Development of recombinant proteins for vaccine candidates against serotypes O and A of Foot-and-Mouth Disease virus in Bangladesh

  1. Salma Akter
  2. M. Shaminur Rahman
  3. M. Rafiul Islam
  4. Masuda Akther
  5. Humaira Anjume
  6. Mafruha Marjia
  7. Md. Mizanur Rahaman
  8. M. Anwar Hossain
  9. Munawar Sultana

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Abstract

Frequent vaccine failure leading to recurrent outbreaks of Foot-and-Mouth Disease (FMD) in livestock populations necessitates the development of a customizable vaccine platform comprising potential antigenic determinants of circulating lineages of FMD viruses. Artificially designed, chimaeric protein-based recombinant vaccines are novel approaches to combat the phylogenetically diverse FMD Virus (FMDV) strains. Among seven recognized serotypes, only serotypes O and A are dominantly circulating in Bangladesh and neighbouring countries of Asia, where transboundary transmission, recurrent outbreaks and emergence of novel lineages of FMDV are highly prevalent. The objective of this study was to develop multi-epitope recombinant proteins, procuring immunogenicity against circulating diverse genotypes of FMDV serotypes O and A. Two chimaeric proteins, named B1 (41.0 kDa) and B3 (39.3 kDa), have been designed to incorporate potential B-cell and T-cell epitopes selected from multiple FMDV strains, including previously reported and newly emerged sub-lineages. After expression, characterization and immunization of guinea pigs with a considerable antigen load of B1 and B3 followed by serological assays revealed the significant protective immunogenicity, developed from the higher (100 µg) doses of both antigens, against most of the currently prevalent serotype O and A strains of FMDV. The efficient expression, antigenic stability, and multivalent immunogenic potency of the chimaeric proteins strongly indicate their credibility as novel vaccine candidates for existing serotypes O and A of FMDV in Bangladesh and surrounding territories.

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  1. Version published to 10.1099/acmi.0.000713.v4 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000713.v3 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    I do agree with your responses.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    The author has addressed my concerns and has thoroughly addressed all the points I raised in my previous feedback. I am pleased with the revisions they have made and believe that the manuscript is now ready for publication.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Access Microbiology

    Comments to Author

    Line 297: please reconsider clustering of isolates in the phylogenetic tree, the bootstrap values are too low to be considered as separate clusters.  Response: Thank you very much for your feedback. We have over 40 protein sequences for VP1. We used a bootstrap value of 1000 to generate the trees. We thoroughly checked the amino acid modifications for additional confirmation and clarity before selecting the appropriate strains for peptide synthesis. If necessary, we can include these analyses for your reference. Suggestion: Please mention the rationale how the clusters were decided, the bootstrap value for each cluster (C1, C2 etc.) is too low. Please follow the journal format for references in the text OR follow the same pattern of citations within text throughout the manuscript. Manuscript need some editorial corrections.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  8. Access Microbiology

    Comments to Author

    The authors have provided a thorough study of the development of using a chimeric platform to develop vaccines to FMDV serotypes O and A. As per my previous review the materials and methods are well described, the results are presently cllearly and the conclusions are supported by the results. The authors have provided a method to develop chimeric vaccines in Bangladesh. The authors have successfully answered the concerns of this reviewer.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  9. Version published to 10.1099/acmi.0.000713.v2 on Access Microbiology
  11. Access Microbiology

    Comments to Author

    Please note my comments in the manuscript; 1. it may be a good idea to mention the target species in the title and the rest of the manuscript. 2. Line 41 - 103: introduction is too long, please introduce the topic with information relevant to the subject of the manuscript. 3. Line 45: reference 2 in only for South Korea, provide other references. 4. Line 170 -172: please rephrase. 5. Line 174: Hig speed centrifugation, please provide details. 6. Line 243 - 246: are these commercially available vaccines? if yes, provide details of the commercial vaccines. If no, provide details how these vaccines were prepared? 7. Line 247 -249: please explain why the authors decided to boost GPs on day 14 (any reference?) and the why the animals were sacrificed on day 28? also mention the method used of euthanasia. 8. Lime 249 -250: serum is not collected by centrifugation of blood samples, centrifugation of blood yields plasma. Please rephrase. 9. Line 250 - 251: please mention the storage conditions of serum until further use. 10: Line 274: Section 2.4.6 Statistical analysis: Ab titre data is non-parametric data, perhaps authors should consider doing statistical analysis with non-parametric tests. Ab titres are often measured on an ordinale scale, and the distribution of such data may not meet tha assumptions pf normality required for parametric tests. 11: Line 297: please reconsider clustering of isolates in the phylogenetic tree, the bootstrap values are too low to be considered as separate clusters. 12. Table 1 and 2: please explain the values in parenthesis in the column header. 13. Lime 365: Section 3.1.2 & 3.1.3: Please provide details in the M&Ms section. 14. Supplementary table 3: Please explain n=24 in the experimental group box.

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    No: methods used for euthanizing animals (guinea pigs) used in the experiment is not explained in the manuscript

  12. Access Microbiology

    Comments to Author

    The manuscript titled "Development of Recombinant Proteins for Vaccine Candidates Against Serotype O and A of Foot and Mouth Disease Virus in Bangladesh" provides a comprehensive overview of the research carried out in developing recombinant proteins as vaccine candidates against serotype O and A of Foot and Mouth Disease Virus (FMDV) in Bangladesh. The manuscript provides a concise background on FMD, its economic impact, and the importance of vaccines in preventing and controlling the disease. The rationale for targeting serotype O and A of FMDV in Bangladesh is explained, highlighting their high prevalence and economic impact. The experimental methods employed in the study are described in detail, allowing for replication of the research. The authors have successfully developed recombinant proteins against serotype O and A of FMDV, using appropriate cloning techniques and expression systems. The immunogenicity of the recombinant proteins has been evaluated through serological assays, and their protective efficacy has been assessed in animal models. The authors have presented clear and concise results, supported by appropriate statistical analysis. The immunogenicity of the recombinant proteins has been evaluated, demonstrating significant levels of antibody production against the targeted serotypes. The protective efficacy studies in animal models have shown promising results, indicating the potential for these recombinant proteins as vaccine candidates against serotype O and A of FMDV in Bangladesh. Overall Assessment: The manuscript is well-written, organized, and scientifically sound. The research methodology is robust, and the conclusions are justified by the results. The manuscript is a significant contribution to the field and has the potential to impact the development of vaccines against serotype O and A of FMDV in Bangladesh and beyond. I recommend that the authors address the following minor points: Comment 1 The authors described the manuscript very well including all the required information was added. However, it would be greatly addition if authors could include one additional figure showing the sequence or structural architecture of selected capsid protein with its corresponding domains. Comment 2 The authors perform a detailed three dimensional modeling building of both chmieric protein B1 and B3 which highlights all the important domains of capsid proteins. It would be helpful if authors performed a addional structure homology modeling to show the three dimension domain orientation have exact same as present in viral capsid protein. Comment 3 It would also be very helpful if authors will perform a molecular dynamics study to further support the stability of three dimensional models.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  13. Access Microbiology

    Comments to Author

    Foot and Mouth Disease (FMD) is an endemic disease in many parts of the globe that is directly linked with food security and heavy economic losses. In the current study the authors have highlighted novel vaccine development strategy to this important disease. I do agree with the investigations on the development of recombinant proteins containing serotype O and A of FMD antigen. For the current study, I have two curies to ask/ clarifications. 1. In the current study, the authors do work on serotype O and serotype A and why not on serotype Asia-1 as the serotype Asia-1 is also reoccurring in the Bangladesh. Moreover, serotype Asia-1 is existing in the neighboring countries as well. 2. Secondly, the author(s) should explain about NS50 that is being used in the manuscript.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  14. Access Microbiology

    Comments to Author

    The purpose of this paper was to develop chimeric protein based recombinant vaccines to types O and A Foot and Mouth Disease circulating in Bangladesh. The authors provide a very detailed and thorough description of their methods that includes how the different lineages, or sub lineages were chosen; how the multiepitope recombinant proteins were incorporated into the procaryotic platform; and also the antibody response derived in guinea pigs inoculated with different concentrations of the recombinant vaccines. Sufficient detail is provided in the material and methods to allow for reproducibility. The results are well organized and presented in logical sequence. Figures are excellent and the figure captions provide appropriate detail to understand the data presented in the figures. Authors on Figure 4 F is the thresh hold line supposed to be at the higher level of 12 or at 2 as are all the other histograms in this figure? In your results line 475, not sure the significance codes are correct please review. The Discussion is very long and laborious to read. You seem to spend much time rehashing the results and methods and not focusing specifically on the data. The Discussion needs to be reduced and rewritten to focus on your data and prior reports that either support or refute your observations. You indicate in the methods that specific coding sequences that activate T-cell responses were included in the recombinant vaccine, however, there is no data presented on the T-cell response in the inoculated guinea pigs? Also you indicate that that level of antibody detected on neutralization assays would provide a protective response to the virus. How did you determine this without doing challenge experiments in the guinea pigs or a natural host? If you cannot verify that a certain level of antibodies is protective, then simple confine your comments to what the data shows (that is a certain level of antibodies).

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  15. Version published to 10.1099/acmi.0.000713.v1 on Access Microbiology