Immunization with a Prefusion SARS-CoV-2 Spike Protein Vaccine (RBMRNA-176) Protects against Viral Challenge in Mice and Nonhuman Primates

Round 1

Reviewer 1 Report

 

This manuscript describes efforts to produce a new mRNA vaccine for COVID-19. The manuscript describes the design and creation of the vaccine, followed by preclinical studies in vitro, in mice, and in non-human primates. The research is well done and advances the state of knowledge and the state of the art of SARS-CoV-2 vaccine production.

 

I recommend a few changes. Mostly grammar and style, mixed in with recommendations for more substantial improvements.

 

RECOMMENDATIONS:

 

 

Please add some sentences at the end of the Discussion as to whether you think this particular vaccine will be a candidate for actual clinical use, given that Omicron descendants BA.4 and BA.5 are currently prevalent and other VOCs are coming. Is this vaccine a precursor or model for a future vaccine, and/or is it an actual candidate for use in humans?

 

You hedge at this with your last sentence, "laying the groundwork for further research to broaden protection and for clinical translation." But that is a bit vague. Please be bold and specific. You are allowed to speculate in the Discussion.

 

Consider adding some citations relating to to evolving VOCs.

 

Try this PuBb Med search: "ba.5 [titl]"

https://pubmed.ncbi.nlm.nih.gov/?term=ba.5%20%5Btitl%5D

 

You might consider citing some of the resulting articles.

 

Maybe including this one:

Callaway E. What Omicron's BA.4 and BA.5 variants mean for the pandemic. Nature. 2022 Jun;606(7916):848-849. doi: 10.1038/d41586-022-01730-y. PMID: 35750920.

 

 

This might be a good one:

Altmann DM, Boyton RJ. COVID-19 vaccination: The road ahead. Science. 2022 Mar 11;375(6585):1127-1132. doi: 10.1126/science.abn1755. Epub 2022 Mar 10. PMID: 35271316.

 

And/or:

Gong W, Parkkila S, Wu X, Aspatwar A. SARS-CoV-2 variants and COVID-19 vaccines: Current challenges and future strategies. Int Rev Immunol. 2022 May 28:1-22. doi: 10.1080/08830185.2022.2079642. Epub ahead of print. PMID: 35635216.

 

you already have "Next-generation COVID-19 vaccines: Opportunities for vaccine development and challenges in tackling COVID-19" cited along these lines, but that editorial is from 2021.

 

_______

 

 

"Immunization with a novel prefusion SARS-CoV-2 spike protein vaccine (RBMRNA-176) protects against viral challenge in mice and nonhuman primates"

 

change Title to

 

"Immunization with a prefusion SARS-CoV-2 spike protein vaccine (RBMRNA-176) protects against viral challenge in mice and nonhuman primates"

 

claims if novelty are unnecessary in scientific articles, as all published science is novel.

 

 

____

 

ABSTRACT

 

"Altogether, these findings suggest the potential of RBMRNA-176 as an effective vaccine candidate in prevention of COVID-19."

 

'suggest', 'potential' and 'candidate' mean the same thing, (and any vaccine must be effective) so just write

 

"These findings support RBMRNA-176 as a vaccine candidate for prevention of COVID-19."

 

____

 

"We hereby report "

 

just write

 

"We report"

 

and delete all the rest of the 'hereby's in the paper. They are not needed.

 

___

 

"range, 1.28×104 to 8.19×105 log dilution"

 

I think you are missing superscripts.

 

___

 

"various mutated spike proteins"

 

delete the word "various"  and make it more specific if you can, like add in the number or kind of mutated spike proteins.

 

__

 

"from 1: 1020 to 1:2894"

 

extra space

 

"from 1:1020 to 1:2894"

 

___

 

"Moreover, significant control of viral replication and histopathology in lungs were observed"

 

->

 

"Moreover, significant control of viral replication and histopathology in lungs was observed"

 

 

____

 

"In nonhuman primates, a boost given by RBMRNA-176 on day 21 after the first prime induced a persistent and sustained IgG response and."

 

->

 

"In nonhuman primates, a boost given by RBMRNA-176 on day 21 after the first prime induced a persistent and sustained IgG response."

 

 

 

___

 

"and substantial lethality (over 5.83 million cases)"

 

should be

 

"and substantial lethality (over 5.83 million deaths)"

 

 

____

 

"Noteworthy, the most recent variant Omicron is more transmissible than Delta (R0Delta:＜7; R0Omicron:10) and has dominated the circulating SARS-CoV-2 strain[7, 8]."

 

By the time your paper is published this statistic will be out of date. Another VOC will come along. 

Also, you do not know R(sub)0. You might know R(sub)effective. You probably do not want to wade into this debate, as it is not important for your paper. The references you cite barely touch on the R(sub)0 debate.

Just write

 

"New VOCs are arising, and transmissibility may increase with some VOCs [7,8]".  

 

You may or may not need to reference 7&8 here. You might just leave that unreferenced, as it is common knowledge.

 

____

 

"validated for use by WHO, including BNT162b2 and mRNA-1273."

 

some readers know these only by manufacturer, so write

 

"validated for use by WHO, including BNT162b2 (Pfizer) and mRNA-1273 (Moderna)."

 

where possible add manufacturers/sponsors for other named vaccines (e.g., NVX-CoV2373)

__

 

"In the present study, we report the preclinical development of RBMRNA-176，an LNP-formulated pseudouridine (Ψ) nucleoside-modified mRNA vaccine that encodes S ectodomain trimer with 2 prolines（K986P/V987P）mutation that stabilize the S Protein in prefusion conformation and amino acid substitutions to prevent protease cleavage at a S1/S2 protease cleavage site and a S2' protease cleave site."

 

Maybe split this into two sentences.  I am not sure what "2 prolines mutation" means. Maybe you want to write "2 mutations that result in prolines"

 

 

__

 

"wild type"

hypenathed when used as an adjective, so

"wild-type"

 

_

 

 

watch for loose commas. I think you have spaces before commas in places. This causes line breaks before commas.

 

also, some periods do not have spaces after them.

____

 

Do not put Results in Introduction.

 

Most of the paragraph starting with "In the present study, we report the preclinical development..." has results in it. These sentences all belong in Results, not Introduction.

 

 

___

 

"The SARS-CoV-2 viruses used in mice studies (Genebank accession no. MT123290.1) and nonhuman primate studies (GISAID accession no. EPI_ISI_402124) were grown in Vero E6 cells"

 

It is a little odd to cite Genbank for the first and GISAID for the second. Can you cite both databases for both? Or if one of these is not in one of the databases, maybe mention that it is absent from that database.

 

Can you add the collection dates, Pango Lineages, NextStrain Clades, and VOC or strain designations for each of these sequences?

 

____

 

"An instrument Software..."

 

What is an "instrument Software"? DO you just mean "Software"?

 

 

 

___

 

"We further evaluation the immunogenicity..."

->

"We further evaluated the immunogenicity..."

 

 

_____

 

"On week 4 after the second shot, mice were intranasally challenged with 5×104 PFU of SARS-CoV-2."

 

check for superscripts throughout the manuscript. You surely do not mean 5x104

 

___

 

"RT-Qpcr"

 

->

 

"RT-qPCR"

 

 

___

 

"At 3- and 5day post infection (dpi.), the"

->

"At 3- and 5day post infection (dpi), the"

 

so that later

 

"swabs were collected once daily from 1dpi. to 7dpi."

->

"swabs were collected once daily from 1 dpi to 7 dpi."

 

remember to put a space between numbers and units throughout 

e.g., 7 dpi, not 7dpi

 

 

 

 

 

___

 

"A total of 1 106 splenocytes (100 μL) were seeded"

 

not sure what number 1    106  refers to. Clarify.

 

 

"1 105 events were collected per sample"

 

same problem

 

these sorts of problems with periods, commas, spaces, and superscripts continue throughout the manuscript. Please fix them all.

 

___

 

Figure 2A legend. 

 

Describe briefly in the legend what you mean by IgG titer. I couldn't find it in the Methods. Is this total IgG or SARS-CoV-2 specific IgG?

 

Figure 2C labels

What is the dose of RBMRNA-176?

 

 

 

Figure 2 legend

"Wuhan strain"

provide a Genbank or sequence accession number or other repository identifier

 

 

somewhere in Methods or the legend of Figure 2, put in the accession numbers for the used Alpha, Beta, Gamma, and Kappa strains.

 

___

 

Figure 5

 

labels

write "Day 3" and "Day 5" rather than D3 and D5 or otherwise make it clear that days is the category

 

legend. Explain a bit better. There is no mention of the D3 bar chart in the legnend.

 

___

 

"7.8E+3 log IgG"

 

 

what does this mean? Be consistent with expression of numbers and powers of ten and scientific notation throughout the mansucript.

 

 

___

 

"2P mutation"

 

maybe call this a "two-proline mutation"

 

2P is too obscure

 

___

 

"The Th1-skewed response is more critical for viral control and is important for the development of SARS-CoV-2 vaccine"

 

since you are not comparing to anything, just write

 

"The Th1-skewed response is critical for viral control and is important for the development of SARS-CoV-2 vaccine"

 

 

___

 

"although this vaccine exhibits slightly reduced (0.85- to 3.33-fold) neutralizing potency to VOCs."

->

"although this vaccine exhibits slightly reduced (0.85- to 3.33-fold) neutralizing potency to these VOCs compared to wild type."

 

---

 

"based on the results obtained from mRNA-vaccinated individual"

I think you mean

"based on the results obtained from mRNA-vaccinated individuals"

 

___

 

"also provide evidence of protection for lower respiratory tract in NHP"

most readers won't recall the meaning of NHP, so write it out

"also provide evidence of protection for lower respiratory tract in non-human primates"

 

 

__

 

"We showed that RBMRNA-176 vaccination could completely neutralize infectious virus in the right lungs of mice."

 

it is better to write

 

"We showed that RBMRNA-176 vaccination could completely neutralize infectious virus in the lungs of mice."

 

____

 

", laying the groundwork for further research into broaden protection and clinical translation."

 

->

 

", laying the groundwork for further research to broaden protection and for clinical translation."

 

Author Response

RECOMMENDATIONS:

1. Please add some sentences at the end of the Discussion as to whether you think this particular vaccine will be a candidate for actual clinical use, given that Omicron descendants BA.4 and BA.5 are currently prevalent and other VOCs are coming. Is this vaccine a precursor or model for a future vaccine, and/or is it an actual candidate for use in humans?

Response: RBMRNA-176 is a vaccine designed against the spike protein of ancestral strains. To date，a large number of studies indicated that COVID-19 vaccines based on earlier strains showed dramatic reduction in protection against Omicron variant，including BA.4 and BA.5. Actually, we also evaluated the cross-neutralization capacity induced by RBMRNA-176 immunization against BA.1 and BA.2 using pseudoviruses neutralization assay in the later study. The results showed a 15 and 12-fold reduction in neutralizing antibodies to BA.1 and BA.2, compared to original COVID-19 (FigureS1B). Considering RBMRNA-176 showed significant reduction in neutralization antibody titers against the Omicron virus, therefore, we will develop an updated mRNA vaccine with efficient neutralization capacity against the SARS-CoV-2 Omicron variant based on our established LNP-mRNA vaccine platform in our future work. We have added this part in Discussion (Line 553-559).

FigureS1. BALB/c mice were vaccinated with two doses of RBMRNA-176(5μg or 20μg) at an interval of 21 days, at week2 after the second dose, the serum was collected and Omicron spike-specific IgG antibodies were tested (n=6). Pseudovirus neutralization assays with wild type SARS-CoV-2, BA.1 and BA.2 variants were performed by using serum collected from mice vaccinated with 20ug RBMRNA-176 (n=3). Geometric mean titers (GMTs) are indicated above the columns.

2. You hedge at this with your last sentence, "laying the groundwork for further research to broaden protection and for clinical translation." But that is a bit vague. Please be bold and specific. You are allowed to speculate in the Discussion.

Response: We agree that this sentence is vague. We found that the neutralizing ability of RBMRNA-176 against Omiron reduced compared to the wild type. Therefore, at this stage, it is not convincing to make a statement that ‘laying the groundwork for further research for clinical translation’. Instead, the further optimization of RBMRNA-176 for higher efficacy against Omicron variants are needed. Together with Reviewer 2’ comments, we reconstruction the conclusion as “Taken together, our study provides insight into the broad neutralization and in vivo protection against SARS-CoV-2 infection with RBMRNA-176 vaccination. Due to RBMRNA-176 evasion by Omicron and the relatively high vaccination dose, it will be necessary to develop a vaccine of the next generation” (Line 572-575).

3. Consider adding some citations relating to to evolving VOCs. Try this PuBb Med search: "ba.5 [titl]" https://pubmed.ncbi.nlm.nih.gov/?term=ba.5%20%5Btitl%5D You might consider citing some of the resulting articles. Maybe including this one:

Callaway E. What Omicron's BA.4 and BA.5 variants mean for the pandemic. Nature. 2022 Jun;606(7916):848-849. doi: 10.1038/d41586-022-01730-y. PMID: 35750920. This might be a good one:

Altmann DM, Boyton RJ. COVID-19 vaccination: The road ahead. Science. 2022 Mar 11;375(6585):1127-1132. doi: 10.1126/science.abn1755. Epub 2022 Mar 10. PMID: 35271316. And/or:

Gong W, Parkkila S, Wu X, Aspatwar A. SARS-CoV-2 variants and COVID-19 vaccines: Current challenges and future strategies. Int Rev Immunol. 2022 May 28:1-22. doi: 10.1080/08830185.2022.2079642. Epub ahead of print. PMID: 35635216. you already have "Next-generation COVID-19 vaccines: Opportunities for vaccine development and challenges in tackling COVID-19" cited along these lines, but that editorial is from 2021.

Response: We have cited these articles in the 1^st paragraph of Introduction (Line 54-55).

4. "Immunization with a novel prefusion SARS-CoV-2 spike protein vaccine (RBMRNA-176) protects against viral challenge in mice and nonhuman primates" change Title to "Immunization with a prefusion SARS-CoV-2 spike protein vaccine (RBMRNA-176) protects against viral challenge in mice and nonhuman primates" claims if novelty are unnecessary in scientific articles, as all published science is novel.

Response: Corrected.

5. ABSTRACT

"Altogether, these findings suggest the potential of RBMRNA-176 as an effective vaccine candidate in prevention of COVID-19." 'suggest', 'potential' and 'candidate' mean the same thing, (and any vaccine must be effective) so just write "These findings support RBMRNA-176 as a vaccine candidate for prevention of COVID-19."

 Response: Corrected (Line 40-41).

6. "We hereby report " just write "We report" and delete all the rest of the 'hereby's in the paper. They are not needed.

 Response: Corrected.

7. "range, 1.28×104 to 8.19×105 log dilution" I think you are missing superscripts.

 Response: Corrected (Line 369).

8. "various mutated spike proteins" delete the word "various" and make it more specific if you can, like add in the number or kind of mutated spike proteins.

Response: This sentence has been rephrased as “RBMRNA-176 vaccination induced pseudovirus-neutralizing antibodies with IC50 ranging from 1:1020 to 1:2894 against SARS-CoV-2 wild-type and mutant spikes, including Alpha, Beta, Gamma, and Kappa” (Line 34-36).

9. "from 1: 1020 to 1:2894" extra space "from 1:1020 to 1:2894"

 Response: Corrected (Line 35).

10. "Moreover, significant control of viral replication and histopathology in lungs were observed" ->"Moreover, significant control of viral replication and histopathology in lungs was observed"

 Response: Corrected (Line 37).

11. "In nonhuman primates, a boost given by RBMRNA-176 on day 21 after the first prime induced a persistent and sustained IgG response and." -> "In nonhuman primates, a boost given by RBMRNA-176 on day 21 after the first prime induced a persistent and sustained IgG response."

 Response: Corrected (Line 38).

12. "and substantial lethality (over 5.83 million cases)" should be "and substantial lethality (over 5.83 million deaths)"

Response: Corrected (Line 47).

13. "Noteworthy, the most recent variant Omicron is more transmissible than Delta (R0Delta:＜7; R0Omicron:10) and has dominated the circulating SARS-CoV-2 strain[7, 8]." By the time your paper is published this statistic will be out of date. Another VOC will come along. Also, you do not know R(sub)0. You might know R(sub)effective. You probably do not want to wade into this debate, as it is not important for your paper. The references you cite barely touch on the R(sub)0 debate. Just write "New VOCs are arising, and transmissibility may increase with some VOCs [7,8]".  You may or may not need to reference 7&8 here. You might just leave that unreferenced, as it is common knowledge.

Response: We strongly agree this suggestion and has rephrased the sentence (Line 54-55) and removed [7] and [8].

14. "validated for use by WHO, including BNT162b2 and mRNA-1273." some readers know these only by manufacturer, so write "validated for use by WHO, including BNT162b2 (Pfizer) and mRNA-1273 (Moderna)." where possible add manufacturers/sponsors for other named vaccines (e.g., NVX-CoV2373)

Response: This part has been rephrased as “validated for use by WHO, including BNT162b2 (Pfizer), mRNA-1273 (Moderna) and NVX-CoV2373 (Novavax)” (Line 73-74).

15. "In the present study, we report the preclinical development of RBMRNA-176，an LNP-formulated pseudouridine (Ψ) nucleoside-modified mRNA vaccine that encodes S ectodomain trimer with 2 prolines（K986P/V987P）mutation that stabilize the S Protein in prefusion conformation and amino acid substitutions to prevent protease cleavage at a S1/S2 protease cleavage site and a S2' protease cleave site." Maybe split this into two sentences.  I am not sure what "2 prolines mutation" means. Maybe you want to write "2 mutations that result in prolines"

Response: This sentence has been split as “In the present study, we report the preclinical development of RBMRNA-176, an LNP-formulated pseudouridine (Ψ) nucleoside-modified mRNA vaccine that encodes S ectodomain trimer with a two-proline mutation（K986P and V987P）. This mutation strategy could stabilize the S protein in its prefusion conformation and prevent protease cleavage at S1/S2 and S2' cleave sites.” (Line 77-81).

16. "wild type" hypenathed when used as an adjective, so "wild-type"

Response: Corrected throughout the entire manuscript.

17. watch for loose commas. I think you have spaces before commas in places. This causes line breaks before commas. also, some periods do not have spaces after them.

Response: Corrected throughout the entire manuscript.

18. Do not put Results in Introduction. Most of the paragraph starting with "In the present study, we report the preclinical development..." has results in it. These sentences all belong in Results, not Introduction.

Response: We have removed sentences with results and replaced with what we intend to do in our study. Please refer to Line 89-92.

19. "The SARS-CoV-2 viruses used in mice studies (Genebank accession no. MT123290.1) and nonhuman primate studies (GISAID accession no. EPI_ISI_402124) were grown in Vero E6 cells". It is a little odd to cite Genbank for the first and GISAID for the second. Can you cite both databases for both? Or if one of these is not in one of the databases, maybe mention that it is absent from that database. Can you add the collection dates, Pango Lineages, NextStrain Clades, and VOC or strain designations for each of these sequences?

Response: Thank you for notifying us of this issue. We sincerely apologize that we didn’t check this information before submission. Actually, the virus strains used in mice and nonhuman primate were the same (GISAID accession no. EPI_ISI_402124), and we have revised as shown in Line 97-98.

20. "An instrument Software..." What is an "instrument Software"? DO you just mean "Software"?

Response: The sentence in Method 2.4 was revised as follows: The ZS XPLORER Software (V1.3.1.7) was used to analyze the light scattering data (Line 131-132).

21. "We further evaluation the immunogenicity..." -> "We further evaluated the immunogenicity..."

Response: Corrected (Line 186).

22. "On week 4 after the second shot, mice were intranasally challenged with 5×104 PFU of SARS-CoV-2." check for superscripts throughout the manuscript. You surely do not mean 5x104

Response: Corrected all related typo in the manuscript.

23. "RT-Qpcr" -> "RT-qPCR"

Response: Corrected.

24. "At 3- and 5day post infection (dpi.), the" -> "At 3- and 5day post infection (dpi), the" so that later "swabs were collected once daily from 1dpi. to 7dpi." -> "swabs were collected once daily from 1 dpi to 7 dpi." remember to put a space between numbers and units throughout  e.g., 7 dpi, not 7dpi

Response: Corrected all related typo in the manuscript.

25. "A total of 1 106 splenocytes (100 μL) were seeded" not sure what number 1    106  refers to. Clarify. "1 105 events were collected per sample" same problem these sorts of problems with periods, commas, spaces, and superscripts continue throughout the manuscript. Please fix them all.

Response: Corrected all related typo in the manuscript.

26. Figure 2A legend. Describe briefly in the legend what you mean by IgG titer. I couldn't find it in the Methods. Is this total IgG or SARS-CoV-2 specific IgG?

Response: This is SARS-CoV-2-specific IgG responses. We have added and improved the legend to make it clearer.

27. Figure 2C labels What is the dose of RBMRNA-176?

Response: We have added the dose in Figure 2C.

28. Figure 2 legend "Wuhan strain" provide a Genbank or sequence accession number or other repository identifier somewhere in Methods or the legend of Figure 2, put in the accession numbers for the used Alpha, Beta, Gamma, and Kappa

Response: We have removed “Wuhan strain” and replaced it with “(GISAID accession no. EPI_ISI_402124)” in Figure 2 legend (Line 392). The accession numbers for Alpha, Beta, Gamma and Kappa have been added (Line 284-288).

29. Figure 5 labels write "Day 3" and "Day 5" rather than D3 and D5 or otherwise make it clear that days is the category legend. Explain a bit better. There is no mention of the D3 bar chart in the legnend.

Response: Corrected.

30. "7.8E+3 log IgG" what does this mean? Be consistent with expression of numbers and powers of ten and scientific notation throughout the mansucript.

Response: Changed to 7.8×10³ (Line 450).

31. "2P mutation" maybe call this a "two-proline mutation" 2P is too obscure

Response: Corrected (Line 79, 494).

32. "The Th1-skewed response is more critical for viral control and is important for the development of SARS-CoV-2 vaccine" since you are not comparing to anything, just write "The Th1-skewed response is critical for viral control and is important for the development of SARS-CoV-2 vaccine"

Response: Corrected (Line 513-514).

33. "although this vaccine exhibits slightly reduced (0.85- to 3.33-fold) neutralizing potency to VOCs." -> "although this vaccine exhibits slightly reduced (0.85- to 3.33-fold) neutralizing potency to these VOCs compared to wild type."

Response: Corrected (Line 540).

34. "based on the results obtained from mRNA-vaccinated individual" I think you mean "based on the results obtained from mRNA-vaccinated individuals"

Response: Corrected (Line 545).

35. "also provide evidence of protection for lower respiratory tract in NHP" most readers won't recall the meaning of NHP, so write it out "also provide evidence of protection for lower respiratory tract in non-human primates"

Response: Corrected.

36. "We showed that RBMRNA-176 vaccination could completely neutralize infectious virus in the right lungs of mice." it is better to write "We showed that RBMRNA-176 vaccination could completely neutralize infectious virus in the lungs of mice."

Response: Corrected.

37. ", laying the groundwork for further research into broaden protection and clinical translation."

-> ", laying the groundwork for further research to broaden protection and for clinical translation.

Response: Due to the vaccine's limitations and the need for further optimization before clinical translation, we have rephrased this sentence as “Due to RBMRNA-176 evasion by Omicron and the relatively high vaccination dose, it will be necessary to develop a vaccine of the next generation.” (Line 572-575).

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript by Ma et al. reported the development of the spike mRNA based COVID-19 vaccine. The authors used ELISA to test the antibody response to the RBMRNA-176 vaccine and pseudovirus and authentic virus to test neutralizing antibody response. With these data, the authors showed this RBMRNA-176 vaccine is capable of eliciting protective antibodies against ancestral wild-type SARS-CoV-2, as well as several merged variants, i.e., Alpha, Beta, Gamma, Kappa, albeit with decreased titers. In addition, the authors also challenged the mice and non-human primate models with the ancestral wild-type SARS-CoV-2 virus. They also showed protection in animals vaccinated with high doses of RBMRNA-176. This study delivered a lot of data regarding the RBMRNA-176 vaccine. However, the study design also has some defects and should be addressed. Since this is closely related to current human medicine, I would also suggest a declaration of limitation of study based on these defects and data unavailability to be included in the main text.

 

1.         several vaccines have been developed based on wild-type and variant SARS-CoV-2, and billions of mRNA shots have been injected into human arms. The study here should provide additional information and comparison with mRNA vaccines already available worldwide. 

 

2.         The authors used the antibody secretion signal tag for the spike protein expression. Hence, the author should compare whether this design would affect any physiological component of the spike protein compared with others' designs on spike protein. Specifically, the protein stability, expression level, oligomer state, and glycosylation pattern of the spike protein expressed by this RBMRNA-176 warrant more investigation.

 

3.         The authors claimed the removal of fusion peptide in their spike protein design to reduce cell-cell fusion. However, the furin and TMPRSS2 cleavage sites have already been mutated to disable the fusion peptide release. So why do the authors have to remove the fusion peptide? Several recent studies showed the fusion peptide is an important epitope site for pan-coronavirus neutralizing antibodies. In short, leaving the fusion peptide in the spike construct may be beneficial in increasing the vaccine's breadth, an urgently needed feature of COVID-19 vaccines.

 

4.         The author should also test the binding and neutralization against Omicron since it is the dominant strain responsible for many break-through infections in vaccinated people. If Omicron strain is not readily available, at least antibody binding to Omicron spike, neutralization against Omicron pseudovirus should be tested. To be specific, whether the RBMRNA-176 is effective to Omicron.

 

5.         The vaccination doses listed in the method section are not the same as those used in experiments and in the results section. Please clarify.

 

6.         The doses used in K18-ACE2 mice and monkeys are much higher than those used in humans, as seen in Moderna and Pfizer vaccines. How could the authors claim the RBMRNA-176 is comparable to the two already commercially available?

 

7.         There are many typos in the main text. I just listed a few here, line 89, "timer"; line 185, “Chanese”; lines 433-435, "Figure 5A", "educed", etc.

 

8.         Reference [33], the BNT162b vaccine paper should be updated with its published one: Vogel AB, Kanevsky I, Che Y et al. BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature 2021; 592:283-289.

 

9.         Several key papers in the field reporting escape mutations and viral resistance to current vaccines have not been cited.

 

10.      In the method section, the formulated LNP was stored at 4-degree until use. Would this be an advantage of this vaccine compared to Moderna and Pfizer ones?

 

11.      In figure 1B, additional SDS-PAGE may show whether the spike proteins are cleaved or not.

 

12.      Figure 8A-B, please specify what gray and red lines represent.

 

13.      In line 537, the authors cite a study in Shan-Lu Liu's group (not "Evans's group"). Please also cite several other key publications in recent two years.

 

14.      I am not convinced by the facts provided in this study to make the conclusion the author declared, "the responses elicited by this vaccine follow a profile consistent with vaccine safety and efficacy, laying the groundwork for further research into broaden protection and clinical translation."  Too much high dose for such protection to be seen in animals, and a lack of clinical trial will preclude the statement of safety and efficacy. Instead, I would add a limitation of the study statement to address these issues.

Author Response

 

1. Several vaccines have been developed based on wild-type and variant SARS-CoV-2, and billions of mRNA shots have been injected into human arms. The study here should provide additional information and comparison with mRNA vaccines already available worldwide. 

Response: We have made comparison among RBMRNA-176 and other mRNA vaccines that encode S protein, including BNT162b2 (Pfizer), mRNA-1273 (Moderna), and NVX-CoV2373 (Novavax), regarding IgG response (Line 521-525), Th1 response (Line 530-532), and protection (Line 563-568) against SARS-CoV-2 infection in vivo.

2. The authors used the antibody secretion signal tag for the spike protein expression. Hence, the author should compare whether this design would affect any physiological component of the spike protein compared with others' designs on spike protein. Specifically, the protein stability, expression level, oligomer state, and glycosylation pattern of the spike protein expressed by this RBMRNA-176 warrant more investigation.

Response: Using signal peptides from human Igκ, IgE or immunoglobin heavy chain instead of the authentic signal peptide of antigen in mRNA vaccines was reported induced significantly higher immune response. Therefore, in RBMRNA-176 the authentic signal peptide of S protein was swapped with IGVH signal peptide. We will assess whether this design would affect any physiological component of the spike protein or improve the immunogenicity in our future work.

3. The authors claimed the removal of fusion peptide in their spike protein design to reduce cell-cell fusion. However, the furin and TMPRSS2 cleavage sites have already been mutated to disable the fusion peptide release. So why do the authors have to remove the fusion peptide? Several recent studies showed the fusion peptide is an important epitope site for pan-coronavirus neutralizing antibodies. In short, leaving the fusion peptide in the spike construct may be beneficial in increasing the vaccine's breadth, an urgently needed feature of COVID-19 vaccines.

Response: A previous study by Y.-M. Kwon et al [1] reported that RSV F glycoprotein with fusion peptide deletion and furin cleavage sites mutation induced higher levels of IgG antibodies, RSV neutralizing activity titers, and effective lung viral clearance after challenge. These results suggest that cleavage site mutations and fusion peptide deletion contributed to structural stabilization of pre-fusion like F conformation. So we used spike protein with fusion peptide deletion and cleavage sites mutation as antigen in our earlier work. Now several recent studies showed that the fusion peptide is an important epitope site for pan-coronavirus neutralizing antibodies [2,3], and it has been suggested that having broader epitope coverage outside of the RBD can improve protection from MERS-CoV and SARS-CoV challenge [4,5]. Therefore, in our later work an updated full-length S-based vaccine against SARS-CoV-2 variants was developed. These views have been added into the 1^st paragraph of Discussion (Line 501-509).

[1] Young-Man K,Youri L,Ki HK, et al. Antigenicity and immunogenicity of unique prefusion-mimic F proteins presented on enveloped virus-like particles. Vaccine 2019; 37(44):6656-6664.

[2] Cherrelle Dacon, Courtney Tucker, Linghang Peng, et al. Broadly neutralizing antibodies target the coronavirus fusion peptide. Science. 2022, 377(6607):728-735.

[3] Jun Siong Low, Josipa Jerak, M. Alejandra Tortorici, et al. ACE2 engagement exposes the fusion peptide to pan-coronavirus neutralizing antibodies. bioRxiv. 2022, doi: https://doi.org/10.1101/2022.03.30.486377

[4] Lingshu Wang, Wei Shi, M Gordon Joyce, et al. Evaluation of candidate vaccine approaches for MERS-CoV. Nat Commun. 2015, 6:7712.

[5] Tong Zhou, Hong Wang, Danlin Luo, et al. An exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies. J Virol. 2004, 78(13):7217-26.

4. The author should also test the binding and neutralization against Omicron since it is the dominant strain responsible for many break-through infections in vaccinated people. If Omicron strain is not readily available, at least antibody binding to Omicron spike, neutralization against Omicron pseudovirus should be tested. To be specific, whether the RBMRNA-176 is effective to Omicron.

Response: We evaluated the Omicron spike-specific IgG antibodies and cross-neutralization capacity induced by RBMRNA-176 immunization against BA.1 and BA.2 using pseudoviruses neutralization assay in our study. IgG titers in serum against the Omicron spike reached five logs (FigureS1A), however, RBMRNA-176 immunized sera showed a 15 and 12-fold reduction in neutralizing antibodies to BA.1 and BA.2, compared to wild type COVID-19 (FigureS1B), indicating an updated mRNA vaccine with efficient neutralization capacity against the Omicron variant are needed. We added this part in Discussion (Line 553-559).

FigureS1. BALB/c mice were vaccinated with two doses of RBMRNA-176(5μg or 20μg) at an interval of 21 days, at week2 after the second dose, the serum was collected and Omicron spike-specific IgG antibodies were tested (n=6).Pseudovirus neutralization assays with wild type SARS-CoV-2, BA.1 and BA.2 variants were performed by using serum collected from mice vaccinated with 20ug RBMRNA-176. Geometric mean titers (GMTs) are indicated above the columns.

5. The vaccination doses listed in the method section are not the same as those used in experiments and in the results section. Please clarify.

Response: Corrected.

6. The doses used in K18-ACE2 mice and monkeys are much higher than those used in humans, as seen in Moderna and Pfizer vaccines. How could the authors claim the RBMRNA-176 is comparable to the two already commercially available?

 Response: We have rediscussed this part (Line 563-570).

7. There are many typos in the main text. I just listed a few here, line 89, "timer"; line 185, “Chanese”; lines 433-435, "Figure 5A", "educed", etc.

 Response: We have corrected these and other typos through the entire manuscript.

8. Reference [33], the BNT162b vaccine paper should be updated with its published one: Vogel AB, Kanevsky I, Che Y et al. BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature 2021; 592:283-289.

 Response: Replaced.

9. Several key papers in the field reporting escape mutations and viral resistance to current vaccines have not been cited.

Response: L452R, F486V and F486V mutations contributed to BA.4/5 escape [1-2]. Two mutations in the RBD of Omicron, K417N and E484A, were the driven factors in vaccine breakthroughs [3]. We have added this part in Discussion (Line 551-553).

[1] Aekkachai Tuekprakhon, Rungtiwa Nutalai, Aiste Dijokaite-Guraliuc, et al. Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum. Cell. 2022, 185(14):2422-2433.e13.

[2] Yunlong Cao, Ayijiang Yisimayi, Fanchong Jian, et al. BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature. 2022, 608(7923):593-602.

[3] Lok Bahadur Shrestha, Charles Foster, William Rawlinson, et al. Evolution of the SARS-CoV-2 omicron variants BA.1 to BA.5: Implications for immune escape and transmission. Rev Med Virol. 2022, e2381.

10. In the method section, the formulated LNP was stored at 4-degree until use. Would this be an advantage of this vaccine compared to Moderna and Pfizer ones?

Response: Yes. Moderna and Pfizer’s vaccines are shipped and stored at -15~-25°C and -60~-90°C respectively. But mRNA-LNP vaccines need to be stored frozen, presenting a considerable obstacle to vaccine distribution. mRNA-LNP vaccines can be stored in refrigerated temperature (2-8°C) or higher temperature are needed. RBMRNA-176 was manufactured in liquid formulation and can be stored at 2°C–8°C for at least 12 months.

11. In figure 1B, additional SDS-PAGE may show whether the spike proteins are cleaved or not.

Response: Thanks. We've added required SDS-PAGE results in Figure 1B. To determine whether the spike proteins are cleaved or not, we performed SDS-PAGE followed by western blot (Figure 1B).As seen in Figure 1B, there were two major bands in wild type S-transfected 293T cells, showing the full-length and cleaved spike proteins. However, no cleaved forms of the spike proteins were observed in RBMRNA-176-transfected cells.

 

Figure 1. Construction of RBMRNA-176 (A). Native PAGE and western blot analysis for the expression of wild-type S protein or S ectodomain trimer in transfected cell lysates (left). SDS-PAGE and western blot analysis for S protein cleavage (right) (B). Cryo-TEM of RBMRNA-176(C). Particle size distribution of RBMRNA-176 was measured in triplicate using dynamic light scattering (DLS) on a Malvern ZETASIZER (D). Biodistribution of LNP-Luciferase 357 mRNA (E). BALB/c mice were administrated intramuscularly (i.m.) with 5 μg LNP-packaged luciferase mRNA and.bioluminescence was measured 3 h post injection in a In-Vivo Xtreme System.

12. Figure 8A-B, please specify what gray and red lines represent.

Response: Red and black lines present median and individual viral load, respectively (Line 475-476).

13. In line 537, the authors cite a study in Shan-Lu Liu's group (not "Evans's group"). Please also cite several other key publications in recent two years.

Response: Replaced as Evans's group. Recent studies have also shown that COVID-19 vaccines based on previous strains provided less protection against the Omicron variants, including BA.4 and BA.5, due to the escape from antibody response. We have cited the 3 literatures (Line 550).

14. I am not convinced by the facts provided in this study to make the conclusion the author declared, "the responses elicited by this vaccine follow a profile consistent with vaccine safety and efficacy, laying the groundwork for further research into broaden protection and clinical translation."  Too much high dose for such protection to be seen in animals, and a lack of clinical trial will preclude the statement of safety and efficacy. Instead, I would add a limitation of the study statement to address these issues.

Response: We agree that, at this stage, it is not convincing to make this statement because there was substantial defects of our study listed as followed: 1) RBMRNA-176 did not have the fusion peptide, but SARS-CoV-2 fusion peptide was favorable for IgG production (Line 504-508); 2) We did not determine cellular immune responses in macaque, evaluate the neutralization of SARS-CoV-2 wild type and variants with macaque serum  (Line 532-534); 3) We haven’t compared the immunogenicity of wild-type S protein with RBMRNA-176  (Line 535); 4) We did not measure RBMRNA-176 vaccination induced-neutralization of SARS-CoV-2 variants by using live viruses; 5) The in vivo protection of RBMRNA-176 vaccination against SARS-CoV-2 variants infection was absent  (Line 569); 6) The overall in vivo protection effect of RBMRNA-176 was inferior to Pfizer BNT162b2, Novavax NVX-CoV2373, and Moderna mRNA-1273 (Line 564-568). Instead, the further optimization of RBMRNA-176 for higher efficacy against Omicron variants are needed. Therefore, we rewrite the conclusion as “Taken together, our study provides insight into the broad neutralization and in vivo protection against SARS-CoV-2 infection with RBMRNA-176 vaccination. Due to RBMRNA-176 evasion by Omicron and the relatively high vaccination dose, it will be necessary to develop a vaccine of the next generation” (Line 572-575).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I want to thank the authors for putting effort to response my comments and revising the manuscript. The revised version is much improved. I only have some minor suggestions and comments on the revised manuscript.

 

1. The Omicron binding and pseudovirus neutralization data are important results that may inform how the vaccine may work against current SARS-CoV-2 variants. It is not usual to report new data in the Discussion section. I will suggest moving these data to the Results section for clarity.

2. Two references the authors cited should be updated to their published version, i.e., ref 33, Science Vol 377, Issue 6607, pp. 735-742, DOI: 10.1126/science.abq2679; ref 40, Nature, 2021 Apr;592(7853):283-289. DOI: 10.1038/s41586-021-03275-y

3. Abstract, “against SARS-CoV-2 wild-type and mutant spikes, including Alpha, Beta, Gamma, and Kappa”, should be “against SARS-CoV-2 spike pseudotyped wild-type and variant viruses, including Alpha, Beta, Gamma, and Kappa”.

Author Response

1. The Omicron binding and pseudovirus neutralization data are important results that may inform how the vaccine may work against current SARS-CoV-2 variants. It is not usual to report new data in the Discussion section. I will suggest moving these data to the Results section for clarity. 

Response: We have combined Figure 2 and the Omicron binding and pseudovirus neutralization data, corrected typo in Figure 2, and modified the legend of Figure 2 accordingly (Line 390-391; Line 393; Line 394-395; Ling 397). Also, we added the corresponding methods (Line 176-179; Line 214-215).

2. Two references the authors cited should be updated to their published version, i.e., ref 33, Science Vol 377, Issue 6607, pp. 735-742, DOI: 10.1126/science.abq2679; ref 40, Nature, 2021 Apr;592(7853):283-289. DOI: 10.1038/s41586-021-03275-y.

Response: Ref 33 has been corrected; the published version of Ref 40 is the Ref 28, which has been corrected. The resulting changes of the Ref number has also been corrected (Ref 41-50 changed to Ref 40-49).

3. Abstract, “against SARS-CoV-2 wild-type and mutant spikes, including Alpha, Beta, Gamma, and Kappa”, should be “against SARS-CoV-2 spike pseudotyped wild-type and variant viruses, including Alpha, Beta, Gamma, and Kappa”.

Response: Corrected (Line 35-36).

Author Response File: Author Response.pdf