigen eliciting protective immunity against SARS-CoV-2 infection in DNA vaccines and Ad26 vectored vaccines (12,13,26). The vectors Sad23L and Ad49L originated from simian adenovirus type 23 and human adenovirus type 49, respectively (22, 23), are novel adenoviral vector. Comparing Ad5-vectored COVID-19, ChAdox1 nCoV-19 and Ad26.COV2.S vaccines (8-12,14,21), three attractive aspects emphasized in this study are highlighted below. Firstly, there is a low-seroprevalence of pre-existing antibodies to Sad23L and Ad49L vectors in humans. According to the investigation of NAb to three types of adenoviruses in Chinese population, the prevalence of both Sad23L and Ad49L NAb was below 10%, while the prevalence of Ad5 NAb was ...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 57 and 44. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.09.28.311480 on bioRxiv