IgG antibodies anti-LcrV of Yersinia pestis: inconsistent responses in confirmed plague patients from Madagascar

IgG antibodies anti-LcrV of Yersinia pestis: inconsistent responses in confirmed plague patients from Madagascar | 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 Short Report IgG antibodies anti-LcrV of Yersinia pestis: inconsistent responses in confirmed plague patients from Madagascar Olifara Herinirina Andriatefy, Anjanirina Rahantamalala, Matthieu Schoenhals, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6222105/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in BMC Research Notes → Version 1 posted 11 You are reading this latest preprint version Abstract Objective: Plague ( Yersinia pestis infection) is a flea-borne zoonotic disease mainly affecting African countries, with most human cases reported from Madagascar and the Democratic Republic of Congo. Although curable, plague can be fatal without prompt treatment, highlighting the importance of reliable diagnostics. Current tests include bacteriological culture, PCR, and anti-F1 ELISA, however the anti-F1 serology has limitations due to the existence of F1-negative virulent strains. To address this, we developed a serological test detecting IgG antibodies against LcrV or V antigen, the main virulence factor of Y. pestis and further evaluated the developed test on clinical samples. Results: V antigen was produced from the culture of V-pGEX-6P-2 clones and purified as GST-LcrV, which was functional for ELISA plate coating. The developed anti-V ELISA showed 60% sensitivity and 93.3% specificity when tested on confirmed plague patients’ serum samples from Madagascar. However, evaluation on convalescent sera collected from Day 1 to Month 3 post-infection revealed inconsistent anti-V antibody production. This suggests the anti-V ELISA is best used as a complementary test for plague diagnosis and during outbreak investigations. In addition, this study provides valuable insight into the humoral response diversity following Y. pestis infection, representing crucial information for plague vaccine development. plague LcrV ELISA antibody Yersinia pestis Madagascar Figures Figure 1 Figure 2 Introduction Plague is a flea-borne zoonosis caused by the gram-negative bacterium Yersinia pestis , still endemic in some parts of the world but mainly Africa. The World Health Organization has classified plague as a re-emerging disease since the 1990s. Between 2013 and 2018, over 90% of global human plague cases were reported from Madagascar and the Democratic Republic of Congo, accounting for 95% of plague-related deaths worldwide [ 1 ]. Although curable by antimicrobial therapy, plague can be fatal without prompt treatment, emphasizing the need for reliable diagnostics, especially in Madagascar, where about 40% of the population is exposed to Y. pestis [ 2 ]. Several diagnostic tools are available for plague diagnosis including rapid diagnostic tests [ 3 ], bacteriology [ 4 ], molecular biology [ 5 ], and serology [ 6 ]. Since 2021, Y. pestis strain isolation by culture or a qPCR positivity for at least two targeted genes is confirmatory for plague [ 7 ]. However, bacteriology is time-consuming and can be hampered by sample quality or prior antibiotic use, while molecular tools require well-trained staff and expensive equipment often unavailable in resource-limited countries. An alternative confirmatory test is the enzyme-linked immunosorbent assay (ELISA) showing a four-fold rise in anti-F1 antibody titers in paired sera [ 7 ]. Yet, this test has limitations: F1 is not essential for Y. pestis virulence, and rare F1-negative strains from confirmed plague cases have already been reported [ 8 , 9 ]. Sera from individuals infected with such strains would not be diagnosed by anti-F1 serology. To address this, detecting antibodies against other Y. pestis markers is necessary. The low-calcium response V-antigen (LcrV or V antigen) is a promising candidate, as it is a key protective antigen required for bubonic and pneumonic plague pathogenesis [ 10 ]. While most anti-V ELISAs have been used for antibody follow-up after experimental vaccination in mice [ 11 , 12 ], here we developed an anti-V ELISA for IgG detection and evaluated the assay in serum samples from confirmed plague patients in Madagascar. Materials and methods LcrV production and visualization Recombinant V antigen was produced from Escherichia coli BL21 clones containing plasmids, which were formed by ligation of the gene encoding LcrV in the pGEX-6-P2 vector [ 13 ]. E. coli clones were kindly provided by the Defence Science and Technology Laboratory (DSTL), Porton Down, UK. After induction of gene expression, proteins were released using different methods to disrupt the bacterial cell wall: enzymatic, mechanical, and thermal. For enzymatic disruption, bacterial pellets were resuspended in PBS with 0.05% DNase (Sigma, 11284932001) and lysozyme (Sigma, 9001-63-2). For thermal disruption, bacterial pellets were washed, resuspended in a lysis buffer containing 1.5M urea (Sigma, U5128), 1% protease inhibitor (Sigma, 539131), 50mM β2-mercaptoethanol (Sigma, M3148), 10% Triton (BioXtra, T9284), and 0.05% DNase, then subjected to freeze-thaw cycles. For mechanical disruption, bacterial pellets were resuspended in the same lysis buffer and homogenized using a dounce homogenizer. Proteins were separated from cellular debris by centrifugation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein solutions were purified on columns specific to the tag (Glutathione-S-transferase or GST) fused with LcrV as previously described [ 13 ], allowing the elimination of other proteins and releasing either the tagged or untagged LcrV antigen depending on the use of the protease lysing the GST-V fusion protein. A LcrV-specific monoclonal antibody, termed MAb 7.3, and reported to protect mice against a fully virulent strain of Y. pestis [ 14 ] was used for the detection of LcrV after protein purification. Anti-V IgG ELISA Detection of anti-V IgG was conducted by ELISA as previously described [ 11 , 12 ] with few modifications. Briefly, microwells were coated overnight at 4°C with 5 µg/ml of LcrV diluted in PBS. After washing with PBS containing 0.02% Tween 20, plates were blocked with 1% PBS-skimmed milk for 1 hour at 37°C and washed again. Patients’ sera diluted 1:1 in 1% PBS-skimmed milk were added in duplicate and incubated for 1 hour. After washing, a secondary antibody coupled with an enzyme HRP diluted at 1:20000 (Sigma-Aldrich, A8419) was added for 1 hour at 37°C and detected after addition of a chromogenic substrate ABTS (KPL). Results were determined by reading the optical density (OD) at 405 nm using an ELx800 spectrophotometer (Biotek, Winooski, VT) after 20 min of incubation. The positive control was a serum sample from a Malagasy confirmed plague case (with Y. pestis strain) collected at Month 12 after the onset of the disease. Negative controls were Malagasy human sera samples without a history plague infection (n = 3). The threshold was predetermined by Receiver Operating Characteristic (ROC) curve analysis using sera from plague-confirmed and non-plague patients, with the best likelihood ratio of 15.38 (data not shown). A sample was considered positive when the ratio (net OD/ mean three negative controls + 3SD) ≥ 2. Sensitivity, specificity and evaluation of the anti-V ELISA The sensitivity of the anti-V ELISA was assessed by testing 30 sera samples from Malagasy confirmed plague patients (positive on PCR, bacteriology and with anti-F1 seroconversion). These sera were collected between Day 21 and Month 3 after clinical symptoms’ onset. The specificity was determined with 30 sera from individuals living in a non- endemic plague area of Madagascar (Taolagnaro District). These samples tested negative for anti- F1 IgGs. The developed anti-V ELISA was evaluated on a total of 63 sera samples collected from 17 plague patients at different time points from the disease onset (Day 1, Day 14, Day 21, Month 3). These patients presented a bubonic form and originated mostly from Ambositra and Manandriana Districts located in the main plague focus in the central highlands of Madagascar. Among the 17 patients, 8 were males and 9 females, with an age range of 4–58 years (median age 12 years). The patients described here present a wider range of outcomes across various plague diagnostic tests (12 sera positive on PCR, bacteriology and anti-F1 serology; 4 sera positive on PCR and anti-F1 serology; 1 serum positive on anti-F1 serology only) compared to those tested in the sensitivity test. This sera assessment also allowed the follow-up of anti-V IgG antibodies kinetics. Statistical analyses Specificity and sensitivity of anti-V IgG ELISA were calculated with 95% confidence intervals using R 3.6.2 [ 15 ]. The kappa coefficient (κ) was also calculated to assess the level of agreement between the index test (anti-V IgG ELISA) and the reference test (anti-F1 IgG ELISA) and interpreted as follows: poor agreement (< 0), slight agreement (0.01–0.20), fair agreement (0.21–0.40), moderate agreement (0.41–0.60), substantial agreement (0.61–0.80), and almost perfect agreement (0.81–1.00). Results LcrV production Visualization of the protein produced with the three different extraction methods, by SDS-PAGE revealed that the thermal method yielded better results than the enzymatic method advised for protein release (Fig. 1 a). In addition, protein purification using SDS-PAGE and following Ponceau staining showed the presence of GST-LcrV at the expected size of 63 kDa (Fig. 1 b). Untagging LcrV using protease digestion was unsuccessful in our hands (without GST) however, it was recognized by the LcrV-specific monoclonal antibody MAb 7.3. Results were consistent with those obtained with the reference untagged V antigen (DSTL, UK) used at the same concentration (data not shown). Therefore, a V antigen still fused to GST was used throughout the study. Characteristics and application of the anti-V IgG ELISA The sensitivity of anti-V ELISA for plague was 60% (95% CI: 42.3%-75.4%). Of the 30 sera collected from individuals living in plague-free area, 28 were both negative on anti-F1 and anti-V IgG ELISA resulting in a specificity of 93.3% (95% CI: 78.7%-98.8%) (Table 1 ). A kappa coefficient of 0.53 (95% CI: 0.3–0.7) was obtained (i.e., moderate agreement). Table 1 Sensitivity and specificity test results of anti-V ELISA for plague diagnosis . Anti-F1 positivity refers to confirmed plague cases (n = 39) and the negative to non-plague patients (n = 30). Anti-F1 ELISA Positive Negative Total Anti-V ELISA Positive 9 2 11 Negative 30 28 58 Total 39 30 69 Among the 63 sera samples from 17 plague patients at different follow-up times, 41 (65.0%) were positive for anti-F1 IgG whereas only 15 (23.8%) were anti-V IgG positive showing that 10/17 patients (58.8%) did not develop detectable antibodies against LcrV during their infection. For sera positive in anti-V IgG, kinetics of anti-V antibodies showed that they increased gradually from Day 1 to Day 14, reached a peak at Day 21 and then decreased progressively but still detectable until Month 3 for 41.2% of them (Fig. 2 a). This trend is similar to anti-F1 IgG kinetics observed in these same patients. However, unlike anti-V IgG, 100% of them still have anti-F1 antibodies at Month 3 after symptoms onset (Fig. 2 b). Discussion We developed an anti-V IgG ELISA using bacterial clones producing the V antigen and further evaluated on suspected plague serum samples from Madagascar, the country most affected by plague. The V antigen used was a tag-fused protein, however previous studies confirmed that its functionality in anti-V ELISA remains intact [ 16 , 17 ]. The GST tag did not interfere with antibody recognition. Compared to plague confirmation tests (qPCR and/or culture), the anti-V ELISA had a sensitivity of 60% (95% CI: 42.3%-75.4%) and specificity of 93.3% (95% CI: 78.7%-98.8%). The kappa coefficient of 0.53 (95% CI: 0.3%-0.7%) shows a moderate agreement between the anti-V ELISA and the positivity to both PCR and bacteriological culture. This low sensitivity indicates 40% risk of the anti-V ELISA to misidentify confirmed plague patients (to give a positive result) while the high specificity supports its reliability in identifying non-plague patients. Although not relevant for plague diagnosis, this anti-V ELISA could still be used for V antigen humoral response assessment. In this study, the evaluation of 17 bubonic plague patients’ sera samples using the developed anti-V ELISA showed that anti-V IgGs were not detected in more than half of these patients. This observation has already been reported in a previous study using a protein microarray technique where 5 out of 7 sera samples from plague patients did not show any detectable anti-V antibodies [ 18 ]. This was also the case for human donors immunized with the live plague vaccine (LPV). Indeed, only 20.6 % o these individuals produced antibodies targeting LcrV [ 19 ]. This positivity rate of anti-V antibodies has even been shown to fall to zero in mice vaccinated with a Y. pseudotuberculosis containing F1 antigen-based vaccine [ 20 ]. Altogether, these results could indicate that in plague immunity, the role of LcrV antigen may be cellular rather than humoral. A T-response-stimulating epitopes within LcrV has already been identified [ 21 ]. The location of the V antigen could be one explanation. Unlike F1, LcrV is predominantly intracellular although it is expressed on the tip of the injectisome during infection and therefore may not promote a humoral response because inaccessible to B cell receptors. However, although anti-LcrV antibodies are not always detectable, this antigen is known to be a major asset in protection against plague, as demonstrated in a recent study [ 22 ]. While most studies on anti-V immune response have been carried out in mice in the context of a plague vaccine development, our study is the first to report the humoral immune response against V antigen within individuals living in a plague-endemic area and where the same biovar of Y. pestis (biovar Orientalis ) has circulated since its introduction. A similar study conducted by Bei Li and colleagues [ 18 ] suggested that the diversity of humoral responses toward LcrV was associated with patients coming from different regions and infected by different biovars of Y. pestis (biovar Antiqua and Orientalis). In conclusion, the IgG anti-V ELISA developed in this study can be used as a complimentary test to anti-F1 ELISA and serve as a mean to investigate humoral responses in plague patients (gain insight into the diversity of the humoral response following Y. pestis infection). It may also help estimate the prevalence of other pathogenic Yersinioses ( Y. enterocolitica , Y. pseudotuberculosis ) since LcrV is a shared antigen among these species. A positive anti-LcrV result in plague-negative samples could indicate infection by these species. Further research is needed to understand the protective role of anti-V and anti-F1 antibodies in plague immunity. Limitations The main limitation of our study could be the unavailability of known human positive controls for IgG anti-V at the beginning of the anti-V ELISA development. They were only identified after the process, possibly affecting assay sensitivity. In addition, the ROC curve was established using plague-confirmed samples as positives, not anti-V positive samples, which may have biased sensitivity and specificity estimates. Abbreviations DSTL: Defence Science and Technology Laboratory; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; GST: Glutathione-S-transferase; OD: Optical density; IgG: Immunoglobulin G; ELISA: enzyme-linked immunosorbent assay; ROC: Receiver Operating Characteristic Declarations Ethics approval and consent to participate The use of human serum samples in this study was approved by the Ethics Committee for Biomedical Research of the Malagasy Ministry of Public Health (N° 086 and 149 MSANP/SG/AMM/CERBM on July 27, 2021, and October 21, 2021, respectively). Informed consent was obtained from each participant or legal guardian. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request Competing interest All authors declare that they have no competing interest. Funding This work was supported in part by the BactiVac Catalyst Project BVNCP5-02, 2021 and the Plague Unit - IPM (PA-14.71) Authors’ contributions OHA contributed to the acquisition, analysis and interpretation of data, and drafted the original manuscript. AR contributed to the design of the work, analysis and interpretation of data, and reviewed the manuscript. MS contributed to the interpretation of data and reviewed the manuscript. RS contributed to the funding acquisition and reviewed the manuscript NJW contributed to the resources and reviewed the manuscript EDW contributed to the resources and reviewed the manuscript. MR contributed to the design of the work, funding acquisition, acquisition, analysis and interpretation of data and reviewed the manuscript. VA contributed to the conception and design of the work, acquisition, analysis and interpretation of data and reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgments We are grateful to the Central Laboratory for Plague of the Ministry of Public Health of Madagascar and the Infectious Diseases Immunology Unit of the Pasteur Institute of Madagascar for providing samples used in this study. We also would like to recognize Mahenintsoa Rakotondrazaka and Solohery Lalaina Razafimahatratra for their technical assistance. References World Health Organization (WHO). Plague around the world in 2019. Wkly. epidemiol. rec. 2019;94:289-292. Chanteau S, Ratsitorahina M, Rahalison L, Rasoamanana B, Chan F, Boisier P, et al. Current epidemiology of human plague in Madagascar. Microbes Infect. 2000;2:25–31. https://doi.org/ 10.1016/S1286-4579(00)00289-6. Chanteau S, Rahalison L, Ratsitorahina M, Mahafaly, Rasolomaharo M, Boisier P, et al. Early diagnosis of bubonic plague using F1 antigen capture ELISA assay and rapid immunogold dipstick. Int J Med Microbiol. 2000;290:279–83. https://doi.org/ 10.1016/S1438-4221(00)80126-5. 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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-6222105","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":430085161,"identity":"66a0b0e7-bff7-435c-958e-f19eb3babc47","order_by":0,"name":"Olifara Herinirina Andriatefy","email":"","orcid":"","institution":"Institut Pasteur de Madagascar","correspondingAuthor":false,"prefix":"","firstName":"Olifara","middleName":"Herinirina","lastName":"Andriatefy","suffix":""},{"id":430085162,"identity":"53a6e6d5-1263-41a0-8641-ff9d89893fb0","order_by":1,"name":"Anjanirina Rahantamalala","email":"","orcid":"","institution":"Institut Pasteur de Madagascar","correspondingAuthor":false,"prefix":"","firstName":"Anjanirina","middleName":"","lastName":"Rahantamalala","suffix":""},{"id":430085164,"identity":"46240946-e07a-426e-bcf6-a1731034723b","order_by":2,"name":"Matthieu Schoenhals","email":"","orcid":"","institution":"Institut Pasteur de Madagascar","correspondingAuthor":false,"prefix":"","firstName":"Matthieu","middleName":"","lastName":"Schoenhals","suffix":""},{"id":430085166,"identity":"678143ae-a91b-43df-bccc-d58ee40f4bb7","order_by":3,"name":"Robin Shattock","email":"","orcid":"","institution":"Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Robin","middleName":"","lastName":"Shattock","suffix":""},{"id":430085168,"identity":"c91e925a-bacd-42d5-b22d-7721bf0f3b5b","order_by":4,"name":"Nicola J. Walker","email":"","orcid":"","institution":"Defence Science and Technology Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Nicola","middleName":"J.","lastName":"Walker","suffix":""},{"id":430085169,"identity":"840ec3e9-e8ff-46a4-b9d8-f96503579937","order_by":5,"name":"Ethel Diane Williamson","email":"","orcid":"","institution":"Defence Science and Technology Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Ethel","middleName":"Diane","lastName":"Williamson","suffix":""},{"id":430085171,"identity":"f57524f7-e9b8-45eb-85c8-6d62e93d27fa","order_by":6,"name":"Minoarisoa Rajerison","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYDACZiD+QLIWxhlAmoc0XTwkadFt5z342HaHnb09e4/ZB8acWsJazA7zJRvnnklO7OE5YzyDcdtxYrTwmEnntjEn8EjkGDMwbjtGpBbLtnp7Hvk3pGhhbDvM2CPBA9JSQ5QWY8PeM8cTe86kFTMkbjtAhJbzZwwf/NxRbc/efngzw8dtdYS1gAFjA5SRwHCYVC0MDMTaMgpGwSgYBSMJAACqqjL/YbzJBQAAAABJRU5ErkJggg==","orcid":"","institution":"Institut Pasteur de Madagascar","correspondingAuthor":true,"prefix":"","firstName":"Minoarisoa","middleName":"","lastName":"Rajerison","suffix":""},{"id":430085172,"identity":"de963f3f-e2cd-4404-b5f5-8146c8bae866","order_by":7,"name":"Voahangy Andrianaivoarimanana","email":"","orcid":"","institution":"Institut Pasteur de Madagascar","correspondingAuthor":false,"prefix":"","firstName":"Voahangy","middleName":"","lastName":"Andrianaivoarimanana","suffix":""}],"badges":[],"createdAt":"2025-03-13 17:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6222105/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6222105/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-025-07315-y","type":"published","date":"2025-07-01T15:58:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79217482,"identity":"aaee3149-6c7d-42ba-a7f1-0e8a4686fa78","added_by":"auto","created_at":"2025-03-25 19:08:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLcrV production using SDS-PAGE and Ponceau staining. a: \u003c/strong\u003eVisualization of protein extracts from the 3 extraction techniques (thermal, mechanical and enzymatic), the thermal method yielded more proteins (thicker band) at 63 kDa (V antigen or LcrV).\u003cstrong\u003e b: \u003c/strong\u003eThe Protein purification also showed the presence of GST-LcrV at the expected size of 63 kDa,\u003cstrong\u003e \u003c/strong\u003eM.W.= Molecular Weight\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6222105/v1/3ac88a4c7891e19f33e1af4d.png"},{"id":79216830,"identity":"dd2ed4ac-0cac-459d-a1f7-2bcec1b8f992","added_by":"auto","created_at":"2025-03-25 19:00:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolution of IgG anti-V (a) and anti-F1 (b) days and month after disease onset\u003c/strong\u003e. Only 47,2% of individuals evaluated showed seropositivity towards IgG anti-V using ELISA, whereas 100% had IgG anti-F1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6222105/v1/893f052126117fce809daafa.png"},{"id":86179889,"identity":"067c26cb-83c6-4086-86c1-cc0abfcaee1f","added_by":"auto","created_at":"2025-07-07 16:20:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":810035,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6222105/v1/6687774e-a5f6-4729-92f5-641e0ac43c96.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"IgG antibodies anti-LcrV of Yersinia pestis: inconsistent responses in confirmed plague patients from Madagascar","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlague is a flea-borne zoonosis caused by the gram-negative bacterium \u003cem\u003eYersinia pestis\u003c/em\u003e, still endemic in some parts of the world but mainly Africa. The World Health Organization has classified plague as a re-emerging disease since the 1990s. Between 2013 and 2018, over 90% of global human plague cases were reported from Madagascar and the Democratic Republic of Congo, accounting for 95% of plague-related deaths worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although curable by antimicrobial therapy, plague can be fatal without prompt treatment, emphasizing the need for reliable diagnostics, especially in Madagascar, where about 40% of the population is exposed to \u003cem\u003eY. pestis\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral diagnostic tools are available for plague diagnosis including rapid diagnostic tests [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], bacteriology [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], molecular biology [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and serology [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Since 2021, \u003cem\u003eY. pestis\u003c/em\u003e strain isolation by culture or a qPCR positivity for at least two targeted genes is confirmatory for plague [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, bacteriology is time-consuming and can be hampered by sample quality or prior antibiotic use, while molecular tools require well-trained staff and expensive equipment often unavailable in resource-limited countries.\u003c/p\u003e \u003cp\u003eAn alternative confirmatory test is the enzyme-linked immunosorbent assay (ELISA) showing a four-fold rise in anti-F1 antibody titers in paired sera [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Yet, this test has limitations: F1 is not essential for \u003cem\u003eY. pestis\u003c/em\u003e virulence, and rare F1-negative strains from confirmed plague cases have already been reported [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Sera from individuals infected with such strains would not be diagnosed by anti-F1 serology.\u003c/p\u003e \u003cp\u003eTo address this, detecting antibodies against other \u003cem\u003eY. pestis\u003c/em\u003e markers is necessary. The low-calcium response V-antigen (LcrV or V antigen) is a promising candidate, as it is a key protective antigen required for bubonic and pneumonic plague pathogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While most anti-V ELISAs have been used for antibody follow-up after experimental vaccination in mice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], here we developed an anti-V ELISA for IgG detection and evaluated the assay in serum samples from confirmed plague patients in Madagascar.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLcrV production and visualization\u003c/h2\u003e \u003cp\u003eRecombinant V antigen was produced from \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 clones containing plasmids, which were formed by ligation of the gene encoding LcrV in the pGEX-6-P2 vector [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e clones were kindly provided by the Defence Science and Technology Laboratory (DSTL), Porton Down, UK. After induction of gene expression, proteins were released using different methods to disrupt the bacterial cell wall: enzymatic, mechanical, and thermal. For enzymatic disruption, bacterial pellets were resuspended in PBS with 0.05% DNase (Sigma, 11284932001) and lysozyme (Sigma, 9001-63-2). For thermal disruption, bacterial pellets were washed, resuspended in a lysis buffer containing 1.5M urea (Sigma, U5128), 1% protease inhibitor (Sigma, 539131), 50mM β2-mercaptoethanol (Sigma, M3148), 10% Triton (BioXtra, T9284), and 0.05% DNase, then subjected to freeze-thaw cycles. For mechanical disruption, bacterial pellets were resuspended in the same lysis buffer and homogenized using a dounce homogenizer. Proteins were separated from cellular debris by centrifugation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein solutions were purified on columns specific to the tag (Glutathione-S-transferase or GST) fused with LcrV as previously described [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], allowing the elimination of other proteins and releasing either the tagged or untagged LcrV antigen depending on the use of the protease lysing the GST-V fusion protein. A LcrV-specific monoclonal antibody, termed MAb 7.3, and reported to protect mice against a fully virulent strain of \u003cem\u003eY. pestis\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] was used for the detection of LcrV after protein purification.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnti-V IgG ELISA\u003c/h3\u003e\n\u003cp\u003eDetection of anti-V IgG was conducted by ELISA as previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] with few modifications. Briefly, microwells were coated overnight at 4\u0026deg;C with 5 \u0026micro;g/ml of LcrV diluted in PBS. After washing with PBS containing 0.02% Tween 20, plates were blocked with 1% PBS-skimmed milk for 1 hour at 37\u0026deg;C and washed again. Patients\u0026rsquo; sera diluted 1:1 in 1% PBS-skimmed milk were added in duplicate and incubated for 1 hour. After washing, a secondary antibody coupled with an enzyme HRP diluted at 1:20000 (Sigma-Aldrich, A8419) was added for 1 hour at 37\u0026deg;C and detected after addition of a chromogenic substrate ABTS (KPL). Results were determined by reading the optical density (OD) at 405 nm using an ELx800 spectrophotometer (Biotek, Winooski, VT) after 20 min of incubation. The positive control was a serum sample from a Malagasy confirmed plague case (with \u003cem\u003eY. pestis\u003c/em\u003e strain) collected at Month 12 after the onset of the disease. Negative controls were Malagasy human sera samples without a history plague infection (n\u0026thinsp;=\u0026thinsp;3). The threshold was predetermined by Receiver Operating Characteristic (ROC) curve analysis using sera from plague-confirmed and non-plague patients, with the best likelihood ratio of 15.38 (data not shown). A sample was considered positive when the ratio (net OD/ mean three negative controls\u0026thinsp;+\u0026thinsp;3SD)\u0026thinsp;\u0026ge;\u0026thinsp;2.\u003c/p\u003e\n\u003ch3\u003eSensitivity, specificity and evaluation of the anti-V ELISA\u003c/h3\u003e\n\u003cp\u003eThe sensitivity of the anti-V ELISA was assessed by testing 30 sera samples from Malagasy confirmed plague patients (positive on PCR, bacteriology and with anti-F1 seroconversion). These sera were collected between Day 21 and Month 3 after clinical symptoms\u0026rsquo; onset.\u003c/p\u003e \u003cp\u003eThe specificity was determined with 30 sera from individuals living in a non- endemic plague area of Madagascar (Taolagnaro District). These samples tested negative for anti- F1 IgGs.\u003c/p\u003e \u003cp\u003eThe developed anti-V ELISA was evaluated on a total of 63 sera samples collected from 17 plague patients at different time points from the disease onset (Day 1, Day 14, Day 21, Month 3). These patients presented a bubonic form and originated mostly from Ambositra and Manandriana Districts located in the main plague focus in the central highlands of Madagascar. Among the 17 patients, 8 were males and 9 females, with an age range of 4\u0026ndash;58 years (median age 12 years). The patients described here present a wider range of outcomes across various plague diagnostic tests (12 sera positive on PCR, bacteriology and anti-F1 serology; 4 sera positive on PCR and anti-F1 serology; 1 serum positive on anti-F1 serology only) compared to those tested in the sensitivity test. This sera assessment also allowed the follow-up of anti-V IgG antibodies kinetics.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eSpecificity and sensitivity of anti-V IgG ELISA were calculated with 95% confidence intervals using R 3.6.2 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The kappa coefficient (κ) was also calculated to assess the level of agreement between the index test (anti-V IgG ELISA) and the reference test (anti-F1 IgG ELISA) and interpreted as follows: poor agreement (\u0026lt;\u0026thinsp;0), slight agreement (0.01\u0026ndash;0.20), fair agreement (0.21\u0026ndash;0.40), moderate agreement (0.41\u0026ndash;0.60), substantial agreement (0.61\u0026ndash;0.80), and almost perfect agreement (0.81\u0026ndash;1.00).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eLcrV production\u003c/h2\u003e \u003cp\u003eVisualization of the protein produced with the three different extraction methods, by SDS-PAGE revealed that the thermal method yielded better results than the enzymatic method advised for protein release (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In addition, protein purification using SDS-PAGE and following Ponceau staining showed the presence of GST-LcrV at the expected size of 63 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUntagging LcrV using protease digestion was unsuccessful in our hands (without GST) however, it was recognized by the LcrV-specific monoclonal antibody MAb 7.3. Results were consistent with those obtained with the reference untagged V antigen (DSTL, UK) used at the same concentration (data not shown).\u003c/p\u003e \u003cp\u003eTherefore, a V antigen still fused to GST was used throughout the study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCharacteristics and application of the anti-V IgG ELISA\u003c/h3\u003e\n\u003cp\u003eThe sensitivity of anti-V ELISA for plague was 60% (95% CI: 42.3%-75.4%). Of the 30 sera collected from individuals living in plague-free area, 28 were both negative on anti-F1 and anti-V IgG ELISA resulting in a specificity of 93.3% (95% CI: 78.7%-98.8%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A kappa coefficient of 0.53 (95% CI: 0.3\u0026ndash;0.7) was obtained (i.e., moderate agreement).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSensitivity and specificity test results of anti-V ELISA for plague diagnosis\u003c/b\u003e. Anti-F1 positivity refers to confirmed plague cases (n\u0026thinsp;=\u0026thinsp;39) and the negative to non-plague patients (n\u0026thinsp;=\u0026thinsp;30).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eAnti-F1 ELISA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eAnti-V ELISA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePositive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNegative\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAmong the 63 sera samples from 17 plague patients at different follow-up times, 41 (65.0%) were positive for anti-F1 IgG whereas only 15 (23.8%) were anti-V IgG positive showing that 10/17 patients (58.8%) did not develop detectable antibodies against LcrV during their infection.\u003c/p\u003e \u003cp\u003eFor sera positive in anti-V IgG, kinetics of anti-V antibodies showed that they increased gradually from Day 1 to Day 14, reached a peak at Day 21 and then decreased progressively but still detectable until Month 3 for 41.2% of them (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This trend is similar to anti-F1 IgG kinetics observed in these same patients. However, unlike anti-V IgG, 100% of them still have anti-F1 antibodies at Month 3 after symptoms onset (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe developed an anti-V IgG ELISA using bacterial clones producing the V antigen and further evaluated on suspected plague serum samples from Madagascar, the country most affected by plague. The V antigen used was a tag-fused protein, however previous studies confirmed that its functionality in anti-V ELISA remains intact [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The GST tag did not interfere with antibody recognition. Compared to plague confirmation tests (qPCR and/or culture), the anti-V ELISA had a sensitivity of 60% (95% CI: 42.3%-75.4%) and specificity of 93.3% (95% CI: 78.7%-98.8%). The kappa coefficient of 0.53 (95% CI: 0.3%-0.7%) shows a moderate agreement between the anti-V ELISA and the positivity to both PCR and bacteriological culture. This low sensitivity indicates 40% risk of the anti-V ELISA to misidentify confirmed plague patients (to give a positive result) while the high specificity supports its reliability in identifying non-plague patients. Although not relevant for plague diagnosis, this anti-V ELISA could still be used for V antigen humoral response assessment.\u003c/p\u003e \u003cp\u003eIn this study, the evaluation of 17 bubonic plague patients\u0026rsquo; sera samples using the developed anti-V ELISA showed that anti-V IgGs were not detected in more than half of these patients. This observation has already been reported in a previous study using a protein microarray technique where 5 out of 7 sera samples from plague patients did not show any detectable anti-V antibodies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This was also the case for human donors immunized with the live plague vaccine (LPV). Indeed, only 20.6 % o these individuals produced antibodies targeting LcrV [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This positivity rate of anti-V antibodies has even been shown to fall to zero in mice vaccinated with a \u003cem\u003eY. pseudotuberculosis\u003c/em\u003e containing F1 antigen-based vaccine [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Altogether, these results could indicate that in plague immunity, the role of LcrV antigen may be cellular rather than humoral. A T-response-stimulating epitopes within LcrV has already been identified [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The location of the V antigen could be one explanation. Unlike F1, LcrV is predominantly intracellular although it is expressed on the tip of the injectisome during infection and therefore may not promote a humoral response because inaccessible to B cell receptors. However, although anti-LcrV antibodies are not always detectable, this antigen is known to be a major asset in protection against plague, as demonstrated in a recent study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile most studies on anti-V immune response have been carried out in mice in the context of a plague vaccine development, our study is the first to report the humoral immune response against V antigen within individuals living in a plague-endemic area and where the same biovar of \u003cem\u003eY. pestis\u003c/em\u003e (biovar \u003cem\u003eOrientalis\u003c/em\u003e) has circulated since its introduction. A similar study conducted by Bei Li and colleagues [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] suggested that the diversity of humoral responses toward LcrV was associated with patients coming from different regions and infected by different biovars of \u003cem\u003eY. pestis\u003c/em\u003e (biovar \u003cem\u003eAntiqua\u003c/em\u003e and \u003cem\u003eOrientalis).\u003c/em\u003e\u003c/p\u003e \u003cp\u003eIn conclusion, the IgG anti-V ELISA developed in this study can be used as a complimentary test to anti-F1 ELISA and serve as a mean to investigate humoral responses in plague patients (gain insight into the diversity of the humoral response following \u003cem\u003eY. pestis\u003c/em\u003e infection). It may also help estimate the prevalence of other pathogenic Yersinioses (\u003cem\u003eY. enterocolitica\u003c/em\u003e, \u003cem\u003eY. pseudotuberculosis\u003c/em\u003e) since LcrV is a shared antigen among these species. A positive anti-LcrV result in plague-negative samples could indicate infection by these species. Further research is needed to understand the protective role of anti-V and anti-F1 antibodies in plague immunity.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThe main limitation of our study could be the unavailability of known human positive controls for IgG anti-V at the beginning of the anti-V ELISA development. They were only identified after the process, possibly affecting assay sensitivity. In addition, the ROC curve was established using plague-confirmed samples as positives, not anti-V positive samples, which may have biased sensitivity and specificity estimates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDSTL: Defence Science and Technology Laboratory; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; GST: Glutathione-S-transferase; OD: Optical density; IgG: Immunoglobulin G; ELISA: enzyme-linked immunosorbent assay; ROC: Receiver Operating Characteristic\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe use of human serum samples in this study was approved by the Ethics Committee for Biomedical Research of the Malagasy Ministry of Public Health (N\u0026deg; 086 and 149 MSANP/SG/AMM/CERBM on July 27, 2021, and October 21, 2021, respectively). Informed consent was obtained from each participant or legal guardian.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by the BactiVac Catalyst Project BVNCP5-02, 2021 and the Plague Unit - IPM (PA-14.71)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOHA contributed to the acquisition, analysis and interpretation of data, and drafted the original manuscript.\u003c/p\u003e\n\u003cp\u003eAR contributed to the design of the work, analysis and interpretation of data, and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eMS contributed to the interpretation of data and reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRS contributed to the funding acquisition and reviewed the manuscript\u003c/p\u003e\n\u003cp\u003eNJW contributed to the resources and reviewed the manuscript\u003c/p\u003e\n\u003cp\u003eEDW contributed to the resources and reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMR contributed to the design of the work, funding acquisition, acquisition, analysis and interpretation of data and reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVA contributed to the conception and design of the work, acquisition, analysis and interpretation of data and reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the Central Laboratory for Plague of the Ministry of Public Health of Madagascar and the Infectious Diseases Immunology Unit of the Pasteur Institute of Madagascar for providing samples used in this study. We also would like to recognize Mahenintsoa Rakotondrazaka and Solohery Lalaina Razafimahatratra for their technical assistance.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization (WHO). Plague around the world in 2019. Wkly. epidemiol. rec. 2019;94:289-292.\u003c/li\u003e\n\u003cli\u003eChanteau S, Ratsitorahina M, Rahalison L, Rasoamanana B, Chan F, Boisier P, et al. Current epidemiology of human plague in Madagascar. Microbes Infect. 2000;2:25\u0026ndash;31. https://doi.org/ 10.1016/S1286-4579(00)00289-6.\u003c/li\u003e\n\u003cli\u003eChanteau S, Rahalison L, Ratsitorahina M, Mahafaly, Rasolomaharo M, Boisier P, et al. Early diagnosis of bubonic plague using F1 antigen capture ELISA assay and rapid immunogold dipstick. Int J Med Microbiol. 2000;290:279\u0026ndash;83. https://doi.org/ 10.1016/S1438-4221(00)80126-5.\u003c/li\u003e\n\u003cli\u003eRasoamanana B, Rahalison L, Raharimanana C, Chanteau S. Comparison of \u003cem\u003eYersinia\u003c/em\u003e CIN agar and mouse inoculation assay for the diagnosis of plague. Trans R Soc Trop Med Hyg. 1996;90:651. https://doi.org/10.1016/S0035-9203(96)90420-4.\u003c/li\u003e\n\u003cli\u003eZhang Y, Wang Z, Wang W, Yu H, Jin M. Applications of polymerase chain reaction‑based methods for the diagnosis of plague (Review). Exp Ther Med. 2022;24. https://doi.org/10.3892/etm.2022.11438.\u003c/li\u003e\n\u003cli\u003eRasoamanana B, Leroy F, Boisier P, Rasolomaharo M, Buchy P, Carniel E, et al. Field evaluation of an immunoglobulin G anti-F1 enzyme-linked immunosorbent assay for serodiagnosis of human plaque in Madagascar. Clin Diagn Lab Immunol. 1997;4:587\u0026ndash;91. https://doi.org/10.1128/cdli.4.5.587-591.1997.\u003c/li\u003e\n\u003cli\u003eWorld Health Organization (WHO). Revision of the international definition of plague cases. Wkly Epidemiol Rec. 2021;96:238\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eAnisimov AP, Lindler LE, Pier GB. Intraspecific Diversity of \u003cem\u003eYersinia pestis\u003c/em\u003e. Clin Microbiol Rev. 2004;17:434\u0026ndash;64. https://doi.org/10.1128/CMR.17.2.434-464.2004.\u003c/li\u003e\n\u003cli\u003eMeka-Mechenko TV. F1-negative natural \u003cem\u003eY. pestis\u003c/em\u003e Strains. Adv Exp Med Biol. 2003;529:379\u0026ndash;81. https://doi.org/10.1007/0-306-48416-1_76.\u003c/li\u003e\n\u003cli\u003eBurrows TW. An antigen determining virulence in \u003cem\u003ePasteurella pestis\u003c/em\u003e. Nature. 1956;177:426\u0026ndash;7. https://doi.org/10.1038/177426b0.\u003c/li\u003e\n\u003cli\u003eWilliamson ED, Stagg AJ, Eley SM, Taylor R, Green M, Jones SM, et al. Kinetics of the immune response to the (F1 + V) vaccine in models of bubonic and pneumonic plague. Vaccine. 2007;25:1142\u0026ndash;8. https://doi.org/10.1016/j.vaccine.2006.09.052.\u003c/li\u003e\n\u003cli\u003eWilliamson ED, Eley SM, Stagg AJ, Green M, Russell P, Titball RW. A sub-unit vaccine elicits IgG in serum, spleen cell cultures and bronchial washings and protects immunized animals against pneumonic plague. Vaccine. 1997;15:1079\u0026ndash;84. https://doi.org/10.1016/S0264-410X(96)00303-9.\u003c/li\u003e\n\u003cli\u003eCarr S, Miller J, Leary SEC, Bennett AM, Ho A, Williamson ED. Expression of a recombinant form of the V antigen of \u003cem\u003eYersinia\u003c/em\u003e pestis, using three different expression systems. Vaccine. 1999;18:153\u0026ndash;9. https://doi.org/10.1016/S0264-410X(99)00214-5.\u003c/li\u003e\n\u003cli\u003eHill J, Leary SEC, Griffin KF, Williamson E, Titball RW. Regions of \u003cem\u003eYersinia\u003c/em\u003e pestis V antigen that contribute to protection against plague identified by passive and active immunization. Infect Immun. 1997;65:4476\u0026ndash;82. https://doi.org/10.1128/iai.65.11.4476-4482.1997.\u003c/li\u003e\n\u003cli\u003eR Studio Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing.2021. https://www.r-project.org/.\u003c/li\u003e\n\u003cli\u003eFlashner Y, Mamroud E, Tidhar A, Ber R, Aftalion M, Gur D, et al. Generation of \u003cem\u003eYersinia pestis\u003c/em\u003e attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates. Infect Immun. 2004;72:908\u0026ndash;15. https://doi.org/10.3390/vaccines11030581.\u003c/li\u003e\n\u003cli\u003eZauberman A, Cohen S, Levy Y, Halperin G, Lazar S, Velan B, et al. Neutralization of \u003cem\u003eYersinia pestis\u003c/em\u003e-mediated macrophage cytotoxicity by anti-LcrV antibodies and its correlation with protective immunity in a mouse model of bubonic plague. Vaccine. 2008;26:1616\u0026ndash;25. https://doi.org/10.1016/j.vaccine.2008.01.033.\u003c/li\u003e\n\u003cli\u003eLi B, Zhou D, Wang Z, Song Z, Wang H, Li M, et al. Antibody profiling in plague patients by protein microarray. Microbes Infect. 2008;10:45\u0026ndash;51. https://doi.org/ 10.1016/j.micinf.2007.10.003.\u003c/li\u003e\n\u003cli\u003eFeodorova VA, Lyapina AM, Khizhnyakova MA, Zaitsev SS, Saltykov YV, Motin VL. \u003cem\u003eYersinia pestis\u003c/em\u003e antigen f1 but not lcrv induced humoral and cellular immune responses in humans immunized with live plague vaccine\u0026mdash;comparison of immunoinformatic and immunological approaches. Vaccines. 2020;8:1\u0026ndash;19. https://doi.org/10.3390/vaccines8040698. \u003c/li\u003e\n\u003cli\u003eDemeure CE, Derbise A, Guillas C, Gerke C, Cauchemez S, Carniel E, et al. Humoral and cellular immune correlates of protection against bubonic plague by a live \u003cem\u003eYersinia pseudotuberculosis\u003c/em\u003e vaccine. Vaccine. 2019;37:123\u0026ndash;9. https://doi.org/10.1016/j.vaccine.2018.11.022. \u003c/li\u003e\n\u003cli\u003eParent MA, Berggren KN, Mullarky IK, Szaba FM, Kummer LW, Adamovicz JJ, et al. \u003cem\u003eYersinia pestis\u003c/em\u003e V protein epitopes recognized by CD4 T cells. Infect Immun. 2005;73:2197\u0026ndash;204. https://doi.org/10.1128/IAI.73.4.2197-2204.2005.\u003c/li\u003e\n\u003cli\u003eAftalion M, Tidhar A, Vagima Y, Gur D, Zauberman A, Holtzman T, et al. Rapid induction of protective immunity against pneumonic plague by \u003cem\u003eYersinia pestis\u003c/em\u003e polymeric F1 and LcrV Antigens. Vaccines. 2023;11:581. https://doi.org/10.3390/vaccines11030581.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"plague, LcrV, ELISA, antibody, Yersinia pestis, Madagascar","lastPublishedDoi":"10.21203/rs.3.rs-6222105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6222105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e Plague (\u003cem\u003eYersinia pestis\u003c/em\u003e infection) is a flea-borne zoonotic disease mainly affecting African countries, with most human cases reported from Madagascar and the Democratic Republic of Congo. Although curable, plague can be fatal without prompt treatment, highlighting the importance of reliable diagnostics. Current tests include bacteriological culture, PCR, and anti-F1 ELISA, however the anti-F1 serology has limitations due to the existence of F1-negative virulent strains. To address this, we developed a serological test detecting IgG antibodies against LcrV or V antigen, the main virulence factor of \u003cem\u003eY. pestis\u003c/em\u003e and further evaluated the developed test on clinical samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e V antigen was produced from the culture of V-pGEX-6P-2 clones and purified as GST-LcrV, which was functional for ELISA plate coating. The developed anti-V ELISA showed 60% sensitivity and 93.3% specificity when tested on confirmed plague patients’ serum samples from Madagascar. However, evaluation on convalescent sera collected from Day 1 to Month 3 post-infection revealed inconsistent anti-V antibody production. This suggests the anti-V ELISA is best used as a complementary test for plague diagnosis and during outbreak investigations. In addition, this study provides valuable insight into the humoral response diversity following \u003cem\u003eY. pestis\u003c/em\u003e infection, representing crucial information for plague vaccine development.\u003c/p\u003e","manuscriptTitle":"IgG antibodies anti-LcrV of Yersinia pestis: inconsistent responses in confirmed plague patients from Madagascar","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-25 19:00:32","doi":"10.21203/rs.3.rs-6222105/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-11T09:54:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-09T14:02:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229316149232895481639225424752057317639","date":"2025-04-04T02:31:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-28T00:29:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156754222060420823067137175156846895925","date":"2025-03-27T15:54:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14243755759263278823726303691730872450","date":"2025-03-27T14:35:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-27T12:26:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-14T12:13:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-14T08:43:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-14T08:41:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-03-13T17:32:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5e99292-d773-4a5a-a680-67cb30bad34d","owner":[],"postedDate":"March 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:12:04+00:00","versionOfRecord":{"articleIdentity":"rs-6222105","link":"https://doi.org/10.1186/s13104-025-07315-y","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2025-07-01 15:58:10","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-03-25 19:00:32","video":"","vorDoi":"10.1186/s13104-025-07315-y","vorDoiUrl":"https://doi.org/10.1186/s13104-025-07315-y","workflowStages":[]},"version":"v1","identity":"rs-6222105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6222105","identity":"rs-6222105","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}