key: cord-0951118-6l7h3zxv
authors: Petrie, Joshua G; Bazzi, Latifa A; McDermott, Adrian B; Follmann, Dean; Esposito, Dominic; Hatcher, Christian; Mateja, Allyson; Narpala, Sandeep R; O’Connell, Sarah E; Martin, Emily T; Monto, Arnold S
title: Coronavirus Occurrence in the HIVE Cohort of Michigan Households: Reinfection frequency and serologic responses to seasonal and SARS coronaviruses
date: 2021-03-23
journal: J Infect Dis
DOI: 10.1093/infdis/jiab161
sha: 6cbe4a51501236eb8c1e7a79be5cd3b52409fae2
doc_id: 951118
cord_uid: 6l7h3zxv

BACKGROUND: We investigated frequency of reinfection with seasonal coronaviruses (HCoV) and serum antibody response following infection over 8 years in the Household Influenza Vaccine Evaluation cohort. METHODS: Households were followed annually for identification of acute respiratory illness with RT-PCR confirmed HCoV infection. Serum collected before and at two time points post infection were tested using a multiplex binding assay to quantify antibody to seasonal, SARS-CoV-1, and SARS-CoV-2 spike proteins and SARS-CoV-2 spike sub-domains and N protein. RESULTS: Of 3418 participants, 40% were followed for ≥3years. A total of 1004 HCoV infections were documented; 303 (30%) were reinfections of any HCoV type. The number of HCoV infections ranged from 1 to 13 per individual. The mean time to reinfection with the same type was estimated at 983 days for 229E, 578 days for HKU1, 615 days for OC43, and 711 days for NL63. Binding antibody levels to seasonal HCoVs were high, with little increase post-infection, and were maintained over time. Homologous, pre-infection antibody levels did not significantly correlate with odds of infection, and there was little cross response to SARS-CoV-2 proteins. CONCLUSIONS: Reinfection with seasonal HCoVs is frequent. Binding anti-spike protein antibodies do not correlate with protection from seasonal HCoV infection.

M a n u s c r i p t 5 Prior to the emergence of SARS-CoV-2, it was recognized that coronaviruses that infect humans (HCoVs) could be separated into the 4 seasonal, or common coronaviruses, 229E, OC43, NL53 and HKU1 which regularly cause mainly mild respiratory illnesses, and SARS-CoV and MERS-CoV viruses which have caused epidemics of severe lower respiratory disease [1] [2] [3] [4] . The current COVID-19 pandemic, the first recognized to be caused by an HCoV, has focused attention on the seasonal coronaviruses in comparison to SARS-CoV-2. Of particular importance are questions around possible cross protection or enhancement of COVID-19 disease from prior seasonal virus infection and duration of infection following SARS-CoV-2 infection [5] [6] [7] . Few have examined antibody response to seasonal infection, antibody waning, or cross response between the 4 seasonal HCoV or with SARS-CoV-2 in large prospective cohort studies [8] [9] [10] .

We have previously reported on 8 years of seasonal HCoV infection among persons in the continuing Household Influenza Vaccine Evaluation (HIVE) study being conducted in Michigan [11] . Over that period, 2010-2018, 1004 infections were detected by RT-PCR. The infections were most frequent in children, but substantial numbers of infections were identified in adults. This, and past studies of HCoVs suggested that these agents, like most respiratory viruses reinfect through life [12, 13] . In this report, we characterize RT-PCR documented, symptomatic reinfections with these viruses and investigate antibody response to infection, including cross-reactivity and persistence.

The complete methods of the HIVE cohort have been published previously [14] . Households with children receiving primary care from Michigan Medicine were recruited from Ann Arbor, MI and surrounding communities beginning in the summer of 2010. Households were retained as long as possible with replacement households enrolled and returning households reengaged in the spring or A c c e p t e d M a n u s c r i p t 6 summer of each year; participant and household characteristics were recorded at this time. Adult participants provided informed consent for themselves and their children, and children ≥7 years provided verbal assent prior to participating. This study was reviewed and approved by the University of Michigan Medical School institutional review board.

Each study year, participants were asked to report all acute respiratory illnesses (ARI) defined by ≥2 symptoms as soon as they occurred [14] . Participants were also actively questioned regarding their illness status via weekly calls or emails. Although year-round surveillance did not begin until the fall of 2014, complete coronavirus epidemics were likely captured in each study year because of their sharp seasonality [11] . Participants with ARI attended an illness visit within 7 days of symptom onset where study staff collected nasal and throat swabs (nasal only in children <3 years) combined in a single vial of viral transport media; asymptomatic infections were not assessed. Specimens were assayed for detection of respiratory viruses, including the 4 seasonal coronavirus types (229E, OC43, HKU1, and NL63). Specimens collected prior to the 2016-2017 study year were tested by singleplex RT-PCR using primers and probes developed by the CDC Division of Viral Diseases, Gastroenteritis, and Respiratory Viruses [15] . Specimens collected in the 2016-2017 and 2017-2018 years were tested using the FTD Respiratory Pathogen 33 multiplex PCR kit (Fast Track Diagnostics). Co-infections were defined ARI associated with the detection of 2 or more HCoV in the same specimen. Reinfection was defined as detection of the same or different HCoV type during an ARI with new onset of symptoms 14 or more days from the onset of a previously reported illness.

A c c e p t e d M a n u s c r i p t 7

Beginning in the fall of 2011, participants age ≥13 years were invited to provide blood specimens for serologic studies in the fall of each year prior to the respiratory virus season, and in the spring or summer following each respiratory virus season. Eligibility for serologic studies was expanded to children ≥6 months in the fall of 2016. We selected all individuals with PCR confirmed common coronavirus infection between 2011 and 2018 who had paired serum collected in the fall or summer prior to their infection and in the spring or summer following their infection for serologic studies. In addition to the pair pre-and post-infection serum specimens, a subsequent post-infection specimen was selected from a later study year for each individual when available.

These serum specimens were tested in a 10-Plex Electro-Chemi-Luminescence Immuno-Assays (ECLIA) (Meso Scale Discovery, Rockville, MD) to measure antibody binding to the following antigens: the 4 seasonal coronavirus spike proteins (229E, OC43, HKU1, and NL63), SARS-CoV-1 spike protein, SARS-CoV-2 spike protein, SARS-CoV-2 spike protein receptor binding domain, SARS-CoV-2 spike protein Nterminal domain, SARS-CoV-2 N protein, and a BSA negative control [16] [17] [18] [19] . On the day of the assay, the plate was blocked for 60 minutes with MSD Blocker A (5% BSA). The blocking solution was washed off and test samples applied to the wells at 4 dilutions (1:100, 1:800, 1:3200 and 1:12,800) and incubated with shaking for two hours. Plates were washed and Sulfo-tag labeled anti IgG antibody applied to the wells and allowed to associate with complexed coated antigen-sample antibody. Plates were washed to remove unbound detection antibody, and a read solution containing ECL substrate was applied. In a MSD Sector instrument, a current was applied to the plate and areas of well surface where sample antibody has complexed with coated antigen and labeled reporter will emit light in the presence of the ECL substrate. A MSD Sector instrument quantitated the amount of light emitted and reported A c c e p t e d M a n u s c r i p t 8 this ECL unit response which is directly proportional to binding antibody. The Area Under Curve (AUC) was calculated using Prism (GraphPad Prism, San Diego, CA).

Antibody binding to the 4 common coronavirus spike proteins was also measured in singleplex ECLIA assays. The correlation between the singleplex and multiplex assays was generally high and patterns of antibody response were similar for the common coronaviruses comparing the mulitplex and singleplex assays (Supplemental Table 1 and Supplemental Table 2 ). Therefore, the results of only the multiplex analysis are presented here.

For ease of calculation in time to event analyses individuals were considered to contribute time at risk from July 1 through June 30 for each study year they were enrolled even though ARI surveillance was not carried out during the summer months in all years. Mean, median, minimum, and maximum times from July 1 of the first study year of enrollment to first infection and re-infection were estimated overall and for specific HCoV types and genera. Kaplan-Meier curves summarizing time to reinfection following each previous infection were also generated. Individuals re-entered the data after each infection with time at risk of reinfection beginning at the day of onset of symptoms of their prior infection (time = 0). Individuals who were lost to follow-up and re-enrolled in a later season were censored during the period during which they were lost to follow-up.

the following year were estimated in Cox proportional hazards models, stratified by year, with respective overall, type, and genus-specific covariates specified as 1 if the individual was infected in the previous study year and 0 otherwise. Results of unadjusted models and models adjusted for age group Table 1 and Supplemental Table 2 ).

The association between antibody binding levels and subsequent infection risk was estimated using a case test-negative design analysis. Log 2 AUC was compared between cases of single infection with a specific HCoV type and controls that were singly infected with the other three HCoV types in logistic generalized estimating equations regression models with exchangeable correlation structure clustered on the individual and adjusted for age, influenza vaccination, and high-risk health status. Inclusion of study year in adjusted models did not substantially change point estimates. Odds ratios estimated from these models were interpreted as the reduction in odds of infection associated with a 2-fold increase in

A c c e p t e d M a n u s c r i p t 10

In total, 3,418 individuals participated for 1 to 8 study years (median [interquartile range]: 2 [1, 4] years) contributing a total of 9,378 person-years observation (Figure 1 ). There were 1378 (40%) individuals who were under study for 3 or more years. Between 8.3% and 16.3% of the cohort had an ARI associated with seasonal HCoV infection each year. In total, 1,004 ARI associated with coronavirus infection were identified. The beta genus OC43 was most common (N=390), followed by the alpha genus NL63 (N=328). Less common were the beta HKU1 (N=194), and alpha 229E (N=152). The mean time from enrollment to first HCoV infection was 542 days. Times from enrollment to first infection for each seasonal HCoV were consistent with their relative incidence i.e. shorter times for the more common OC43 and NL63, and longer for the less common HKU1 and 229E (Supplemental Table 3 ).

Of the 1004 HCoV-associated ARI, there were 53 (5.3%) instances coinfection with 2 HCoV types detected from the same specimen and 3 (0.3%) in which 3 different types were detected. Combinations of all 4 HCoV types were observed in these co-infections, but alpha and beta genus co-infections were most common ( were of the same HCoV type potentially representing prolonged shedding (range of days between illnesses: 14-152 days). Considering any type reinfections, the mean time to reinfection was 505 days.

The mean time to same type reinfection was estimated at 983 days for 229E, 578 days for HKU1, 615 A c c e p t e d M a n u s c r i p t 11 days for OC43, and 711 days for NL63 (Supplemental Table 3 ). Distributions of HCoV type pairings in consecutive any type reinfections were similar to those for coinfections with consecutive alpha and beta genus infections most common (Figure 2 C & D) .

Overall, the hazard of infection with any coronavirus was over twice as high among subjects with documented infection of any type in the immediately prior study year relative to those who were not (HR: 2.2; 95% CI: 1.7, 2.7). Similarly, the hazard of reinfection with a beta genus HCoV was over twice as high among subjects infected with a beta genus HCoV in the previous study year; this effect was consistent for both beta genus viruses, HKU1 and OC43 (Table 1) (Table 3 ). With the exception of the SARS-CoV-2 N protein, antibody levels were low against these targets and did not substantially change from pre-to post-infection with each of the 4 seasonal coronaviruses. While much lower than antibody levels to the seasonal coronaviruses, the preinfection geometric mean AUC for the SARS-CoV-2 N protein was higher than for other SARS targets.

Modest, but not statistically significant, increases in geometric mean AUC for SARS-CoV-2 N protein were observed for those infected with 229E where nearly 20% had ≥4 fold rise in log 2 AUC.

During an influenza pandemic, past experience is useful in many areas of planning, but the novel COVID-19 pandemic has left us with few precedents for a number of critical subjects, particularly duration of immunity post-infection or vaccination [20] . Prior experience with epidemic HCoVs offers little help; SARS-CoV-1 was eliminated and MERS has not spread widely [21, 22] . As a result, we have looked to the 4 seasonal HCoV viruses causing typically mild disease to gain insights on how SARS-CoV-2 might behave going forward. We have previously demonstrated that these HCoVs are truly seasonal, transmitting mainly in the months between November-May, peaking in December-March, and only time will tell if the SARS-CoV-2 occurrence will begin to follow the same pattern as immunity increases in the population [11] .

A c c e p t e d M a n u s c r i p t 14 While it was clear from studies conducted years ago that reinfection with seasonal HCoVs, like other common respiratory viruses occurred through life, their frequency had not been a matter of great interest [12, 13] . This has become more urgent currently given the importance of knowing how long immunity might last after SARS-CoV-2 infection and vaccination. Given the age structure of our cohort, and high levels of pre-existing antibody, nearly all infections observed in this study are likely reinfections. However, nearly a third of all identified infections were confirmed reinfections during enrollment with an average duration of 505 days between infections. In primary analyses, we observed that the risk of infection with beta coronaviruses was higher if an individual had a beta coronavirus infection in the immediately prior study year. This effect was attenuated in sensitivity analyses conditioning only on those with a prior infection suggesting that this result is likely due to confounding by unmeasured shared risk factors for infection rather than a specific biological effect. Regardless, this finding underscores the frequency of reinfection in this cohort.

While there have been several recent studies on antibody to the seasonal HCoVs in those infected with SARS-CoV-2, there have been few looking at the antibody response of RT-PCR confirmed seasonal infection [23, 24] . We used a multiplex binding assay that included SARS-CoV-2, SARS-CoV-1, seasonal coronavirus prefusion-stabilized spike protein constructs made using techniques developed for studying vaccine response [19, [25] [26] [27] . We found high levels of antibody binding the spike protein of the seasonal viruses and very low levels binding SARS-CoV-2 antigens in this pre-Covid-19 population. Individuals in this study were infected with seasonal HCoV despite these high levels of binding antibody, and had very modest increases in antibody following infection suggesting a ceiling effect. Consistent with this observed infection despite high antibody levels, there was no significant evidence that homologous binding antibody correlated with protection. This analysis was limited by lack of antibody measurements A c c e p t e d M a n u s c r i p t 15 in a completely uninfected control group, and by a relatively narrow distribution of pre-infection antibody binding levels in this population. We also did not measure neutralizing antibody which is likely to be a better correlate of protection. While binding assays for SARS-CoV-2 have correlated well with neutralizing antibodies in recent studies of response to infection or vaccination with the novel virus [26, [28] [29] [30] [31] , it is possible that is not the case with the repeat infection that occurs with the seasonal viruses.

We also found that antibody was remarkably persistent over time with little indication of waning. This stability of antibody is in contrast to several studies that have observed rapidly waning immunity following SARS-CoV-2 infection [8, 28, 32, 33] ; although other studies do suggest a more persistent antibody response [29, 30, 34] . As with correlates of protection, it may be the case that persistence of neutralizing antibody differs from that of binding antibodies.

There were few responses in SARS-CoV-2 antibodies following seasonal HCoV infection, indicating the cross response is infrequent. An exception was higher pre-infection antibody to the SARS-CoV-2 N protein, known to be more conserved among the HCoVs [35] . Measuring antibody directed to the SARS-CoV-2 N protein has been suggested as a way to distinguish individuals who are infected vs vaccinated as both will exhibit spike directed antibody. The antigen we used was the full N protein, using epitopes restricted to SARS-CoV-2 may be more specific for this strategy.

This study has demonstrated that seasonal HCoV reinfection frequently occurs over a relatively short time period and is possibly affected by the prior infecting virus type. However, we have not determined precisely how frequently this occurs in a broad population nor is it evident whether this will apply to A c c e p t e d M a n u s c r i p t 16 SARS-CoV-2 reinfection or infection after vaccination. Still, it does suggest that duration of immunity will probably be limited by either waning immunity or viral antigenic drift, at least in some of the population, and supports careful determination of the need for booster vaccinations as time from original immunization increases. Careful monitoring of the ways in which repeated vaccination and reinfection shape the development of immunity are also warranted. This has implications for the use of immune assays as it is possible that the close relation between binding and neutralizing antibodies for SARS-CoV-2 relates to the novelty of the virus. The need for periodic revaccination should not be viewed with alarm, since it has been the practiced for many years with influenza. Such booster immunizations, through updated reformulation, could also address possible antigenic changes, now becoming a major concern as novel variants continue to be identified globally.

M a n u s c r i p t M a n u s c r i p t 24 Table 2 . Geometric mean AUC levels of antibodies binding seasonal coronavirus spike protein at pre-infection, and at 2 post-infection time points for individuals infected with 229E, HKU1, NL63, OC43; and the proportion of individuals who demonstrated a ≥2-fold or ≥4-fold rise in binding antibody following infection. Table 3 . Geometric mean AUC levels of antibodies binding SARS-CoV-1 and SARS-CoV-2 antigens at pre-infection, and at 2 post-infection time points for individuals infected with 229E, HKU1, NL63, OC43; and the proportion of individuals who demonstrated a ≥2-fold or ≥4-fold rise in binding antibody following infection.

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Abbreviations: GM-AUC, geometric mean area under the curve; NTD, N terminal domain; RBD, receptor binding domain