Synergistic Effect of Co-Administered SARS-CoV-2 Vaccines Improves Immune Responses in BALB/c Mice: A Preliminary Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall a well written and conclusive piece of work.  My suggestions below are meant to help improve some slight points of readability / clarity for the eventual reader. 

One thing that I was concerned about as I read your discussion was no mention of the possibility that higher levels of antibody response with combined vaccines could be a function of the antigenic mass presented to the immune system.  The suggestion is made that different vaccine platforms provide different stimuli for receptor binding moieties from those platforms, and in the case of the attenuated vaccines, multiple antigens, and this is certainly true, but an additional factor is that there are two to three times the dose of an antigen, depending on the combination given.  Would a more proper control to demonstrate superiority of combined vaccines have been a doubling or even tripling of the individual platform doses?

Thank you for acknowledging in the conclusion that ELISA data does on represent specific protective efficacy, which would have been better demonstrated by plaque neutralization or challenge-type studies.  

I had a more difficult time interpreting the statistical significance brackets in Figures 1 and 2.  I was not sure whether a bracket was inclusive of all of the bars located below the bracket, or whether these were one-way comparisons of ANOVA where just the two conditions at the end of the brackets were being highlighted for statistically significant difference (which I suspect was the meaning). But then in Figure 1A, and B there is a bracket that covers all of the combination regimens and is significantly different from what (the unvaccinated group or the single vaccine group or something else?). If those could be somehow improved or really better described, it would make the reader's experience easier.

In your figure 2C with the Pearson Correlation analysis, I would move the legend at the top where you define the symbols to a place away from the graphic, or certainly not right above it because at least as I read it at first, it seemed that there were two data points very high on the Y-axis and low on the X-axis values, which took me a while to reconcile until I understood that these were legend items.  I am not sure whether drawing a regression line would also not help drive the points home better as well.

In Figure 1C, is there a bar missing for statistical significance between the D14 and D28 IgG levels for the Vectored/Inactivated combined vaccine group?

Some specfic language to consider: 

Line 47 - I think of vaccines not as "treatments" but as "prophylaxis" so consider changing that term.

Lines 57-60 - you make generalities about the duration of a humoral immune response (presumably a protective level) and it might make your case a bit stronger to add specific data in the general time intervals observed for duration until waning immunity below protective level for each of the vaccine platforms, now that there has been such a body of information gathered.

Line 61 - for historical purposes, you may want to mention the Clade(s) that were associated with this 95% efficacy, because this number seemed to change as evolutionary changes in the virus altered these observations. 

Lines 75-80 - It may be beneficial to cite other heterologous prime-boost or coadministered approaches used for other pathogens, like influenza.  

Line 132 - I think you mean the word "synthesized" instead of "synthesis"

Line 333 - do you mean a wider "repertoire" of spike specific IgG antibodies?

Line 335 - I would phrase this as mechanisms involved in the "longevity" of antibody response.

Comments on the Quality of English Language

I have no concerns about the quality of the English language use in this manuscript.  

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Nshimirimana et. al. investigated the synergistic effect of co-administered SARS-CoV-2 vaccines on the immune responses in mice. Instead of sequential prime-booster administration, this study combined and co-administered different SARS-CoV-2 vaccines. The study found mRNA/Vector/Inactivated group induced elevated IL-6 and IFN-γ mRNA expression levels compared to immunization with individual vaccines. Histopathological analysis of the heart and liver tissues from mice after 28 days post immunization showed normal architecture of liver and heart tissues but a mild necrosis was observed in liver. The authors conclude that co-administration of different kinds of vaccines is a safe and effective strategy to counter the decline in effectiveness of single vaccine regimen.   

Although the goal of the study is interesting and critical in designing combination vaccines against SARS-CoV-2, the observations made are preliminary for a publication at this stage.  Safety is always a major concern in adopting different strategies of vaccination.  This study shows analyses of liver and heart 28 days after vaccination which is a very short observation window to conclude the safety of a vaccine strategy considering a mild necrosis in the liver.  Therefore, a long-term toxicity study is required.  Further, comparing neutralizing and cross-reactive capacity of antibodies induced by different vaccine combinations will strengthen the manuscript.

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

In their study, Nshimirimana et al. explore various combinations of SARS-CoV-2 vaccines, particularly focusing on those targeting the spike protein. The authors investigate the impact of combining different types of vaccines on antibody production and cell-mediated immune responses. Their findings reveal that immunization with mRNA/Vector/Inactivated vaccines results in slightly higher anti-spike IgG levels and a significant increase in IL6 and IFN-γ mRNA expression levels.

While the data presented suggest a potential enhancement in efficacy through combination strategies compared to single-vaccine immunization, certain aspects require further scrutiny to solidify the conclusions.

The use of OD levels in ELISA measurements poses a limitation, as OD values lack quantitativeness and can vary significantly between assays and ELISA methodologies. Consequently, direct comparison with other studies becomes challenging. To address this issue, incorporating absolute quantification through the inclusion of a standard control, such as serial dilution with purified spike IgG, would enhance the reliability and comparability of the results.

Regarding the cell-mediated response, it's crucial to note that IL-6 serves as a general inflammation marker, potentially induced by vaccine adjuvants alone. Thus, the direct linkage of IL-6 production to antigen-specific responses may not be definitive. A reframing of this aspect in the manuscript is warranted for clarity and accuracy.

Furthermore, considering the paper's focus on long-term interactions between different vaccination strategies, it is important to discuss potential interactions with antiviral drugs, a common clinical scenario. Notably, a significant subset of patients treated with antiviral drugs experiences a COVID rebound phenomenon, characterized by a return of symptoms or positive test results shortly after initial recovery. Recent research suggests a negative impact of antiviral treatment on the establishment of long-term immune responses post-immunization or infection (PMID: 36946379). Given the clinical relevance of this effect, its mention in the introduction would enrich the discussion and contextualize the findings within the broader clinical landscape.

Author Response

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Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors claim it as a preliminary study and therefore the manuscript can be published as a preliminary study as long as the journal policy permits it.