Differential sars-cov-2 antigen specificity of the humoral response in inactivated virus-vaccinated, convalescent, and breakthrough subjects

Analytical methods for the differential determination between natural infection with SARS- CoV-2 vs. immunity elicited by vaccination or infection after immunization (breakthrough cases) represent attractive new research venues in the context of the ongoing COVID-19 pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). Herein, we set out to compare humoral responses against several SARS-CoV-2 structural and non-structural proteins in infected unvaccinated (convalescent), vaccinated, as well as vaccinated and infected (breakthrough) individuals. Our results indicate that immunization with an inactivated SARS-CoV-2 vaccine (CoronaVac) induces significantly higher levels of IgG antibodies against the membrane (M) protein of SARS-CoV-2 as compared to convalescent subjects both, after the primary vaccination schedule and after a booster dose. Moreover, we found that CoronaVac-immunized individuals, after receiving a third vaccine shot, display equivalent levels of N-specific IgG antibodies as convalescents subjects. Regarding non-structural viral proteins, for the two viral proteins ORF3a and NSP8, IgG antibodies were produced in more than 50% of the convalescent subjects. Finally, a logistic regression model and a receiver operating characteristic (ROC) analysis show that combined detection of M and N proteins may be useful as a biomarker to differentiate breakthrough cases from vaccinated and convalescent individuals that did not receive prior vaccination. Taken together, these results suggest that multiple SARS-CoV-2 antigens may be used as differential biomarkers for distinguishing natural infection from vaccination. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT04651790 ### Funding Statement PATH facilitated sera panels donations for this work. The work was also supported by the Bill &; Melinda Gates Foundation () via grants INV-021239, and INV-016821. Under the grant conditions of the foundation, a Creative Commons Attribution 4.0 generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Laboratories providing samples used in this work: Microbial Pathogenesis Laboratory and Molecular Biomedical Immunology Laboratory at FCB-UC and accessed through the Washington Covid-19 Biorepository: The Everett Clinic and Bloodworks Northwest. The scientific-clinical studies that generated serum samples evaluated in this report (CoronaVac03CL) were supported by the Ministry of Health, Government of Chile; The Confederation of Production and Commerce, Chile; the Consortium of Universities for Vaccines and Therapies against COVID-19, Chile; the Chilean Public Health Institute (ISP); the Millennium Institute on Immunology and Immunotherapy and Sinovac Life Sciences Co. AMK, SMB and PAG are supported by the National Agency for Research and Development (ANID) through the Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT) grants 1190830, 1170964, and 1190864, respectively. The Millennium Institute on Immunology and Immunotherapy (ICN09\_016/ICN 2021\_045; former P09/016-F) supports AMK, SMB and PAG; The Innovation Fund for Competitiveness FIC-R 2017 (BIP Code: 30488811-0) supports AMK, SMB and PAG. BDV is supported by ANID through the National Doctoral Scholarship, fellow #21221163. LFD is supported by ANID through the Postdoctoral FONDECYT grant # 3210473. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Blood samples were obtained from volunteers recruited in the clinical trial CoronaVac03CL ([clinicaltrials.gov][1] #[NCT04651790][2]) carried out in Chile starting in November 2020. The study was approved by the sponsoring institution Ethical Committee (ID 200708006), each Institutional Ethical Committee of the other sites, and the Public Health Institute of Chile (ISP Chile, number 24204/20). 1. Full names and affiliations: Comite Etico Cientifico Ciencias de la Salud UC, Pontificia Universidad Catolica de Chile, Santiago, Chile Comite Etico Cientifico Universidad de Los Andes, Santiago, Chile Comite Etico Cientifico Facultad de Medicina Clenica Alemana, Universidad del Desarrollo, Santiago, Chile Comite Etico Cientefico Hospital Clinico Felix Bulnes, Santiago, Chile Comite Etico Cientifico Servicio de Salud Valparaiso-San Antonio, Valparaiso, Chile Comite Etico Cientifico Servicio de Salud Metropolitano Sur Oriente, Santiago, Chile 2. Decision of all CEC was: Approval. Execution of the trials was conducted according to the current Tripartite Guidelines for Good Clinical Practices, the Declaration of Helsinki and local regulations. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as [ClinicalTrials.gov][3]. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript. [1]: http://clinicaltrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT04651790&atom=%2Fmedrxiv%2Fearly%2F2022%2F07%2F02%2F2022.07.01.22277165.atom [3]: http://ClinicalTrials.gov