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  1. Author Response

    Reviewer #1 (Public Review):

    In this manuscript, the authors investigated that vaccine which is designated RNA replicons delivered by lipid inorganic nanoparticles (LION) exhibited the protective immune response against SARS-CoV2 variants by heterologous challenging. They also provide the evidence its significant efficacy to assess pathological analysis in the lung using hamster model. However, this study presented descriptive data with a few mechanistic studies in the immune response. Concerns with the manuscript are related to data describing the relevant of protective effects in vivo and the data supporting the interpretation of vaccination efficacy against multiple SARS-CoV2 strains.

    Specific concerns In previous study (Erasmus et al, 2020a), the authors developed a novel vaccine and demonstrated that this novel vaccine harboring an alphavirus-derived repRNA induced antibody production responses in mice and macaques. In this manuscript, the authors demonstrated that this novel vaccine harboring SARS-CoV2 variant derived repRNA with pre-fusion type has significant cross-neutralization activity and protective immunity against SARS-CoV2 variant.

    1. The antibodies which are produced after immunization by repRNA expressing pre-fusion stabilized spike protein/LION can bind to S1 or S2 or RBD? Please define the reactivity of antibodies and also compare to those from native form.

    We thank the reviewer for this suggestion, we have updated figure 2 of the manuscript with this additional data.

    1. The authors demonstrated that this novel vaccine has significant efficacy with heterologous neutralizing activities. Please provide some evidence for reasons. F.i. this novel vaccine (with pre-fusion type) can induce the production of cross-reactive antibodies against SARS-CoV variant? And also it would be better to define the epitopes of these antibodies.

    We propose/postulate the cross-protective efficacy is due to cross-reactive neutralizing antibodies as shown in figure 3. Although in some combinations of vaccine vs challenge the neutralizing titers are diminished. Additionally, thanks to your suggestion to characterize within S domain-binding antibodies, we also noted that the apparent increase in breadth of the B.1.1.7 vaccine did appear to correlate with this vaccine’s ability to drive higher S1-binding antibody responses relative to the A.1 vaccine.

    1. In the hamster model, this novel vaccination showed the significant protective effects on lung pathology. Please provide some data that a novel vaccination induce T cell responses in hamster by the frequency of antigen specific CD4 or CD8 T cell and cytokines.

    We chose to focus on neutralizing antibodies as these appear to be the primary correlate of protection against disease (https://www.nature.com/articles/s41591-021-01377-8). In this study, we therefore did not collect samples to measure T-cell responses to the vaccine. However, we have shown induction of T-cell responses in mice and non-human primates receiving the A.1-targeted vaccine (https://pubmed.ncbi.nlm.nih.gov/32690628/), suggesting T-cell responses may have been induced in hamsters. We have included an updated discussion section with discussion on this limitation of our study.

    Minor concerns Miss-labeling in Figure 5, B, D, F in the manuscript. Please correct it.

    We thank the reviewer for catching this error and have corrected the text in the manuscript.

    Reviewer #2 (Public Review):

    This paper aims to develop second-generation vaccines that protect against multiple SARS-CoV-2 variants of concern. For this purpose, the authors developed new vaccine candidates composed of SARS-CoV-2 spike protein derived from B.1.1.7 (alpha) and B.1.351 (beta) variants. The essential backbone of the vaccines they used contains alphavirus-derived sequences to be self-amplifying, and one containing spike protein of the Wuhan strain is already in clinical trials. They demonstrated no significant difference in virus removal and pathogenesis in the lower respiratory tract. However, the titer of in vitro neutralizing activity and virus removal ability in the upper respiratory tract were decreased against the strains different from the vaccine strain.

    Overall, their data are convincing and valuable as a platform for a new vaccine against SARS-CoV-2 VoC in the future. Besides, I have some comments to strengthen their argument.

    1. The challenge experiments in Figure 4, Figure 5, Figure 6, and Figure 7 lack data on infection protection against B.1.617.2 (delta strain). It is better to add B.1.617.2 to the challenge experiments and neutralizing assay in Figure 3. The addition of data against B.1.1.529 (Omicron) is ideal.

    We appreciate the reviewer’s suggestion. However, these studies were completed prior to having a working B.1.617.2-stock which was difficult to achieve due to mutations arising in tissue culture. Nevertheless, we believe the neutralizing titers achieved against B.1.617.2 in figure 3 would suggest significant efficacy against B.1.617.2 infection. We found neutralizing titers against B.1.617.2 by the three vaccines were similar or greater than seen against B.1.351, a VoC for which we still saw near complete protection. Additionally, the complete replacement of the B.1.617.2 VoC with B.1.1.529 now makes further testing against B.1.617.2 of limited benefit. Efficacy testing against the B.1.1.529 VoC is still ongoing.

    1. There are no data on T cell responses to vaccines, even in mice. If their vaccine can also induce T-cell responses, it would be more attractive. At least, it would be better to discuss the potential contribution of T-cell responses since alphavirus-based replicating RNA vaccines could be one of the nice vaccine platforms to elicit T-cell responses, according to previous works. (For example, McKay PF et al., Nat Commun. 2020 Jul 9;11(1):3523.)

    As mentioned in response to reviewer 1, we chose to focus on neutralizing antibodies as these appear to be the primary correlate of protection against disease (https://www.nature.com/articles/s41591-021-01377-8). We agree that data on T-cell responses would be helpful but we did not collect samples to evaluate T-cell responses during the course of the studies presented here. We have shown induction of T-cell responses in mice and non-human primates receiving the A.1-targeted vaccine (https://pubmed.ncbi.nlm.nih.gov/32690628/). We have included an updated discussion section with discussion on this limitation of our study.

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  2. Evaluation Summary:

    This manuscript aims to develop second-generation vaccines that protect against multiple SARS-CoV2 variants. The overall experimental design, the data, and the importance to the SARS-CoV2 pandemic are very nice, although some concerns are raised by the reviewers.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    In this manuscript, the authors investigated that vaccine which is designated RNA replicons delivered by lipid inorganic nanoparticles (LION) exhibited the protective immune response against SARS-CoV2 variants by heterologous challenging. They also provide the evidence its significant efficacy to assess pathological analysis in the lung using hamster model. However, this study presented descriptive data with a few mechanistic studies in the immune response. Concerns with the manuscript are related to data describing the relevant of protective effects in vivo and the data supporting the interpretation of vaccination efficacy against multiple SARS-CoV2 strains.

    Specific concerns:

    In previous study (Erasmus et al, 2020a), the authors developed a novel vaccine and demonstrated that this novel vaccine harboring an alphavirus-derived repRNA induced antibody production responses in mice and macaques. In this manuscript, the authors demonstrated that this novel vaccine harboring SARS-CoV2 variant derived repRNA with pre-fusion type has significant cross-neutralization activity and protective immunity against SARS-CoV2 variant.

    1. The antibodies which are produced after immunization by repRNA expressing pre-fusion stabilized spike protein/LION can bind to S1 or S2 or RBD? Please define the reactivity of antibodies and also compare to those from native form.

    2. The authors demonstrated that this novel vaccine has significant efficacy with heterologous neutralizing activities. Please provide some evidence for reasons. F.i. this novel vaccine (with pre-fusion type) can induce the production of cross-reactive antibodies against SARS-CoV variant? And also it would be better to define the epitopes of these antibodies.

    3. In the hamster model, this novel vaccination showed the significant protective effects on lung pathology. Please provide some data that a novel vaccination induce T cell responses in hamster by the frequency of antigen specific CD4 or CD8 T cell and cytokines.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    This paper aims to develop second-generation vaccines that protect against multiple SARS-CoV-2 variants of concern. For this purpose, the authors developed new vaccine candidates composed of SARS-CoV-2 spike protein derived from B.1.1.7 (alpha) and B.1.351 (beta) variants. The essential backbone of the vaccines they used contains alphavirus-derived sequences to be self-amplifying, and one containing spike protein of the Wuhan strain is already in clinical trials. They demonstrated no significant difference in virus removal and pathogenesis in the lower respiratory tract. However, the titer of in vitro neutralizing activity and virus removal ability in the upper respiratory tract were decreased against the strains different from the vaccine strain.

    Overall, their data are convincing and valuable as a platform for a new vaccine against SARS-CoV-2 VoC in the future. Besides, I have some comments to strengthen their argument.

    1. The challenge experiments in Figure 4, Figure 5, Figure 6, and Figure 7 lack data on infection protection against B.1.617.2 (delta strain). It is better to add B.1.617.2 to the challenge experiments and neutralizing assay in Figure 3. The addition of data against B.1.1.529 (Omicron) is ideal.

    2. There are no data on T cell responses to vaccines, even in mice. If their vaccine can also induce T-cell responses, it would be more attractive. At least, it would be better to discuss the potential contribution of T-cell responses since alphavirus-based replicating RNA vaccines could be one of the nice vaccine platforms to elicit T-cell responses, according to previous works. (For example, McKay PF et al., Nat Commun. 2020 Jul 9;11(1):3523.)

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  5. SciScore for 10.1101/2021.12.10.472134: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Animal experiments were approved by the corresponding institutional animal care and use committee and performed by experienced personnel under veterinary oversight.
    Sex as a biological variableMouse studies: For mouse studies, 6-8 week old female C57BL/6 mice (Jackson laboratory) received 1μg of each vaccine, as outlined in Table 1, via intramuscular injections on days 0 and 28.
    RandomizationHamsters were randomly assigned to study groups and acclimatized for several days prior to vaccination.
    BlindingSections were scored by certified pathologists who were blinded to study groups.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Using GenScript U864YFA140-4/CB2093 NP-1 (1:1000) specific anti-CoV immunoreactivity was detected using the Vector Laboratories ImPress VR anti-rabbit IgG polymer (# MP-6401) as secondary antibody.
    NP-1
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    In addition, VeroE6-TMPRSS2 and Vero-hACE2-TMPRSS2 cells were cultured in the presence of 1mg/ml G418 and 10ug/ml puromycin, respectively.
    Vero-hACE2-TMPRSS2
    suggested: None
    Virus stocks generated were tittered on VeroE6-TMPRSS2 cells.
    VeroE6-TMPRSS2
    suggested: None
    Infectious virus titration: Infectious virus in swabs or tissues was quantified by tissue-culture infectious dose 50 assay (TCID50) on Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Experimental Models: Organisms/Strains
    SentencesResources
    For in vitro assays: Vero USAMRIID (a gift from Ralph Baric, UNC-Chapel Hill), VeroE6-TMPRSS2 (JCRB1819, JCRB Cell Bank, NIBIOHN), and Vero-hACE2-TMPRSS2 (a gift from Michael Diamond, Washington Univ) cells were cultured at 37C in DMEM supplemented with 10% FBS, and 100U/ml of penicillin-streptomycin.
    VeroE6-TMPRSS2
    suggested: None
    Mouse studies: For mouse studies, 6-8 week old female C57BL/6 mice (Jackson laboratory) received 1μg of each vaccine, as outlined in Table 1, via intramuscular injections on days 0 and 28.
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical Analyses: Statistical analyses as described in the figure legends were performed using Prism 8.4.3 (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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: We did not find any issues relating to colormaps.


    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.

    Results from scite Reference Check: We found no unreliable references.


    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.

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