Development of monoclonal antibodies targeting the conserved fragment of hexon protein to detect different serotypes of human adenovirus

ABSTRACT Human adenovirus (HAdV) infects the respiratory system, thus posing a threat to health. However, immunodiagnostic reagents for human adenovirus are limited. This study aimed to develop efficient diagnostic reagents based on monoclonal antibodies for diagnosing various human adenovirus infections. Evolutionary and homology analyses of various human adenoviral antigen genes revealed highly conserved antigenic fragments. The prokaryotic expression system was applied to recombinant penton, hexon, and IVa2 conserved fragments of adenovirus, which were injected into BALB/c mice to prepare human adenovirus-specific monoclonal antibodies. Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and Western blotting were used to determine the immune specificity of the monoclonal antibodies. Indirect ELISA showed that monoclonal antibodies 1F10, 8D3, 4A1, and 9B2 were specifically bound to HAdV-3 and HAdV-55 and revealed high sensitivity and low detection limits for various human adenoviruses. Western blotting showed that 1F10 and 8D3 specifically recognized various human adenovirus types, including HAdV-1, HAdV-2, HAdV-3, HAdV-4, HAdV-5, HAdV-7, HAdV-21, and HAdV-55, and 4A1 specifically recognized HAdV-1, HAdV-2, HAdV-3, HAdV-5, HAdV-7, HAdV-21, and HAdV-55. IFAs showed that 1F10, 8D3, and 4A1 exhibited highly selective localization to A549 cells infected with HAdV-3 and HAdV-55. Finally, two antibody pairs that could detect hexon antigens HAdV-3 and HAdV-55 at low concentrations were developed. The monoclonal antibodies developed in this study show potential for detecting human adenoviruses. IMPORTANCE In this study, we selected the three most conserved antigenic fragments of human adenovirus to prepare a murine monoclonal antibody for the first time, and human adenovirus antigenic fragments with heretofore unheard of degrees of conservatism were isolated. The three monoclonal antibodies with the ability to recognize human respiratory adenovirus over a broad spectrum were screened by hybridoma and monoclonal antibody preparation. Human adenovirus infections are serious; however, therapeutic drugs and diagnostic reagents are scarce. Thus, to reduce the serious consequences of human viral infections and adenovirus pneumonitis, early diagnosis of infection is required. The present study provides three monoclonal antibodies capable of recognizing a wide range of human adenoviruses, thereby offering guidance for subsequent research and development.


Introduction:
Use human adenovirus or HAdV throughout the manuscript Lines 60, 63, 66 and others: Currently subgenus/subgroup of HAdV is designated as species.Use species throughout the manuscript.Line 84: Write HAdV-2 Line 112: Write HAdV Lines 118-123: The rationale for the development of monoclonal antibodies is not clear.Is there any targeted antiviral against HAdVs?If any, please mention.
Materials and method: Lines 148-150: Write sequences of which types were obtained from the Genbank with their accession number.Use more types of respiratory HAdVs for alignment as mentioned in the introduction and make a figure of alignment of the include conserved region.Lines 148-160: provide web site address of all the editing tools.Mention why you selected core protein HAdV IVa2 in this study.Line 187: Write BALB/c mice Result: Line 285-91: HAdV-2, HAdV-4,  are included for alignment and phylogenesis.Currently, HAdV-3 is a mostly recovered respiratory isolate.It seems HAdV-3 is not included in alignment or phylogenesis.Use all types of respiratory HAdVs (including 3) for alignment as mentioned in the introduction and make a figure of alignment that include conserved regions.
Discussion: HAdV-3, HAdV-4, HAdV-5, HAdV-7 and HAdV-55 are used to check the binding activity of mAbs.I am not sure why other types are not tested.Overall, the writing of the manuscript is not clear.There are many minor mistakes The experimental part also needed to revise.The authors can use professional editing services for improvement.
Reviewer #2 (Comments for the Author): This paper details the generation of cross-reactive mAbs against specific epitopes within HAdV proteins hexon and IVa2.It is more of a technical report, with little science in terms of hypothesis etc.The mAbs generated could perhaps be useful for HAdV diagnostics but that wasn't directly tested or benchmarked against more sensitive techniques like qPCR, and only a small subset of HAdV types were tested via western blot and ELISA.
Reviewer #3 (Comments for the Author): In the current manuscript, Wu-Lin-Yin et al. have described the production of 6 mAbs, out of which 3 mAbs bind specifically to Hexon protein on the surface of AdV particles.The data presented appears robust and the applicability of broad-spectrum hexon antibody would be great.I have a few specific suggestions experimentally mentioned below.
1.The authors need to clarify if the hexon mAbs are functional under native or denaturing conditions in western blots.This will give hints as to where these antibodies bind.They should also check in immunofluorescent whether the antibody works before or after AdV replication.To add to that, one of the important uses of highly specific hexon antibodies (e.g., 9C12) is their ability to be used during virus entry studies.The authors should hence test in parallel with 9C12 whether incoming AdVs can be detected with either of these antibodies.In any case, 9C12 antibody should be included as a comparison for hexon mAbs.2. Can any of the 3 hexon antibodies promote AdV neutralisation?This simple experiment (i.e., pre-incubating the antibodies with virions before binding to cells etc.) would clarify if any of these bind to the surface exposed regions.3. The manuscript can be improved in terms of the language of the paper which needs a strong reevaluation and sentences in several places need to be appended.4. Include statistical tests in Figs 4 and 5.

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The manuscript (Spectrum01816-23) en tled "Development of monoclonal an bodies targe ng the conserved fragment of Hexon protein for broadly detec ng different serotypes of adenovirus" by Linfan Wu et al.In their study, the authors developed monoclonal an bodies that can detect a few human adenoviruses.The development of diagnos c methods against human adenoviruses is important as there are many recombinant types included in the last few years.My queries regarding the manuscripts are the following:

Abstract:
The abstract does not match clearly with the experiment.Rewri ng is necessary.

Introduc on:
Use human adenovirus or HAdV throughout the manuscript Lines 60, 63, 66 and others: Currently subgenus/subgroup of HAdV is designated as species.Use species throughout the manuscript.
Line 84: Write HAdV-2 Line 112: Write HAdV Lines 118-123: The ra onale for the development of monoclonal an bodies is not clear.
Is there any targeted an viral against HAdVs?If any, please men on.

Materials and method:
Lines 148-150: Write sequences of which types were obtained from the Genbank with their accession number.
Use more types of respiratory HAdVs for alignment as men oned in the introduc on and make a figure of alignment of the include conserved region.
Lines 148-160: provide web site address of all the edi ng tools.
Men on why you selected core protein HAdV IVa2 in this study.
Line 187: Write BALB/c mice
Overall, the wri ng of the manuscript is not clear.There are many minor mistakes The experimental part also needed to revise.The authors can use professional edi ng services for improvement.
Dear editor Jérôme Le Goff and dear reviewers, Re: Manuscript ID: Spectrum01816-23 and Title: Development of monoclonal antibodies targeting the conserved fragment of Hexon protein for broadly detecting different serotypes of human adenovirus.

Editor Comments:
The manuscript requires extensive modifications before it can be considered for publication in Microbiology Spectrum.According to the reviewers' comments, there is room for improvement.In addition to significant scientific revisions, language editing is imperative.Response: Thank you very much for your comments and professional advice.These opinions help to improve the academic rigor of our article.Based on your suggestion and request, we have corrected the revised manuscript's modifications.Meanwhile, we have carefully polished the language of our manuscript as suggested.It is hoped that this version of the article will be accepted by the Microbiology Spectrum Reviewer(s)' Comments to Author: Reviewer 1# Comments for the Author The manuscript (Spectrum01816-23) entitled "Development of monoclonal antibodies targeting the conserved fragment of Hexon protein for broadly detecting different serotypes of adenovirus" by Linfan Wu et al.In their study, the authors developed monoclonal antibodies that can detect a few human adenoviruses.The development of diagnostic methods against human adenoviruses is important as there are many recombinant types included in the last few years.My queries regarding the manuscripts are the following: Response: Thanks for your time and the insightful comments about our study.
1.Title: Include human adenovirus；Abstract: The abstract does not match clearly with the experiment.Rewriting is necessary.Response: Thanks for your time and comments on our manuscript, we have revised the title, and the abstract has been rewritten (lines 2-40).

2.Introduction:
Use human adenovirus or HAdV throughout the manuscript.Lines 60, 63, 66 and others: Currently subgenus/subgroup of HAdV is designated as species.Use species throughout the manuscript.Line 84: Write HAdV-2 Line 112: Write HAdV Lines 118-123: The rationale for the development of monoclonal antibodies is not clear.Is there any targeted antiviral against HAdVs?If any, please mention.Response: Thank you for your scrutiny and suggestions.We are very sorry for our carelessness.We have changed the adenovirus in the manuscript to human adenovirus or HAdV and subgenus changed to species (lines 65-75).Then, we have corrected the correction of line 84 (lines 88-89) and line 112 (line 120).At the same time, we have added the basic principle of monoclonal antibody development (lines 126-136).In addition, we answered question about anti-human adenovirus drugs (lines 139-144).

3.Materials and method:
Lines 148-150: Write sequences of which types were obtained from the GenBank with their accession number.Use more types of respiratory HAdVs for alignment as mentioned in the introduction and make a figure of alignment of the include conserved region.Response: Thanks for your insightful comments and suggestions.We added more human adenovirus types and their accession numbers from GenBank, as shown in Table 1 (lines 199-200).Meanwhile, we have drawn a sequence alignment analysis diagram containing conserved regions, as shown in Figure .1 of the results.
4. Lines 148-160: provide web site address of all the editing tools.Mention why you selected core protein HAdV IVa2 in this study.Line 187: Write BALB/c mice.Response: Thanks for your insightful comments and suggestions.In this manuscript, we have edited the website address of all the tools used (lines 185, 188, 195-196, 374).In the previous work, we compared all human adenovirus sequences.Finally, we selected the three most conserved antigens, one of which is IVa2 (lines 192-193), and as shown in Figure .1 of the results.Furthermore, we've supplemented all the BALB/c mice, as shown in supplementary Fig. 2 of the Supplementary Materials.

Result: Line 285-291:
HAdV-2, HAdV-4, HAdV-5, HAdV-7, HAdV-11 and HAdV-21 are included for alignment and phylogenesis.Currently, HAdV-3 is a mostly recovered respiratory isolate.It seems HAdV-3 is not included in alignment or phylogenesis.Use all types of respiratory HAdVs (including 3) for alignment as mentioned in the introduction and make a figure of alignment that include conserved regions.Response: Thanks for your insightful comments and suggestions.We are sorry for our negligence.We have supplemented the homology analysis and conserved region alignment of HAdV-3, HAdV-1 and HAdV-55 (lines 379-382), as shown in Fiture.1 of the results.

Discussion:
HAdV-3, HAdV-4, HAdV-5, HAdV-7 and HAdV-55 are used to check the binding activity of mAbs.I am not sure why other types are not tested.Overall, the writing of the manuscript is not clear.There are many minor mistakes The experimental part also needed to revise.The authors can use professional editing services for improvement.Response: Thanks for your insightful comments and suggestions.We added the specific detection of monoclonal antibodies and HAdV-1, HAdV-2, HAdV-21 by Western blot (lines 473-476).At present, HAdV-1, HAdV-2, HAdV-3, HAdV-4, HAdV-5, HAdV-7, HAdV-21 and HAdV-55 have been successfully isolated and cultured in the Third People's Hospital of Shenzhen, Guangdong Province.Therefore, we only used the above 8 types of human respiratory adenovirus detection.At last, we tried our best to improve the manuscript and polish the language of our manuscript.We appreciate for your warm work earnestly.

Reviewer 2#
Comments for the Author 1.This paper details the generation of cross-reactive mAbs against specific epitopes within HAdV proteins Hexon and IVa2.It is more of a technical report, with little science in terms of hypothesis etc. Response: We sincerely thank you for your careful reading.In our work, by analyzing and comparing the gene sequences of all human adenoviruses, the three most conserved antigen fragments were selected.By expressing the antigens, immunizing mice, preparing mouse-derived monoclonal antibodies and identifying the specificity of monoclonal antibodies to HAdVs.In this process, our hypothesis is relatively small.However, the monoclonal antibodies we discovered are innovative and have certain scientific value, and the discovery of these monoclonal antibodies is conducive to the diagnosis and treatment of human adenovirus.
2.The mAbs generated could perhaps be useful for HAdV diagnostics but that wasn't directly tested or benchmarked against more sensitive techniques like qPCR, and only a small subset of HAdV types were tested via western blot and ELISA.Response: Thanks for your insightful comments and suggestions.We also added the specific detection of monoclonal antibodies and HAdV-1, HAdV-2, HAdV-21 by Western blot.A total of 8 types of human respiratory adenovirus were detected (lines 678-680), as shown Figure .7 of the results (lines 696).In addition, we supplemented the double antibody sandwich ELISA test (lines 426-444).At the same time, qPCR was used to detect human adenovirus, and the results were compared with those of double antibody sandwich ELISA (lines 734-763).Finally, we appreciate your suggestion earnestly.

Reviewer 3# Comments for the Author
In the current manuscript, Wu-Lin-Yin et al. have described the production of 6 mAbs, out of which 3 mAbs bind specifically to Hexon protein on the surface of AdV particles.The data presented appears robust and the applicability of broad-spectrum Hexon antibody would be great.I have a few specific suggestions experimentally mentioned below.Response: Thanks for your time and the insightful comments about our study.
1.The authors need to clarify if the Hexon mAbs are functional under native or denaturing conditions in western blots.This will give hints as to where these antibodies bind.They should also check in immunofluorescent whether the antibody works before or after AdV replication.To add to that, one of the important uses of highly specific Hexon antibodies (e.g., 9C12) is their ability to be used during virus entry studies.The authors should hence test in parallel with 9C12 whether incoming AdVs can be detected with either of these antibodies.In any case, 9C12 antibody should be included as a comparison for Hexon mAbs.Response: Thanks for your insightful comments and suggestions.By Western blotting, we detected the specificity of monoclonal antibodies against human adenovirus antigens under natural or denatured conditions.As previously reported, monoclonal antibodies could only detect human adenovirus antigens under denatured conditions by Western bolt (lines 482-485).As suggested by the reviewer, we have supplemented the indirect immunofluorescence assay (lines 308-338).The results suggest that monoclonal antibodies work after HAdV replication (lines 500-509).We have reviewed many articles on highly specific Hexon antibodies, but we have not found the relevant gene sequences, and couldn't express and compare the corresponding mouse antibodies (lines 584-587).However, we detected human adenovirus by highly sensitive qPCR and compared it with our results, as shown in Figure 6 of the results.
2. Can any of the 3 Hexon antibodies promote AdV neutralization?This simple experiment (i.e., pre-incubating the antibodies with virions before binding to cells etc.) would clarify if any of these binds to the surface exposed regions.Response: Thanks for your insightful comments and suggestions.We performed neutralization tests on these three monoclonal antibodies but failed to show satisfactory results (lines 572-574).
3. The manuscript can be improved in terms of the language of the paper which needs a strong reevaluation and sentences in several places need to be appended.Response: Thanks for your insightful comments and suggestions.We have tried our best to modify the details and polish the language of the manuscript.Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
We appreciate the authors for their responses to reviewers' comments aimed at improving the manuscript.The document requires revision by a native speaker specializing in scientific article editing.
Additionally, the introduction, discussion, and conclusion need modification.While specific HAdV antibodies can confirm that individuals have been infected by HAdV, they cannot be used to confirm an ongoing infection and cannot establish a direct link with symptoms.Since the antibodies developed in this study are non-type-specific, they cannot be employed to determine the HAdV type by which an individual could be infected.Such antibodies could be of interest in describing the natural history of HAdV infection and understanding how HAdV concentrations in body fluids correlate with protection or the severity of infection Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, notify me immediately so that the manuscript may be formally withdrawn from consideration by Spectrum.

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Sincerely, Jérôme Le Goff Editor Microbiology Spectrum 4. Include statistical tests in Figs4 and 5. Response: Thanks for your insightful comments and suggestions.Figure4and Figure 5 in the original manuscript are now Figure 2 and 3, respectively.We have passed the statistical tests and replotted, as shown in Figure 2 and Figure 3 of the results.Thank you very much for your attention and time.Look forward to hearing from you.1st Revision -Editorial Decision Re: Spectrum01816-23R1 (Development of monoclonal antibodies targeting the conserved fragment of Hexon protein for broadly detecting different serotypes of human adenovirus) Dear Dr. Chenguang Shen:

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