Centenarians and extremely old people living with frailty can elicit durable SARS-CoV-2 spike specific IgG antibodies with virus neutralization functions following virus infection as determined by serological study
In December 2019, a novel coronavirus was identified in Wuhan, Hubei China, which has led to a devastating pandemic (WHO pandemic) [
]. Coronavirus disease 2019 (COVID-19) is used to define the clinical symptoms that are associated with infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . The SARS-CoV-2 is an enveloped positive-sense, single-stranded RNA genome and belongs to the Coronaviridae family in the genus Betacoronavirus, sharing the same lineage (Lineage B) as the 2002 SARS-CoV [
Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study.
]. It is suspected that age-related changes to the immune system, including immunosenescence and ‘inflammaging’ as well as the increased susceptibility to co-morbidities, may contribute to increased risk of severe COVID-19 in older individuals [
Immunosenescence and its hallmarks: how to oppose aging strategically? a review of potential options for therapeutic intervention.
]. We now know constant inflammation or “inflammaging” is a signficant contributor to aberrant immune responses in older people. Additionally, senescent cells can contribute to inflammation and inflammaging, where inflammaging is defined as a constant low level release of inflammatory mediators (C Reactive Protein (CRP) and the inflammatory cytokines IL-6 and IL-8) above baseline contributing to the reduction of antigen-specific immunity [
]. The aberrant levels of inflammatory mediators in older adults is hypothesized to lead to heightened inflammatory responses and immunopathology in this age group during a viral infection such as with SARS-CoV-211. Additionally, those at the highest risk are older adults residing in long-term care homes [
Covid-19 and immunity in aging populations – a new research agenda.
Here, we conducted a serological study of a cohort of extreme old residents in a long-term care home that experienced a COVID-19 outbreak in Nova Scotia, Canada. We focused on a group of centenarians and nonagenarians who were infected with SARS-CoV-2. The humoral response and durability of antibody production following SARS-CoV-2 infection in these older individuals is currently ill-defined although multiple reports have documented a spectrum of responses in several cohorts. The role of age as a cofactor in establishing protection against future SARS-CoV-2 re-exposure and reinfection is of considerable interest in aged individuals. We investigated the antibody responses to SARS-CoV-2 infection in a small group of extreme aged individuals to better understand how the aged immune system works to protect against SARS-CoV-2 infection.
There were 15 residents of extreme ages that participated in our study. The majority were female (12 women and 3 men) which is consistent with the literature for nonagenarians and centenarians (Table 1) [
Centenarians in nursing homes during the COVID-19 pandemic.
]. In total there were 8 centenarian included, representing 9 of the 12 centenarians in the LTC facility at the time of the outbreak and all of the centenarians approached for consent. Additional residents of the LTC facility were not approached for consent due to resource limitations. The additional 6 residents were selected out of the 107 residents enrolled (of 125 approached for consent) in a larger study as they were the next 6 residents enrolled who were, at the time of consent, deemed to be exposed but not yet infected while still in the oldest old of the cohort. Fig. 1 shows the study outline.
The study descriptive characteristics are in found in Table 1. The mean age of the population included in this analysis was 96.9 and the ages of the SARS-CoV-2 negative and positive groups were similar. The SARS-CoV2 positive participants were all women and most participants were asymptomatic. In both the SARS-CoV2 positive and negative groups, two participants had either a fever or cough. None of the centenarians had respiratory symptoms or fever. Three of the SARS-CoV-2 NP PCR positive residents were centenarians, with ages of 100, 102, and 102 years. The ages of the positive non-centenarians were 93, 97, and 94. The average age of the infected centenarians and non-centenarians were 101 (+/−1.15 SD) and 94.7 (+/- 2.08 SD), respectively. The age average for the COVID-19 negative centenarians and non-centenarians were 101 (+/−1.5 SD) and 89 (+/−2.82 SD), respectively. There were no differences in the level of frailty (Clinical Frailty Scale of 6.2 versus 6.3) [
A global clinical measure of fitness and frailty in elderly people.
], or comorbidities (dementia, hypertension, chronic obstructive pulmonary disease, etc.) in those who were SARS-CoV-2 positive and SARS-CoV-2 negative, respectively (Table 1). None of the participants enrolled required supplemental oxygen for hypoxia or dyspnea, were transferred to hospital or died in the 30 days following SARS-Cov-2 swab.
For plasma sample collection, the mean time from PCR diagnosis with COVID-19 to sample collection was 30 days. For residents with a second blood sample collected, the mean time between the first and second blood collection was 30 days. Due to the small numbers of nonagenarians and centenarians were we only able to determine trends for the clinical outcome of SARS-CoV-2 infection and were not able to perform multivariable analysis to identify factors that would influence clinical outcome.
For plasma sample collection, the mean time from PCR diagnosis with COVID-19 to sample collection was 30 days. For residents with a second blood sample collected, the mean time between the first and second blood collection was 30 days. Due to the small numbers of nonagenarians and centenarians were we only able to determine trends for the clinical outcome of SARS-CoV-2 infection and were not able to perform multivariable analysis to identify factors that would influence clinical outcome
An enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of antibodies directed toward the SARS-CoV-2 S protein present in the first blood collection for each resident enrolled in the study. Subsequently, the isotype and subclasses of S antibodies were identified by ELISA as well. SARS-CoV-2 infected centenarians had high titres of S-directed IgG in their plasma, with endpoint titers ranging from 1:6400–1:52,400 (Fig. 2Ai and C). We were not able to detect S-directed IgG in the plasma of SARS-CoV-2 NP PCR negative participants (Fig. 2Aii and C). Non-centenarians who tested PCR positive also had significant anti-S IgG endpoint titers (1:800) (Fig. 2Bi and C). Antibody IgG subclass analysis indicated that both IgG1 and IgG3 S-directed antibodies were present in centenarian residents 862 and 916 and the non-centenarian residents (Supplemental Figs. 1 and 2). Further isotype characterization revealed that all SARS-CoV-2 PCR positive centenarians had a detectable anti-S IgM response, with titres ranging from 1:400–1:6400 (Fig. 3Ai) similar to positive non-centenarians with the exception of one positive non-centenarian who did not have detectable anti-S IgM (Fig. 3Aii) summarized in Fig. 3C. To complete the isotype analysis, S directed IgA antibodies were also measured in plasma samples where IgA1 anti-S levels were greater than IgA2 (Supplemental Figs. 2 and 3). The SARS-CoV-2 positive centenarians were also positive for S IgA (1:800- 1:25,600) (Fig. 4). To determine if the humoral responses to the SARS-CoV-2 virus were similar in magnitude between infected centenarian and non-centenarian older residents, we directly compared if the titres of S directed IgG, IgM, and IgA of the two groups. The titers of S directed IgG, IgM, and IgA were not statistically different between the groups, demonstrating that advanced age did not alter S-directed antibody elicitation to the SARS-CoV-2 virus.
To investigate the durability and function of antibodies elicited toward SARS-CoV-2, we analyzed a second blood sample collected approximately 30 days after the first draw to determine levels of S antibodies and whether antibodies had viral neutralization capacity. For the durability assay, resident 916 had consistently high levels of anti-S IgG titres with levels of 1:52,400 still observed in their second plasma sample (Fig. 5A). Conversely, the titer of resident 976 decreased from 1:6400 to 1:3200 at the second draw. When evaluating anti-spike IgM, both residents had decreased responses (Fig. 5B). Initial titers for both centenarians were 1:400 and decreased in the follow-up sample, with the titer in resident 916 IgM decreasing to 1:200. There was no detectable IgM for resident 976 at either time point. Decreases in antibody titer were also observed at the second timepoint for spike-directed IgA (Fig. 5C). SARS-CoV-2 virus neutralization assays were performed to assess the functional neutralization capabilities of the plasma antibodies elicited by the COVID-19 advanced aged cohort. The plasma samples collected at the first sampling time point were used in standard virus neutralization assays performed in Vero76 cells with SARS-CoV-2 (strain SARS-CoV-2/Canada/ON/VIDO-01–2020) at 10 TCID50. All of the SARS-CoV-2 NP PCR and S IgG positive residents had detectable neutralizing antibody titers, ranging from 1:20 to 1:160 for centenarians and 1:20 to 1:40 for non-centenarians (Fig. 6). Together, our data illustrates that centenarians infected with SARS-CoV-2 were able to elicit SARS-CoV-2 S-directed neutralizing antibodies, demonstrating an intact humoral immune response.
To our knowledge, this is the first study investigating seroconversion in SARS-CoV-2 infected extreme aged adults, some over 100 years, residing in a long-term care facility. Here, a group of highly aged, frail residents (the majority being female), living in a long-term care facility survived infection with SARS-CoV-2, is described. The centenarians were able to elicit a successful S directed antibody response which was able to functionally neutralize the native SARS-CoV-2 virus. This work demonstrates that the extreme aged immune system is capable of responses during SARS-CoV-2 virus infection which are classically associated with convalescence and recovery from acute respiratory virus infection.
The older residents examined in this study represent a particularly vulnerable group in the ongoing global SARS-CoV-2 pandemic. Estimations of infection fatality risk (IFR) of SARS-CoV-2 in Switzerland demonstrated that people older than 65 years had an IFR of 5.6%, compared to 0.0092% in those aged 20–49 y [
Serology-informed estimates of SARS-CoV-2 infection fatality risk in Geneva, Switzerland.
]. Moreover, risk of death from COVID-19 has been estimated to be 630 times greater for those over 85 y compared to 19–29 y/o . Older adults, especially those living with frailty, are considered disproportionately susceptible to COVID-19 as they have increased likelihood of chronic health issues and immune dysfunction [
SARS-CoV-2, immunosenescence and inflammaging: partners in the COVID-19 crime.
]. The aberrant immune function of older adults compared to younger adults led to concern with their ability to mount a successful humoral immune response to SARS-CoV-2 infection considering their increased susceptibility to the virus. With these examples in mind, it is also recognized that people of extreme ages (nonagenarians and centenarians) often display profiles of healthy aging and are equipped to respond in a manner that is beneficial leading to positive outcomes after pathogen infection. As this does not describe all extreme older individuals, some experts in aging hypothesize that nonagenarians and centenarians may be categorized as high-performing or low-performing where those that are high-performing have genetic or immunological signatures that are advantageous for healthy aging [
SARS CoV2 infection _the longevity study perspectives.
]. These characteristics such as decreased inflammation and inflammatory mediators (such as decreased IL-6), increased telomere length, and deceased redox reactions are associated with the beneficial immune responses and improve the high-performing centenarian outcome to viral infection [
Functional measures, inflammatory markers and endothelin-1 as predictors of 360-day survival in centenarians.
]. Immune responses and immune mediators including NK cell activity, neutrophil microbicidal activity, IL-1bet,a and TNF-alpha, which may be inflammatory as well, have also been associated with positive outcomes for centenarians [
Telomere length and telomerase activity in T cells are biomarkers of high-performing centenarians.
]. Although we have found that the older ages in our study all recovered from SARS-CoV-2 infection without complications, all our residents had high frailty scores suggesting they would be low performers. To extend our analysis, future studies should investigate the immune mediator levels in these surviving centenarians to determine if these residents represent high-performing centenarians despite their high frailty score. Evidence from an epidemiological study out of Italy indicated that the oldest old (nonagenarians and centenarians) had similar or greater mortality and morbidity profiles as those within the old age group of 50 to 80 [
COVID-19 mortality in Lombardy: the vulnerability of the oldest old and the resilience of male centenarians.
]. It would be important to analyze the case fatality rates of high-performing and the low-performing centenarians within this Italian study as well as our own as none of our infected residents succumbed to COVID-19.
An extraordinarily large amount of work has been done over a short amount of time to characterize the evolution of the immune response to SARS-CoV-2 in healthy adults. Antibodies (humoral immunity) and T cells (cell mediated immunity) are essential for eliminating viruses from hosts. Evaluating antibody levels and the antibody isotype are the easiest ways to investigate the involvement of the humoral B cell response and its maturity level, respectively. Antibodies elicited after SARS-CoV-2 infection in healthy adults have been shown to be polyfunctional specifically with virus neutralizing capabilities, ADCC (Antibody-Dependent Cellular Cytotoxicity) activation properties, and complement deposition functions [
Asymptomatic and symptomatic SARS-CoV-2 infections elicit polyfunctional antibodies.
]. When investigating the evolution of the antibody response, one study found that RBD (receptor binding domain)-specific IgG and IgA levels correlated more strongly than IgM levels in patients shortly after seroconversion [
]. Although in our study we only investigated two centenarians over time, we found relative stability in the spike-specific IgG antibodies at 60 days post symptom onset whereas the IgM and IgA levels were more variable. In another study, investigation of the three arms of adaptive immune memory kinetics identified a coordinated response among CD4+ T cells, CD8+ T cells, and antibody longevity over 8 months to be correlated with mild COVID-1935. Interestingly, the authors of this study found that loss of SARS-CoV-2 specificity in any of the immune arms to be more prominent in those over 65 and those experiencing more severe disease [
Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity.
]. It would be of interest to investigate the three arms of immune memory in the oldest old age group to determine if there is evidence of immunological stability as suggested by Lio and colleagues in their discussion of immunity in nonagenarians and centenarians [
SARS CoV2 infection _the longevity study perspectives.
Here, we showed that after an average of 30 days after a positive SARS-CoV-2 NP PCR test, some extremely old LTC residents mounted high SARS-CoV-2 S-directed antibody responses across relevant immunoglobulin isotypes. The highest titers were observed with IgG, where centenarian titres ranged from 1:6400 to 1:52,400. All individuals positive for spike-IgG had neutralizing antibody titers ranging from 1:20 to 1:160. Other studies have established ELISA IgG titres of 1:320 as moderate and titres of 1:960 and above as high [
Robust neutralizing antibodies to SARS-CoV-2 infection persist for months.
], demonstrating that the centenarians in this study induced a robust anti-spike IgG response after SARS-CoV-2 infection. Interestingly, one study by Klein and colleagues indicated that serum SARS-CoV-2 antibody titers increased with age and male sex in COVID-19 patients [
Sex, age, and hospitalization drive antibody responses in a COVID-19 convalescent plasma donor population.
]. Since most all of our advanced aged participants were female, our data suggests that extreme aged females also elicit high SARS-CoV-2 antibody titers. In younger COVID-19 cohorts, by four weeks post symptom onset, over 90% of participants had neutralizing activity; the older residents in our study appear to have had a similar humoral response [
SARS-CoV-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic COVID-19.
], agreeing with our results showing centenarians with anti-S IgG titers were also able to neutralize the virus.
The identified S-directed IgG response was durable and remained high at a second sample collection which was approximately 60 days after an initial diagnostic positive SARS-CoV-2 NP PCR test. Despite media concerns surrounding longevity of the humoral response for SARS-CoV-2 infection, our data, although collected from a small study group, suggest that S IgG antibody levels only slightly decrease 2 months after infection, even in extremely old people. A study investigating the durability of anti-S IgG has shown peak S-directed IgG antibodies 4 weeks after COVID-19 symptom onset which then remained high for 6 months [
Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients.
In contrast to our IgG anti-S findings, we found a robust early increase in S-specific plasma IgA in SARS-CoV-2 infected people studied at 30 days post positive PCR test which declined. Few studies have characterized the onset of IgA after SARS-CoV-2 infection in plasma, most likely due to the mucosal association typically considered for the IgA isotype. One study that examined COVID-19 convalescent plasma found a correlation of S-specific IgG with S-specific IgA in plasma [
Asymptomatic SARS-CoV-2 infection in Belgian long-term care facilities.
]. Older people, especially those who are frail and have dementia, often develop subtle and atypical illness presentations that present challenges for symptom detection and lead to difficulty in identifying infection [
Influenza surveillance case definitions miss a substantial proportion of older adults hospitalized with laboratory-confirmed influenza: a report from the Canadian Immunization Research Network (CIRN) serious outcomes surveillance (SOS) network.
]. Within the cohort studied here, all but two residents were living with dementia and most residents were at least moderately frail. This may have contributed to the rapid spread of the virus through the long-term care facility. Frequent serological investigation and PCR testing for SARS-CoV-2 antibodies and virus, respectively, provide mechanisms for identifying an outbreak in extreme older individuals who may be unaware of their health status which is confounded by an atypical COVID-19 clinical picture.
Another concern surrounding the immune response to SARS-CoV-2 in adults is the concept of cross-reactivity to the virus due to infection with other coronaviruses previously. Studies using highly-sensitive flow-cytometry based methods have shown that people who were not exposed to SARS-CoV-2 had detectable IgG antibodies against the spike protein of the SARS-CoV-2 virus. These antibodies were especially prevalent in children and adolescents [
Preexisting and de novo humoral immunity to SARS-CoV-2 in humans.
]. Other investigations have also found that unexposed people have pre-existing CD4+ memory T cells that are reactive to common cold coronaviruses such as NL63, HKU1, and OC43, as well as the current pandemic coronavirus SARS-CoV-2 [
Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans.
]. As the participants studied were highly aged and had likely been exposed to many viruses over their lifetime, it was hypothesized that some cross-reactivity to the SARS-CoV-2 spike protein or virus may be observed due to previous infection. In our study, residents who did not test positive for SARS-CoV-2 RNA did not have any detectable plasma IgG against the SARS-CoV-2 spike protein. Our method of detection using indirect ELISA for IgG may suggest that cross-reactive antibodies from previous circulating common cold coronaviruses may not be an issue when screening individuals for serological evidence of a SARS-CoV-2 infection. A previous study investigated the TCR (T cell receptor) repertoire changes across life associated development and aging which included newborns and centenarians [
Dynamics of individual T cell repertoires: from cord blood to centenarians.
]. The data indicated that repertoires are more similar among individuals at birth and increase in diversity overtime. It would be of interest to conduct BCR (B cell receptor) repertoire sequencing in our cohort to determine clonal expansion, somatic hypermutation, and isotype switching of the BCR and therefore antibodies. This information may inform if previously established clonotypes from common cold coronavirus exposures were expanded during the infection in SARS-CoV-2. This analysis would also give insight into the specificity of the humoral response in older individuals.
Our study was limited by a small sample size, lack of continued plasma sampling, additional age groups, and uncertain SARS-CoV-2 inoculation/exposure date. The low sample size reflects a low incidence rate in Nova Scotia, even among extremely old people living with frailty (cumulative confirmed cases were 177 per million on April 1 and 1057 by May 15, the period of initial data collection) . Even so, the information gathered here is extremely valuable because of the rare occurrence of centenarians infected with SARS-CoV-2. The second timepoint for blood collection was not available for all residents studied, making it difficult to form more robust and broad conclusions from our data. Although more frequent blood collection would have been beneficial to better model the dynamics of the antibody response following infection in highly aged people and their subsequent outcomes, access to blood samples in this age group will remain a difficult task due to their vulnerability and the pragmatics of venipuncture. Since all of our participants had similar short-term outcomes, follow up analysis may not indicate differences in humoral response correlation to outcome. Long-term follow-up in this cohort may provide a different result for analysis with antibody levels. Additionally, having several age groups for analysis that were infected with SARS-CoV-2 in the long-term care facility as well as in the community setting would have been an important comparator. At the time of the study initiation, we focused on people in their 90 s and 100 s in the long-term care facility as the outcome of infection in these age groups was of particular importance due the epidemiological modeling indicating increased case fatality rates. Since immune mediators are also of interest it would have been important to identify the profiles of specific cytokines such as IL-6 and TNF-alpha in these residents [
Functional measures, inflammatory markers and endothelin-1 as predictors of 360-day survival in centenarians.
]. Due to the limited amount of sample we were able to receive from these frail individuals, we were not able to complete additional immune profiling. Finally, as with most viral infections, pinpointing the exact inoculation date or event for an infection is difficult in people. With increased studies following contact tracing and symptom onset as well as studies such as ours describing time from symptom onset and development of SARS-CoV-2 antibodies, a clearer clinical picture of disease progression and immune responses for extreme older adults as well as other age groups will be better defined.
The current COVID-19 pandemic has highlighted the often marked frailty of residents in long-term care which is evident by the high COVID-19 case numbers, COVID-19 fatalities, and SARS-CoV-2 outbreaks in these facilities across several countries [
Serological survey following SARS-COV-2 outbreaks at long-term care facilities in metro Vancouver, British Columbia: implications for outbreak management and infection control policies.
]. The cause of this may be multifaceted, which may include the inadequacies of infection control measures and gaps in education as well as the difficulty identifying atypical clinical manifestations of COVID-19 in older people. Our study shows that even the oldest people can elicit a strong humoral response to SARS-CoV-2 and recover from infection. These findings are important for developing serological testing protocols as well as investigating the poor COVID-19 outcomes associated with LTC facilities.
Declaration of Competing Interest
Mary K. Foley – Ms. Foley has nothing to disclose.
Samuel D. Searle – Dr. Searle received PhD and fellowship funding from the Canadian Frailty Network, Dalhousie Medical Research Foundation, Dalhousie University Internal Medicine Research Foundation and the QE II Innovation Fund.
Ali Toloue – Toloue has nothing to disclose.
Ryan Booth -Ryan Booth has nothing to disclose.
Alec Falkenham – Dr. Falkenham has nothing to disclose.
Darryl Falzarano – Dr. Falzarano has nothing to disclose.
Salvatore Rubino – Dr. Rubino has nothing to disclose.
Magen E. Francis – Ms. Francis has nothing to disclose.
Mara McNeil – Ms. McNeil has nothing to disclose.
Christopher Richardson – Dr. Richardson has nothing to disclose.
Jason LeBlanc – Dr. LeBlanc has nothing to disclose.
Sharon Oldford – Dr. Oldford has nothing to disclose.
Volker Gerdts – Dr. Gerdts has nothing to disclose.
Melissa K. Andrew – Dr. Andrew reports grants from Sanofi, grants from GSK, grants from Pfizer, grants from Canadian Frailty Network, grants from CIHR, grans from Public Health Agency of Canada, personal fees from Sanofi, from Pfizer, personal fees from Seqirus, outside the submitted work.
Shelly A. McNeil – Dr. McNeil reports grants, personal fees and other from Pfizer, other from Medicago, personal fees and other from Sanofi, grants, personal fees and other from GlaxoSmithKline, grants personal fees and other from Merck, other from CanSino, other from IMV, other from Janssen, outside the submitted work.
Barry Clarke – Dr. Clarke has nothing to disclose.
Kenneth Rockwood – Dr. Rockwood reports personal fees from Clinical Cardio Day – Cape Breton University, personal fees from CRUIGM-Montreal, from Speaker at Jackson Lab, Bar Harbor MA, personal fees from Speaker at MouseAge Rome Italy, personal fees from Frontemporal Dementia Study Group, personal fees from SunLife Insurance Japan, outside the submitted work and Kenneth Rockwood is President and Co-founder of Ardea Outcomes, which in the last three years (as DGI Clinical) has contracts with pharma and device manufacturers (Shire, Hollister, Nutricia, Roche, Otsuka) on individualized outcome measurement. Otherwise any personal fees are for invited guest lectures and academic symposia, received directly from event organizers, chiefly for presentations on frailty. He is Associate Director of the Canadian Consortium on Neurodegeneration in Aging, which is funded by the Canadian Institutes of Health Research, and with additional funding from the Alzheimer Society of Canada and several other charities. He receives career support from the Dalhousie Medical Research Foundation as the Kathryn Allen Weldon Professor of Alzheimer Research, and research support from the Canadian Institutes of Health Research, The Canadian Frailty Network, the QEII Health Science centre Foundation, the Nova Scotia Health Research Fund and the Fountain Family Innovation Fund of the QEII Health Science centre Foundation.
David J. Kelvin – Dr. Kelvin has nothing to disclose.
Alyson A. Kelvin – Dr. A. Kelvin has nothing to disclose.