key: cord-0945955-q7bnkg6u authors: A, Atti; M, Ferrari; J, Castillo-Olivares; EJM, Monk; R, Gopal; M, Patel; K, Hoschler; MJ, Cole; A, Semper; J, Hewson; AD, Otter; S, Foulkes; J, Islam; M, Mirfenderesky; S, Jain; J, Murira; C, Favager; E, Nastouli; MA, Chand; CS, Brown; JL, Heeney; T, Brooks; VJ, Hall; S, Hopkins; M, Zambon title: Serological profile of first SARS-CoV-2 reinfection cases detected within the SIREN study date: 2021-09-30 journal: J Infect DOI: 10.1016/j.jinf.2021.09.019 sha: e8d98f934990f357752a5bb8a1584f41f8f7e18d doc_id: 945955 cord_uid: q7bnkg6u OBJECTIVES: To describe the serological profile of first two SARS-CoV-2 confirmed reinfections in the national healthcare worker cohort study SARS-CoV-2 Immunity and Reinfection Evaluation (SIREN) and potentially identify correlates of protection against reinfection. METHODS: In addition to routine testing within the SIREN study, viral culture, sequencing and phylogenetic analysis were performed. Total antibody testing (Anti-SARS-CoV-2 nucleocapsid and Anti-SARS-CoV-2 spike) were complemented by receptor binding domain indirect ELISA and neutralising antibody assays. RESULTS: The first two SARS-CoV-2 confirmed reinfections had mild symptomatic illness episodes from which infectious virus was recovered at the time of reinfection. The recovered viruses and their sequences were closely related to viruses circulating locally during the time of reinfection and serology was consistent with reinfection. Prior to reinfection, both cases had ELISA and immunoblot detectable anti-N antibodies, but lacked live virus neutralising antibody. Within days following reinfection, neutralising antibodies became detectable and anti-N and anti-S binding antibodies were boosted. CONCLUSIONS: We hypothesise that titres of neutralising antibody can be used as a correlate of protection against reinfection. Further analysis using a case-control design is essential in order to confirm this hypothesis. To describe the serological profile of first two SARS-CoV-2 confirmed reinfections in the national healthcare worker cohort study SARS-CoV-2 Immunity and Reinfection Evaluation (SIREN) and potentially identify correlates of protection against reinfection. In addition to routine testing within the SIREN study, viral culture, sequencing and phylogenetic analysis were performed. Total antibody testing (Anti-SARS-CoV-2 nucleocapsid and Anti-SARS-CoV-2 spike) were complemented by receptor binding domain indirect ELISA and neutralising antibody assays. The first two SARS-CoV-2 confirmed reinfections had mild symptomatic illness episodes from which infectious virus was recovered at the time of reinfection. The recovered viruses and their sequences were closely related to viruses circulating locally during the time of reinfection and serology was consistent with reinfection. Prior to reinfection, both cases had ELISA and immunoblot detectable anti-N antibodies, but lacked live virus neutralising antibody. Within days following reinfection, neutralising antibodies became detectable and anti-N and anti-S binding antibodies were boosted. We hypothesise that titres of neutralising antibody can be used as a correlate of protection against reinfection. Further analysis using a case-control design is essential in order to confirm this hypothesis. Key words: COVID-19; SARS-CoV-2; Reinfection; COVID-19 Serological Testing; Neutralising Antibodies A study published by Hanrath and colleagues in this Journal found no SARS-CoV-2 reinfection cases between the first two waves of the pandemic in a cohort of healthcare workers. However, several SARS-CoV-2 reinfection cases have been reported during the second wave, although reinfection definitions are not consistent. 1, 2 It is crucial to understand whether SARS-CoV-2 antibody titres could be used as a correlate of protection in assessment of disease susceptibility. In the SIREN study, a large national longitudinal cohort of more than 44,000 healthcare workers, participants are followed for at least 12 months using fortnightly symptom and exposure questionnaires and nucleic acid amplification testing (NAAT), with monthly antibody testing against SARS-CoV-2. 3 Potential reinfections are flagged when meeting the following criteria: two positive RT-PCR tests at least 90 days apart (with no additional intervening positives) or a new RT-PCR positive test at least four weeks after a positive SARS-CoV-2 antibody test. Additional total antibody testing is performed at Public Health England laboratory using the semi-quantitative Elecsys Anti-SARS-CoV-2 nucleocapsid (N) protein assay and fully quantitative Elecsys Anti-SARS-CoV-2 spike (S) protein assay which targets the receptor binding domain (RBD) (Roche Diagnostics). 4 We here describe two reinfection cases in which additional serological assays were performed: in-house recombinant SARS-CoV-2 IgG spike (S) protein RBD indirect ELISA, 5 live virus microneutralisation using SARS-CoV-2 isolate England/02/2020 6 and pseudovirus neutralisation. 7 Semi-automated multiplexed immuno-blotting assay was performed to detect RBD-, N-, S1-, S2-and S-specific IgG, IgA and IgM antibodies. 7 A 45-year-old female nurse, with history of asthma and treated breast cancer, was SARS-CoV-2 antibody positive on 7 th August 2020. She reported COVID-19 symptoms in March (dry cough, fever, headache and myalgia, followed by anosmia and ageusia), however RT-PCR was not performed. On 10 th October, during a nosocomial outbreak of SARS-CoV-2, she became SARS-CoV-2 PCR positive, however asymptomatic at the time of testing. Four days later, she reported headache followed by sore throat, myalgia, arthralgia, ageusia and a productive cough. She reported milder symptoms during the second episode. A 37-year-old female administrator had SARS-CoV-2 antibodies on 28 th August 2020. She Previous exposure to SARS-CoV-2 was evident from the immuno-blot (Figure 2b) . Thus, IgG and IgA specific to the Nucleocapsid protein, but not other antigens, could be detected prior to the onset of symptoms. Two weeks later, an increase in antibody responses was observed, against the N antigen, Spike antigen, RBD and S1 sub-domains. IgM was detected against N and S1, albeit weaker against the latter antigen. The mechanisms of failure of immune protection from reinfection have not been clearly elucidated. In these two cases, an anamnestic antibody response was observed using virus neutralisation, antibody binding and immuno-blotting assays. All investigations showed an increase in antibody levels following the onset of symptoms in both cases and low or absent levels of neutralising antibodies at time of reinfection. This might be due to a lack of effective antibody response after the first infection or decrease in neutralising antibody titres over time, as observed in other studies. 8 Our data are consistent with the hypothesis that absence or low levels of neutralising antibody titres are likely correlate with a lack of protection against SARS-CoV-2 reinfection. There is strong evidence that neutralising antibodies play a critical protective role.Was estimated that neutralising antibody titres can offer an accurate prediction of immune protection, with neutralisation level for 50% protection being 54 U/mL, which equates to a titre of 1:10 or 1:30 in most virus neutralisation assays. 9 The sera from both cases had virus neutralising antibody levels below this threshold at the time of SARS-CoV-2 reinfection. There are some limitations in this study: no samples from the first infection episodes were available, thus no comparative genomic analysis of infecting viruses was possible. However, the sequences obtained in October were genetically distant from SARS-CoV-2 viruses from March 2020 but closely related to viruses circulating locally at time of reinfection. Secondly, symptoms from the first infection episodes are subject to recall bias. Finally, our analysis was restricted to two cases; therefore, our hypothesis will require support from more extensive studies. Further analysis using a case-control design is essential to clarify the potential role of neutralising antibodies in SARS-CoV-2 reinfection. None. IgG IgA IgM Lessons for COVID-19 Immunity from Other Coronavirus Infections Seasonal coronavirus protective immunity is short-lasting SIREN protocol: Impact of detectable anti-SARS-CoV-2 on the subsequent incidence of COVID-19 in 100,000 healthcare workers: do antibody positive healthcare workers have less reinfection than antibody negative healthcare workers? Antibody response to first BNT162b2 dose in previously SARS-CoV-2-infected individuals Infection and transmission of SARS-CoV-2 in London care homes reporting no cases or outbreaks of COVID-19: prospective observational cohort study High prevalence of SARS-CoV-2 antibodies in care homes affected by COVID-19: Prospective cohort study Towards Internationally standardised humoral Immune Correlates of Protection from SARS-CoV-2 infection and COVID-19 disease. medRxiv Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection The authors would like to thank all local SIREN research teams for their supporting investigation of potential reinfections up to the date of this publication. The authors also gratefully acknowledge the two SIREN participants mentioned on this article as cases, for their valuable contribution and availability. The authors would like to thank the Francis Crick Institute who contributed with RT-PCR results for cases investigation and one of the sequences as part of the staff testing programme, the UCLH APDU team and the PHE Colindale team for conducting rapid sequencing and phylogenetic analysis.