Bridging Gaps in Antibody Responses and Animal Welfare: Assessing Blood Collection Methods and Vaginal Immunity in Mice Immunized with Intranasal  Gonococcal Vaccines

Bridging Gaps in Antibody Responses and Animal Welfare: Assessing Blood Collection Methods and Vaginal Immunity in Mice Immunized with Intranasal Gonococcal Vaccines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bridging Gaps in Antibody Responses and Animal Welfare: Assessing Blood Collection Methods and Vaginal Immunity in Mice Immunized with Intranasal Gonococcal Vaccines Abhishek Chanda, Yujuan Song, Junaid Nazir, Chenwei Lin, Alicia Cheng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6241509/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Assessing antibody titers and functional responses is essential for evaluating vaccine efficacy, yet the impact of blood collection methods on these immunological assessments remains unclear. Retro-orbital (RO) blood collection is commonly used but significant complications can occur. Increasingly, investigators have adopted alternative blood collection approaches, such as saphenous vein (SV) sampling to improve laboratory animal welfare. This study compared RO and SV sampling in the development of a Neisseria gonorrhoeae (Ng) vaccine, evaluating Adhesin Complex Protein (ACP) and multiple transferable resistance (Mtr) E protein (MtrE) as antigen candidates. Epitope mapping revealed that ACP and MtrE possess multiple, highly accessible B-cell and T-cell epitope clusters, reinforcing their immunological potential. Following intranasal immunization with rACP, rACP+CpG, and rMtrE+CpG, we assessed the specificity, magnitude, kinetics, and functional quality of immune responses elicited by the immunization regimens. Out of 45 comparisons, only eight significant differences were detected in antibody titers, while the human serum bactericidal assays revealed no differences between RO and SV in antigen-immunized groups. However, antibodies elicited by rACP alone or ACP+CpG in SV samples restored 30.05% and 75.2% of human lysozyme hydrolytic activity compared to 19.3 and 59.9 % in RO, respectively suggesting that SV sampling may be more reliable for assessing functional antibody responses. Beyond its immunological advantages, SV sampling reduces stress, minimizes ocular trauma, and improves animal welfare, making it a viable alternative to RO collection. Given its widespread use in vaccine research, standardizing SV sampling could improve data reliability, ethical compliance, and translational relevance in preclinical studies. Biological sciences/Biological techniques Biological sciences/Computational biology and bioinformatics Biological sciences/Immunology Biological sciences/Microbiology retroorbital bleeding saphenous vein blood collection gonorrhea Neisseria gonorrhoeae MtrE ACP mice immunization epitope prediction ELISA immunoblotting serum bactericidal assay lysozyme activity functional blocking antibodies Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryInforSRComp.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 31 Oct, 2025 Reviews received at journal 04 Oct, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviews received at journal 23 Apr, 2025 Reviewers agreed at journal 21 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Editor assigned by journal 26 Mar, 2025 Editor invited by journal 26 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 17 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6241509","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":443511463,"identity":"23fcd126-e84b-4922-8472-42da5ca5f726","order_by":0,"name":"Abhishek Chanda","email":"","orcid":"","institution":"Oregon State University","correspondingAuthor":false,"prefix":"","firstName":"Abhishek","middleName":"","lastName":"Chanda","suffix":""},{"id":443511467,"identity":"e2637f2c-f646-47bf-9990-ffb8f6dc6f12","order_by":1,"name":"Yujuan Song","email":"","orcid":"","institution":"Oregon State University","correspondingAuthor":false,"prefix":"","firstName":"Yujuan","middleName":"","lastName":"Song","suffix":""},{"id":443511468,"identity":"dd6ae71c-f292-45b7-ac72-ce0405037d05","order_by":2,"name":"Junaid Nazir","email":"","orcid":"","institution":"Lovely Professional University","correspondingAuthor":false,"prefix":"","firstName":"Junaid","middleName":"","lastName":"Nazir","suffix":""},{"id":443511469,"identity":"2530744a-d4c2-4559-ba7f-7fface8ffa5d","order_by":3,"name":"Chenwei Lin","email":"","orcid":"","institution":"Fred Hutchinson Cancer Research Center","correspondingAuthor":false,"prefix":"","firstName":"Chenwei","middleName":"","lastName":"Lin","suffix":""},{"id":443511470,"identity":"1891d210-76bc-42cb-a23e-e4d9260ce7ed","order_by":4,"name":"Alicia Cheng","email":"","orcid":"","institution":"Oregon State University","correspondingAuthor":false,"prefix":"","firstName":"Alicia","middleName":"","lastName":"Cheng","suffix":""},{"id":443511471,"identity":"29ec2ebe-5720-469d-b85f-a25052464dd6","order_by":5,"name":"Jennifer Sargent","email":"","orcid":"","institution":"Oregon State University","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Sargent","suffix":""},{"id":443511472,"identity":"b8dd73d6-fa52-424c-9213-0266f808e2a3","order_by":6,"name":"Aleksandra E. 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Retro-orbital (RO) blood collection is commonly used but significant complications can occur. Increasingly, investigators have adopted alternative blood collection approaches, such as saphenous vein (SV) sampling to improve laboratory animal welfare. This study compared RO and SV sampling in the development of a Neisseria gonorrhoeae (Ng) vaccine, evaluating Adhesin Complex Protein (ACP) and multiple transferable resistance (Mtr) E protein (MtrE) as antigen candidates. Epitope mapping revealed that ACP and MtrE possess multiple, highly accessible B-cell and T-cell epitope clusters, reinforcing their immunological potential.\nFollowing intranasal immunization with rACP, rACP+CpG, and rMtrE+CpG, we assessed the specificity, magnitude, kinetics, and functional quality of immune responses elicited by the immunization regimens. Out of 45 comparisons, only eight significant differences were detected in antibody titers, while the human serum bactericidal assays revealed no differences between RO and SV in antigen-immunized groups. However, antibodies elicited by rACP alone or ACP+CpG in SV samples restored 30.05% and 75.2% of human lysozyme hydrolytic activity compared to 19.3 and 59.9 % in RO, respectively suggesting that SV sampling may be more reliable for assessing functional antibody responses.\nBeyond its immunological advantages, SV sampling reduces stress, minimizes ocular trauma, and improves animal welfare, making it a viable alternative to RO collection. Given its widespread use in vaccine research, standardizing SV sampling could improve data reliability, ethical compliance, and translational relevance in preclinical studies.","manuscriptTitle":"Bridging Gaps in Antibody Responses and Animal Welfare: Assessing Blood Collection Methods and Vaginal Immunity in Mice Immunized with Intranasal Gonococcal Vaccines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 19:07:02","doi":"10.21203/rs.3.rs-6241509/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-31T08:32:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-04T17:29:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108522947167798452881593062858205654081","date":"2025-09-12T16:32:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-23T14:20:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316284478024546947649835396261735241214","date":"2025-04-21T16:03:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29998058369708433633887110984058785079","date":"2025-04-01T21:07:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T09:59:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-26T11:54:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-26T08:17:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-25T11:22:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-17T06:20:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"37f699f9-488c-4293-b0ad-7f3d93b7ff3a","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47219409,"name":"Biological sciences/Biological techniques"},{"id":47219410,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":47219411,"name":"Biological sciences/Immunology"},{"id":47219412,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-23T16:09:19+00:00","versionOfRecord":{"articleIdentity":"rs-6241509","link":"https://doi.org/10.1038/s41598-026-44505-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-17 15:59:09","publishedOnDateReadable":"March 17th, 2026"},"versionCreatedAt":"2025-04-21 19:07:02","video":"","vorDoi":"10.1038/s41598-026-44505-5","vorDoiUrl":"https://doi.org/10.1038/s41598-026-44505-5","workflowStages":[]},"version":"v1","identity":"rs-6241509","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6241509","identity":"rs-6241509","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}