Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms

Reviewer #1 (Public Review):

Summary:

The authors constructed a live-attenuated vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in the key coding regions. They showed that intranasal inoculation with the candidate vaccine-induced humoral and cellular immune responses in Syrian hamsters without apparent tissue damage in the lungs and protected against a wild-type SARS-CoV-2 strain with D614G mutation and the latest Omicron subvariant (BA.5) strain. The neutralizing antibodies induced by BK2102 persisted for the long term (up to 364 days). Furthermore, they confirmed the safety of the proposed vaccine using transgenic (Tg) mice expressing human ACE2 (hACE2).

Strengths:

The authors followed a robust methodology to establish the proposed vaccine's protective effect and safety profile in the hamsters and transgenic mice expressing human ACE2.

Weaknesses:

(1) A comparative safety assessment of the available m-RNA and live attenuated vaccines will be necessary. The comparison should include details of the doses, neutralizing antibody titers with duration of protection, tissue damage in the various organs, and other risks, including virulence reversal.

(2) The vaccine's effect on primates is doubtful. The study fails to explain why only two of four monkeys developed neutralizing antibodies. Information about the vaccine's testing in monkeys is also missing: What was the level of protection and duration of the persistence of neutralizing antibodies in monkeys? Were the tissue damages and other risks assessed?

(3) The vaccine's safety in immunosuppressed individuals or individuals with chronic diseases should be assessed. Authors should make specific comments on this aspect.

(4) The candidate vaccine has been tested with a limited number of SARS-CoV-2 strains. Of note, the latest Omicron variants have lesser virulence than many early variants, such as the alfa, beta, and delta strains.

(5) Limitations of the study have not been discussed.

Reviewer #2 (Public Review):

Summary:

In this manuscript "Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms" by Suzuki-Okutani et al., the authors evaluate the attenuation, immunogenicity, and protection efficacy of a live-attenuated SARS-CoV-2 vaccine candidate (BK2102) against SARS-CoV-2.

Strengths:

The authors demonstrate that intranasal inoculation of BK2102 is safe and able to induce humoral and cellular immune responses in hamsters, without apparent signs of damage in the lungs, that protects against homologous SARS-CoV-2 and Omicron BA.5 challenge. Safety of BK2102 was further confirmed in a new hACE2 transgenic mouse model generated by the authors.

Weaknesses:

No major weaknesses were identified, however, this reviewer notes the following:

The authors missed the opportunity to include a mRNA vaccine to demonstrate that the immunity and protection efficacy of their live attenuated vaccine BK2102 is better than a mRNA vaccine.

One of the potential advantages of live-attenuated vaccines is their ability to induce mucosal immunity. It would be great if the authors included experiments to assess the mucosal immunity of their live-attenuated vaccine BK2102.

Reviewer #3 (Public Review):

Summary:

Suzuki-Okutani and collogues reported a new live-attenuated SARS-CoV-2 vaccine (BK2102) containing multiple deletion/substitution mutations. They show that the vaccine candidate is highly attenuated and demonstrates a great safety profile in multiple animal models (hamsters and Tg-Mice). Importantly, their data show that single intranasal immunization with BK2102 leads to strong protection of hamsters against D614G and BA.5 challenge in both lungs and URT (nasal wash). Both humoral and cellular responses were induced, and neutralization activity remained for >360 after a single inoculation.

Strengths:

The manuscript describes a comprehensive study that evaluates the safety, immunogenicity, and efficacy of a new live-attenuated vaccine. Strengths of the study include (1) strong protection against immune evasive variant BA.5 in both lungs and NW; (2) durability of immunity for >360 days; (3) confirmation of URT protection through a transmission experiment.

While first-generation COVID-19 vaccines have achieved much success, new vaccines that provide mucosal and durable protection remain needed. Thus, the study is significant.

Weaknesses:

Lack of a more detailed discussion of this new vaccine approach in the context of reported live-attenuated SARS-CoV-2 vaccines in terms of its advantages and/or weaknesses.

Antibody endpoint titers could be presented.

Lack of elaboration on immune mechanisms of protection at the upper respiratory tract (URT) against an immune evasive variant in the absence of detectable neutralizing antibodies.

Author response:

We extend our sincere gratitude to the editor and three reviewers for their invaluable feedback, which not only included positive comments but also provided constructive suggestions for enhancing the quality of our manuscript.

Of potential interest to you is our forthcoming investigation into vaccine efficacy, where we will compare the effectiveness of our live-attenuated vaccine with an mRNA-based alternative.

Moreover, we acknowledge and fully endorse the recommendation to elucidate why immunization with our live-attenuated vaccine confers protection against viral challenge, even in the absence of sufficient neutralizing antibodies. As pointed out by the reviewers, this phenomenon may be attributed to mucosal immunity. Consequently, we have outlined plans to investigate whether the attenuated live vaccine elicits mucosal immunity as part of our ongoing research.

We are currently working to gather the necessary data to address these inquiries comprehensively, and are aiming to resubmit our manuscript at the earliest opportunity.

Reviewer #1: We sincerely appreciate the insightful comments provided by Reviewer #1. In response to this feedback, we will conduct a comparative analysis of efficacy between our live-attenuated vaccine and an mRNA-based alternative. Furthermore, we will thoroughly examine and delineate the advantages and limitations of this/our live-attenuated vaccine in our discussion.

Reviewer #2: We express our sincere appreciation to Reviewer #2 for invaluable suggestions. In light of the insightful observation concerning the weakness of our study, related to the poor assessment/evaluation of the induction of mucosal immunity by our vaccine candidate, we have resolved to undertake a comprehensive analysis in this regard.

Furthermore, we will take into account this reviewer's recommendation to compare BK2102 results with those of an mRNA vaccine. We are currently in the process of planning additional experiments to thoroughly address this aspect.

Reviewer #3: We are very grateful to Reviewer #3 for the positive feedback and invaluable suggestions. In order to further explore the immune mechanisms underlying the protection against the Omicron variant in the absence of detectable neutralizing antibodies, we are currently devising plans for experiments focused on evaluating mucosal immunity.

Moreover, in accordance with Reviewer #3's suggestion, we are considering the incorporation of an ELISPOT assay experiment. However, we acknowledge uncertainties regarding the feasibility of establishing an experimental system for this purpose.