Evaluation of the immune status of dogs vaccinated against rabies by an enzyme-linked immunosorbent assay using crude preparations of insect cells infected with a recombinant baculovirus encoding the rabies virus glycoprotein gene

A. K. Santosh; Deepak Kumar; Charanpreet Kaur; Priya Gupta; Pagala Jasmeen; L. Dilip; G. Kavitha; Suresh Basagoudanavar; Madhusudan Hosamani; V. Balamurugan; R. Sharada; D. Rathnamma; K. M. Sunil; Nagendra R. Hegde; Shrikrishna Isloor

doi:10.1371/journal.pone.0314516

Peer Review History

Original SubmissionApril 19, 2024
7 Jun 2024 Decision Letter - Jian Xu, Editor PONE-D-24-15752Evaluation of the immune status of dogs vaccinated against rabies by an enzyme-linked immunosorbent assay using crude preparations of insect cells infected with a recombinant baculovirus encoding the rabies virus glycoprotein genePLOS ONE Dear Dr. Isloor, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. As you can see the comments from our reviewers, they pointed out several major and minor issues for your manuscript. The editor also suggests that the figures (e.g., Fig.1 and Fig.2) need significant improvement to meet the standards and quality requirements of this journal. Please provide also the raw gel images and data points used for all figures. 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When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The article by Santosh et al., entitled "Evaluation of the immune status of dogs vaccinated against rabies by an enzyme-linked immunosorbent assay using crude preparations of insect cells infected with a recombinant baculovirus encoding the rabies virus glycoprotein gene," addresses the challenge of evaluating rabies vaccination effectiveness in dogs by proposing an ELISA as a practical alternative to the currently recommended tests, RFFIT and FAVN, which involve the handling of live rabies virus. The authors developed an ELISA using partially purified extracts of insect cells infected with baculovirus expressing the rabies virus glycoprotein (RV G). This method was evaluated against sera from vaccinated dogs, showing good concordance with RFFIT, with sensitivity and specificity of 90.48% and 80.00%, respectively. The proposed system offers a quick screening tool to determine the presence and approximate level of antibodies, and it holds potential for monitoring mass vaccination programs and certifying dogs for international travel. The study is well-conceived and contributes valuable insights into rabies vaccination monitoring. Thus, the manuscript can be accepted after minor revisions. Minor Revisions 1. Introduction: Include the new species name Lyssavirus rabies according to ICTV (https://ictv.global/report/chapter/rhabdoviridae/rhabdoviridae/lyssavirus). 2. Sentence Improvement: Improve the sentence “However, there is no means to verify the success of the vaccination programmes other than keeping a record of the vaccination.” to provide a clearer context. 3. Cell Line Description: On page 10, line 206, specify which cell line was used and provide a detailed description. 4. Figure 1: Ensure the order of the description of the wells matches the order shown in the image to avoid confusion for the readers. 5. Figure 2C: Clarify which fraction was submitted by specifying “One of the fractions (indicate which) was submitted...” 6. Table 3: Include a footnote indicating that the kappa (κ) value of 0.61–0.80 is considered “substantial agreement” and not “almost perfect agreement.” 7. ELISA Antigen Explanation: Provide a better explanation as to why the purified G protein was not used to perform the ELISA, and describe the difficulties encountered in the purification step. 8. Comparison with Other Studies: Include a comparison with values found in other studies: - Cliquet F, McElhinney LM, Servat A, Boucher JM, Lowings JP, Goddard T, Mansfield KL, Fooks AR (2004) Development of a qualitative indirect ELISA for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats. J Virol Methods 117:1–8. https://doi.org/10.1016/j.jviromet.2003.12.001. - Cliquet F, Müller T, Mutinelli F, Geronutti S, Brochier B, Selhorst T, Schereffer JL, Krafft N, Burow J, Schameitat A, Schlüter H, Aubert M (2003) Standardisation and establishment of a rabies ELISA test in European laboratories for assessing the efficacy of oral fox vaccination campaigns. Vaccine 21:2986–2993. https://doi.org/10.1016/S0264-410X(03)00102-6. - Cliquet F, Sagné L, Schereffer JL, Aubert MF (2000) ELISA test for rabies antibody titration in orally vaccinated foxes sampled in the fields. Vaccine 18:3272–3279. https://doi.org/10.1016/S0264-410X(00)00127-4. - Wasniewski M, Cliquet F. Evaluation of ELISA for detection of rabies antibodies in domestic carnivores. J Virol Methods. 2012 Jan;179(1):166-75. doi: 10.1016/j.jviromet.2011.10.019. - Zajac MD (2019) Development and evaluation of a rabies enzyme-linked immunosorbent assay (ELISA) targeting IgM and IgG in human sera. https://doi.org/10.13140/RG.2.2.32365.54242. - Zhao R, Yu P, Shan Y, Thirumeni N, Li M, Lv Y, Li J, Ren W, Huang L, Wei J, Sun Y, Zhu W, Sun L (2019) Rabies virus glycoprotein serology ELISA for measurement of neutralizing antibodies in sera of vaccinated human subjects. Vaccine 37:6060–6067. https://doi.org/10.1016/j.vaccine.2019.08.043. 9. Limitations of the Study: The authors should include a sentence in the discussion topic that reports the limitations of the study. 10. In the study by Kramps et al. (1994), the authors demonstrated that their blocking ELISA was superior to a commercially available indirect ELISA and to the 24-hour virus neutralization test in detecting low antibody levels in serum. Additionally, this blocking ELISA was capable of detecting specific antibodies in serum as early as 7 days post-infection. Thus, it would be beneficial to evaluate whether the newly developed ELISA here can detect antibodies as early as 7 days post-infection, similar to Kramps et al. studies. Kramps JA, Magdalena J, Quak J, Weerdmeester K, Kaashoek MJ, Maris-Veldhuis MA, Rijsewijk FA, Keil G, van Oirschot JT. A simple, specific, and highly sensitive blocking enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus 1. J Clin Microbiol. 1994 Sep;32(9):2175-81. doi: 10.1128/jcm.32.9.2175-2181.1994. Kramps JA, Banks M, Beer M, Kerkhofs P, Perrin M, Wellenberg GJ, Oirschot JT. Evaluation of tests for antibodies against bovine herpesvirus 1 performed in national reference laboratories in Europe. Vet Microbiol. 2004 Sep 8;102(3-4):169-81. doi: 10.1016/j.vetmic.2004.07.003. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. 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Revision 1
28 Oct 2024 Author Response Responses to reviewers We thank the reviewer for a very detailed scrutiny of the manuscript. The suggestions have significantly improved the manuscript. Provided below are point by point responses to the suggestions. Reviewer #1 1. Introduction: Include the new species name Lyssavirus rabies according to ICTV (https://ictv.global/report/chapter/rhabdoviridae/rhabdoviridae/lyssavirus). We have also changed the abbreviation of rabies virus from RV to RABV throughout the manuscript, in accordance with ICTV. Response: This has been included right in the beginning of the Introduction (see line 47 in the clean copy of the revised manuscript). 2. Sentence Improvement: Improve the sentence “However, there is no means to verify the success of the vaccination programmes other than keeping a record of the vaccination.” to provide a clearer context. Response: Context has been provided (see lines 55-58 in the clean copy of the revised manuscript)). 3. Cell Line Description: On page 10, line 206, specify which cell line was used and provide a detailed description. Response: Cell line specification and description are now provided (see lines 247-250 in the clean copy of the revised manuscript)). 4. Figure 1: Ensure the order of the description of the wells matches the order shown in the image to avoid confusion for the readers. Response: We thank the reviewer for pointing this out. The description has now been modified for all the three panels for clarity (see lines 298, 306 and 309 in the clean copy of the revised manuscript). 5. Figure 2C: Clarify which fraction was submitted by specifying “One of the fractions (indicate which) was submitted...” Response: It was pooled fraction 5-8, and this has been specified now (see line 337 in the clean copy of the revised manuscript). 6. Table 3: Include a footnote indicating that the kappa (κ) value of 0.61–0.80 is considered “substantial agreement” and not “almost perfect agreement.” Response: A foot note has been added to the Table (see lines 391-392 in the clean copy of the revised manuscript). It may be noted that we had stated it to be ‘good’ agreement in the description (see line 388 in the clean copy of the revised manuscript). 7. ELISA Antigen Explanation: Provide a better explanation as to why the purified G protein was not used to perform the ELISA, and describe the difficulties encountered in the purification step. Response: A sentence is now included in the Discussion (see lines 419-423 in the clean copy of the revised manuscript). 8. Comparison with Other Studies: Include a comparison with values found in other studies: - Cliquet F, McElhinney LM, Servat A, Boucher JM, Lowings JP, Goddard T, Mansfield KL, Fooks AR (2004) Development of a qualitative indirect ELISA for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats. J Virol Methods 117:1–8. https://doi.org/10.1016/j.jviromet.2003.12.001. - Cliquet F, Müller T, Mutinelli F, Geronutti S, Brochier B, Selhorst T, Schereffer JL, Krafft N, Burow J, Schameitat A, Schlüter H, Aubert M (2003) Standardisation and establishment of a rabies ELISA test in European laboratories for assessing the efficacy of oral fox vaccination campaigns. Vaccine 21:2986–2993. https://doi.org/10.1016/S0264-410X(03)00102-6. - Cliquet F, Sagné L, Schereffer JL, Aubert MF (2000) ELISA test for rabies antibody titration in orally vaccinated foxes sampled in the fields. Vaccine 18:3272–3279. https://doi.org/10.1016/S0264-410X(00)00127-4. - Wasniewski M, Cliquet F. Evaluation of ELISA for detection of rabies antibodies in domestic carnivores. J Virol Methods. 2012 Jan;179(1):166-75. doi: 10.1016/j.jviromet.2011.10.019. - Zajac MD (2019) Development and evaluation of a rabies enzyme-linked immunosorbent assay (ELISA) targeting IgM and IgG in human sera. https://doi.org/10.13140/RG.2.2.32365.54242. - Zhao R, Yu P, Shan Y, Thirumeni N, Li M, Lv Y, Li J, Ren W, Huang L, Wei J, Sun Y, Zhu W, Sun L (2019) Rabies virus glycoprotein serology ELISA for measurement of neutralizing antibodies in sera of vaccinated human subjects. Vaccine 37:6060–6067. https://doi.org/10.1016/j.vaccine.2019.08.043. Response: We appreciate the reviewer for suggesting and drawing our attention to this. We have now added an entire paragraph in the Discussion on this (lines 471-503 in the clean copy of the revised manuscript), but have focussed on those studies which have tested for sera from animals, particularly carnivores. Although we agree with the importance of application of such tests in humans, in the context of our assay, studies using human sera only have been omitted. 9. Limitations of the Study: The authors should include a sentence in the discussion topic that reports the limitations of the study. Response: Two sentences have been added at the end of Discussion (lines 508-511 in the clean copy of the revised manuscript). 10. In the study by Kramps et al. (1994), the authors demonstrated that their blocking ELISA was superior to a commercially available indirect ELISA and to the 24-hour virus neutralization test in detecting low antibody levels in serum. Additionally, this blocking ELISA was capable of detecting specific antibodies in serum as early as 7 days post-infection. Thus, it would be beneficial to evaluate whether the newly developed ELISA here can detect antibodies as early as 7 days post-infection, similar to Kramps et al. studies. Kramps JA, Magdalena J, Quak J, Weerdmeester K, Kaashoek MJ, Maris-Veldhuis MA, Rijsewijk FA, Keil G, van Oirschot JT. A simple, specific, and highly sensitive blocking enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus 1. J Clin Microbiol. 1994 Sep;32(9):2175-81. doi: 10.1128/jcm.32.9.2175-2181.1994. Kramps JA, Banks M, Beer M, Kerkhofs P, Perrin M, Wellenberg GJ, Oirschot JT. Evaluation of tests for antibodies against bovine herpesvirus 1 performed in national reference laboratories in Europe. Vet Microbiol. 2004 Sep 8;102(3-4):169-81. doi: 10.1016/j.vetmic.2004.07.003. Response: Although we understand the argument for detection of antibodies as early as possible, we are not clear about the context of the studies suggested by the reviewer. They refer to bovine herpesvirus 1, and not to rabies. In addition, the context of the ELISA that we have developed is for monitoring antibodies after vaccination, whereas the mentioned studies about BHV-1 are after infection, which is impractical or inconsequential for rabies. The fundamental difference in these two cases is that the one for BHV-1 is for diagnostic purposes and the one for rabies is for monitoring purposes. Furthermore, early detection in case of rabies may not have a bearing on the potential to protect since early responses are dominated by IgM. It is thought that IgM is not as efficient as IgG against rabies virus due to its inability to penetrate interstitial tissue that has been impregnated with high levels of virus through the saliva in the bite of a rabid animal (Kennedy et al. 2007; Fooks 2018). Indeed, driving sustained IgG production is one of the main targets of vaccination against rabies (Flamand et al. 1993; Dorfmeier et al. 2013). Although we agree that a time-lapse experiment on the appearance of potentially protective antibodies is required, we feel that this discussion would be deviant from the focus of the manuscript. References: 1. Dorfmeier, C.L.; Shen, S.; Tzvetkov, E.P.; McGettigan, J.P. 2013. Reinvestigating the role of IgM in rabies virus postexposure vaccination. J. Virol. 87:9217-9222. 2. Flamand, A.; Raux, H.; Gaudin, Y.; Ruigrok, R.W. 1993. Mechanisms of rabies virus neutralization. Virology 194:302-313. 3. Fooks, A. 2018. Rabies (Infection with RABV and Other Lyssaviruses). In Manual of Diagnotics Tests and Vaccines for Terrestrial Animals; World Organisation for Animal Health (OIE), Paris, France, 2018; pp. 578–612. 4. Kennedy, L.J.; Lunt, M.; Barnes, A.; McElhinney, L.; Fooks, A.R.; Baxter, D.N.; Ollier, W.E. 2007. Factors influencing the antibody response of dogs vaccinated against rabies. Vaccine 25:8500-8507. Relevant Editorial Comments: 1. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Response: The phrase has been removed (see line 292 in the revised manuscript). 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. Response: Images of raw gels/blots are provided as a Supplementary file. It may be noted that panels 1B and 2A are already full images. The Supplementary file hence contains the images for panels 1A, 1B, 2B and 2C. Attachments Attachment Submitted filename: Responses to reviewers.docx https://doi.org/10.1371/journal.pone.0314516.r002
12 Nov 2024 Decision Letter - Jian Xu, Editor Evaluation of the immune status of dogs vaccinated against rabies by an enzyme-linked immunosorbent assay using crude preparations of insect cells infected with a recombinant baculovirus encoding the rabies virus glycoprotein gene PONE-D-24-15752R1 Dear Dr. Isloor, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jian Xu, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The author has addressed all the questions and made the necessary revisions in the manuscript, which is now ready for acceptance in PLOS ONE. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No ******** https://doi.org/10.1371/journal.pone.0314516.r003
Formally Accepted
20 Nov 2024 Acceptance Letter - Jian Xu, Editor PONE-D-24-15752R1 PLOS ONE Dear Dr. Isloor, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Jian Xu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0314516.r004

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