REVIEW ARTICLE Silent battles: immune responses in asymptomatic SARS-CoV-2 infection Nina Le Bert 1 ✉ and Taraz Samandari 1 © The Author(s), under exclusive licence to CSI and USTC 2024 SARS-CoV-2 infections manifest with a broad spectrum of presentations, ranging from asymptomatic infections to severe pneumonia and fatal outcomes. This review centers on asymptomatic infections, a widely reported phenomenon that has substantially contributed to the rapid spread of the pandemic. In such asymptomatic infections, we focus on the role of innate, humoral, and cellular immunity. Notably, asymptomatic infections are characterized by an early and robust innate immune response, particularly a swift type 1 IFN reaction, alongside a rapid and broad induction of SARS-CoV-2-speci fi c T cells. Often, antibody levels tend to be lower or undetectable after asymptomatic infections, suggesting that the rapid control of viral replication by innate and cellular responses might impede the full triggering of humoral immunity. Even if antibody levels are present in the early convalescent phase, they wane rapidly below serological detection limits, particularly following asymptomatic infection. Consequently, prevalence studies reliant solely on serological assays likely underestimate the extent of community exposure to the virus. Keywords: COVID-19; asymptomatic; cellular immunity; antibody Cellular & Molecular Immunology ; https://doi.org/10.1038/s41423-024-01127-z INTRODUCTION Within the fi rst few months of the COVID-19 pandemic, it had become clear that the novel virus SARS-CoV-2 was far more transmissible than SARS-CoV-1 that emerged in the 2003 outbreak. By eight months, the SARS-CoV-1 epidemic had been controlled after approximately 8,100 infections in limited geographic areas [1]. By contrast, within the same timeframe, the World Health Organization reported 3.8 million con fi rmed cases of SARS-CoV-2. There was no end in sight to the control of the globally widespread pathogen, and most experts were convinced that there was a huge undercount of deaths and cases of COVID-19. Other than insuf fi cient global testing, the agonizing realization was that a far higher proportion of SARS-CoV-2 than SARS-CoV-1 infections went undetected due to minimal or absent symptoms among cases [2]. Asymptomatic persons can have similar levels of viral replication in their upper respiratory tracts when compared to symptomatic individuals [3, 4]. This similarity in viral load led to the rapid spread of the virus since it is extremely dif fi cult to isolate asymptomatic persons in a timely manner and thereby contain the pandemic [3]. Hence, asymptomatic infections contributed to the high reproduction number (R 0 ) of SARS-CoV-2, with meta- analyses estimating that asymptomatic persons contributed 15 – 50% of all transmissions [5, 6]. The role of asymptomatic infections in the spread of SARS-CoV- 2 is an important public health challenge. However, they also offer opportunities to examine the immune response of asymptomatic persons to understand better how future coronavirus pandemics – and perhaps other pandemic viruses – could be more effectively countered by vaccines or immunotherapies to prevent both, illness and viral transmission. In this review article, we summarize what is currently known and not known about the human immune response to SARS-CoV- 2 in asymptomatic persons. Key questions include: ● What is the epidemiology of asymptomatic infection? ● Does the innate immune response of asymptomatic persons differ from symptomatic individuals? ● Does the humoral immunity of asymptomatic persons differ from symptomatic individuals? ● Does the cellular immunity of asymptomatic persons differ from symptomatic individuals? ● Does the mucosal immunity of asymptomatic persons differ from symptomatic individuals? ● Can our knowledge of asymptomatic infections be translated to improvements in vaccines and immunotherapeutics against SARS-CoV-2 and other coronaviruses? Although there may be genetic and environmental differences between asymptomatic and symptomatic persons, we do not directly explore these aspects of SARS-CoV-2 infection in this review. THE EPIDEMIOLOGY OF ASYMPTOMATIC INFECTION WITH SARS-COV-2 Early during the pandemic, careful clinical studies were opportu- nistically conducted in closed cohorts. It was quickly appreciated Received: 17 December 2023 Revised: 3 January 2024 Accepted: 3 January 2024 1 Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore. ✉ email: nina@duke-nus.edu.sg www.nature.com/cmi 1234567890();,: that some individuals did not remain asymptomatic and were therefore labeled “ pre-symptomatic, ” consistent with what was already known about the incubation period of pathogenic microbes. Those who did not develop illness after 14 days ’ isolation, were considered truly asymptomatic. Although it has been documented that some patients without symptoms actually harbored pulmonary disease consistent with COVID-19 [7], such individuals remained without symptoms and – for the purposes of this paper – are not excluded from the asymptomatic category. In February of 2020, passengers aboard the cruise ship Diamond Princess were quarantined at Yokohama port in Japan. Among 3,711 passengers and crew, 711 were infected, of whom some were pre-symptomatic, but 18% remained symptom-free until viral clearance [8]. In this outbreak, it was noted that the risk of developing symptoms increased with age, as did the risk of delayed resolution of infection [9]. A study of nursing home residents – a cohort obviously enriched for the elderly – showed a 6% asymptomatic rate with 50% pre-symptomatic [10]. In a much more youthful population of military personnel aboard an aircraft carrier, 45% of those infected remained asymptomatic [11]. A narrative review published in 2021 that summarized fi ndings from 16 such studies concluded that asymptomatic persons “ account for approximately 40% to 45% of SARS-CoV-2 infections, and they can transmit the virus to others for an extended period, perhaps longer than 14 days ” [12]. More consistently, it was reported that asymptomatic individuals could harbor a comparable viral load to those exhibiting symptoms and, therefore, transmit the virus. On average, however, they tend to clear the infection faster [13 – 15]. A population view of infection came early on from China where, by using polymerase chain reaction (PCR) testing, a longitudinal cross-sectional study of 9,500 individuals in Wuhan between April and December 2020 showed that 80% of infected persons were asymptomatic [16]. While the high proportion of asymptomatic infections was cause for alarm due to the rapid but largely silent spread of the pandemic, public health of fi cials drew some consolation from the observation that – as a consequence – the case fatality rate of SARS-CoV-2 was less than 1% [17]. Such cohorts were critical to our initial understanding of asymptomatic infections and their role in the spread of SARS-CoV- 2. The subsequent deployment of serologic assays yielded new insights into the extent and spread of the contagion at the population level. Such data have been posted for downloading and analysis by researchers and public health institutions on websites such as https://covid19serohub.nih.gov/. A review and meta-analysis of serosurveys from multiple countries summarized the global spread of the virus by May 2022 [18]. It was found that by September 2021, global SARS-CoV- 2 seroprevalence from infection or vaccination was 59.2%. Only three months later, by December 2021, due to infection, seroprevalence had steeply increased in Africa to 86.7% and to 95.9% in high-income European countries due to the combination of vaccination and infection. A discrepancy between the reported cumulative incidence of COVID-19 and the median seroprevalence was noted and ranged from 1:2 in the Americas and European high-income countries to over 1:100 in African countries. US serologic data described the inexorable spread of the virus through its population [19], although evidence to support the bene fi ts of personal, community, and environmental non- pharmaceutical interventions also accumulated in multiple geo- graphic areas [20, 21]. In countries such as France, presumably due to the successful shielding of older members of the population, seropositivity was lower with increasing age [22]. An examination of seroprevalence versus case noti fi cations documented a huge unrecorded spread of the virus in resource-constrained regions from January to December 2020 [23] (Fig. 1), which suggested an extensive asymptomatic spread of the novel virus. Given that licensed US vaccines are based on the SARS-CoV-2 spike protein only, a distinction could be made between antibodies against the vaccines and natural infection after December 2020, the latter being evidenced by persons with anti-nucleocapsid (N) antibodies. Using samples from blood donors, the CDC showed that by September 2022, eight months after the emergence of the Omicron variant of concern, “ 96.4% of persons aged ≥ 16 years in a longitudinal blood donor cohort had SARS-CoV-2 antibodies from previous infection or vaccination, including 22.6% from infection alone and 26.1% from vaccination alone; 47.7% had hybrid immunity ” [24]. Hybrid immunity is de fi ned as immunity engendered by the combination of vaccines and natural infection [25] and has been found to be superior in protecting against infection and severe disease [26 – 28]. The documentation of asymptomatic infections in a population is important for several reasons. Firstly, using 2021 data, it was estimated that together, asymptomatics and persons with mild illness contributed to 95% of all infections [29]. Secondly, when more humans are infected, there is an increase in the opportunity for viral mutations and, thereby, the emergence of new and potentially more dangerous variants [30]. Thirdly, asymptomatic individuals contributed 50-75% of SARS-CoV-2 transmissions [5, 31], and as asymptomatic individuals have a similar amount of viral shedding as those with symptoms [3], this transmission would silently endanger the lives of the vulnerable. This leads to fourthly, the seroprevalence data revealed that the infection- fatality ratio (IFR) was lower than initially perceived. A team from the Institute for Health Metrics and Evaluation modeled IFR using data from around the world and concluded that “ population age structure accounted for 74% of [the] variation in IFRs ” (Fig. 2). After Central Europe, Eastern Europe, Central Asia (n=0) High-income (n=41) Latin America, Caribbean (n=3) North Africa, Middle East (n=2) South Asia (n=2) Southeat Asia, East Asia, Oceania (n=0) Sub-Saharan Africa (n=1) 0 200 400 600 800 Ratio seroprevalence/cumulative cases no matching data avaialble no matching data avaialble Fig. 1 Ratio between the prevalence of antibodies and the total number of reported cases in different geographical regions. The median ratio between corrected seroprevalence estimates from national studies and the corresponding cumulative incidence of SARS-CoV-2 infection from nine days prior. Includes studies ( n = 49) from January to December 2020. Data from (Bobrovitz et al. PLoS One. 2021) [23] N. Le Bert and T. Samandari 2 Cellular & Molecular Immunology age standardization, the countries with the highest IFR were Peru, Portugal, Oman, Spain, and Mexico, while “ Sub-Saharan African countries and Asian countries generally had the lowest all-age and age-standardized IFRs ” [32]. The latter observation suggested that in Africa and Asia, a low IFR was not entirely explained by their youthful age structures. A fi fth point is that having a prior infection – whether symptomatic or asymptomatic – renders the individual at a reduced risk of re-infection, although for a limited duration [33, 34]. The proportion of asymptomatic infections varied most notably according to age, region, and viral variant (Fig. 3). In addition to the reports described above, differences in symptomaticity according to age group have been prominently noted since the beginning of the pandemic: in China, 43% of children aged 0-17 remained asymptomatic compared to 13% of 18-39 years old, 7% of 40-59 years old, 3% of 60-79 years old and non among those above 80 years old (Fig. 3A) [35]. Even children displayed differences among age groups: in North Carolina, 71% of children 6-13 years of age were asymptomatic as compared to 52% of children under 6 years or 40% of adolescents[36]; in Singapore, 25% of children 0-4 years old were asymptomatic, compared to 47% of 5-9 years old and 36% of 10-16 years old (Fig. 3B) [37]. A global and systematic review and meta-analysis, which included 38 studies that reported asymptomatic rates by age, with a total of 14,850 cases prior to the rollout of vaccines, concluded that the asymptomatic proportion was 44.1% [38]. Their mathematical modeling predicted the highest rates in children aged 13.5 years, which gradually decreased by age, and was lowest at 90.5 years of age [38]. Like variations in IFR, the ratio of asymptomatic cases in con fi rmed SARS-CoV-2 infections varied signi fi cantly across geographical regions. A comprehensive review and meta-analysis, comprising 130,123 infections across 241 studies published before the end of 2020, revealed the highest rate of asymptomatic cases in Africa (64.3%), followed by America (40.0%), Europe (28.1%), and Asia (18.1%) (Fig. 3C) [39]. Infections, including asymptomatic infection, were more common with SARS-CoV-2 variants subsequent to the ancestral strain, as the virus adapted to enhance its transmissibility. In several vaccine ef fi cacy trials, it was possible to examine the proportions of asymptomatic infections in a cohort. From such systematically screened cohorts, we learned about differences in the prevalence of asymptomatic infection during periods of dominance of several SARS-CoV-2 variants. When the ancestral strain was circulating, fewer than 2% of asymptomatic individuals enrolled for immunization were PCR-positive for the virus [40 – 42]. When the B.1.351 (Beta) and B.1.617.2 (Delta) variants circulated, the rate was higher at 2.4% in a vaccine cohort [43]. At the time of the emergence of the Omicron (B.1.1.529) variant, 16% of an assembled cohort had an asymptomatic infection at baseline, and after the emergence of a subsequent Omicron subvariant, this proportion rose to 23% [44]. The demonstration of higher proportions of asymptomatic infection in such cohorts is consistent with what has been learned about the increased transmission ef fi ciency of each successive dominant SARS-CoV-2 variant [45]. A dif fi culty with interpreting these patterns of asymptomatic infections since 2020, is that not only do persons with a history of prior SARS-CoV-2 infection have a reduced risk of subsequent infection [46] including among those vaccinated (also known as “ breakthrough ” infections) [47], but the likelihood of natural infection has also increased exponentially 0 20 40 60 80 100 0.001 0.01 0.1 1 10 100 Age in years Infection–fatality ratio (%) 0 20 40 60 80 100 0 10 20 30 40 50 Age in years Infection–fatality ratio (%) Central Asia Central Europe Eastern Europe Australasia Asia Pacific North America Southern Latin America Western Europe Andean Latin America Caribbean Central Latin America Tropical Latin America North Africa and Middle East South Asia East Asia Oceania Southeast Asia Central sub-Saharan Africa Eastern sub-Saharan Africa Southern sub-Saharan Africa Western sub-Saharan Africa 0.0 0.5 1.0 1.5 2.0 Infection-fatality ratio (%) - all age - Central Europe, eastern Europe, and central Asia High income Latin America and Caribbean North Africa and Middle East South Asia Southeast Asia, east Asia, and Oceania Sub-Saharan Africa Fig. 2 COVID-19 infection – fatality ratio by age and geography. A COVID-19 infection – fatality ratio estimates by age (linear scale). B COVID- 19 infection – fatality ratio estimates by age (log scale). C COVID-19 infection – fatality ratio by location; 1. January 2021. All data from (COVID-19 Forecasting Team. Lancet. 2022) [32] N. Le Bert and T. Samandari 3 Cellular & Molecular Immunology during this period. Three years after the initial detection of the novel virus, truly naïve populations are likely nonexistent. IMMUNE RESPONSES INDUCED BY ASYMPTOMATIC SARS- COV-2 INFECTION Identifying and tracking asymptomatic infections poses an obvious challenge, as they can only be detected by employing regular screening through PCR and/or serological methods, or by monitoring individuals in close contact with infected cases. Moreover, since they do not cause any disease, immunologists and clinicians usually focus on studying immune responses in symptomatic and more severe cases with the aim of learning about underlying mechanisms. Yet, with the realization that a signi fi cant proportion of SARS-CoV-2 infections manifest as asymptomatic, new questions arose: 1. Do individuals with a silent infection mount an adaptive immune response that protects them from re-infection and/ or from more severe outcomes upon a new exposure? 2. What are the features of an immune response that controls SARS-CoV-2 infection without triggering any pathological processes? 3. Can we design vaccines that boost immune mechanisms associated with asymptomatic infection to increase their effectiveness against disease? THE INNATE IMMUNE RESPONSE IN ASYMPTOMATIC PERSONS Innate immunity is the fi rst defense against viral infections and plays a critical role in determining the spread and severity of infection. Most cells can contribute to the innate immune response by producing innate cytokines, particularly the type 1 IFNs, which trigger intracellular mechanisms also in neighboring cells to combat infections. Once SARS-CoV-2 infects a cell and starts its replication cycle, the viral RNA gets detected via multiple cellular pattern recognition receptors [48]. This triggers an in fl ammatory cascade, including a broad interferon response limiting viral spread and controlled cell death of infected cells. A delayed interferon response results in exacerbated cytokine production in the later phase, which can lead to severe COVID-19 with tissue and organ damage [49]. Genetic studies have linked innate immune de fi ciencies with severe cases of COVID-19. This includes uncommon genetic defects related to TLR3 and/or TLR7, which affect type 1 IFN immunity. Moreover, autoantibodies that neutralize type 1 IFNs are frequently found in patients with severe pneumonia [50, 51]. These studies underscore the crucial importance of type 1 IFNs in providing protective immunity against SARS-CoV-2 infection. That the type 1 IFN response might play a critical role in effectively controlling SARS-CoV-2 infections without the devel- opment of symptoms derived from studies of healthcare workers who were regularly blood sampled and PCR tested early on during the pandemic. An early type 1 IFN response, detectable prior to PCR positivity, was reported in those with asymptomatic and mild SARS-CoV-2 infection [52]. Moreover, upregulation of the interferon-inducible gene IFI27 was also seen in a subset of individuals with repetitive exposure to SARS-CoV-2, but who never tested PCR positive nor seroconverted [53]. This suggests that an early innate immune response might hinder the virus from effectively replicating, which results in an abortive infection. Both studies found that this early innate immune response was accompanied by the expansion of SARS-CoV-2-speci fi c T cells, which are likely contributing to the fi nal control of the infection [52, 53]. That the rapid kinetics and not the strength of the type 1 IFN response is de fi ning asymptomatic infections was also concluded from a single-cell RNA sequencing study that analyzed long- itudinally collected PBMC samples from 37 patients with diverse disease outcomes, ranging from asymptomatic to severe COVID- 19. Those who remained asymptomatic mounted an early and effective response, while those who showed longer and more enhanced expression of interferon-stimulated genes, controlled the virus less effectively and became symptomatic [54]. This study also showed differences in the NK cell populations and their function. While patients with severe disease had high frequencies of CD56 dim CD16 + NK cells that are associated with cytolytic activity, CD56 bright CD16 − /dim NK cells, which are ef fi cient cytokine producers equipped with immunoregulatory properties, were enriched in individuals with asymptomatic infections. Both NK cell subsets from these asymptomatic individuals showed upregulated transcription of the anti-viral cytokine IFNG [54], suggesting a rather regulatory than cytolytic role of NK cells in asymptomatic viral control. Most other studies that focused on innate immune responses in asymptomatic SARS-CoV-2 infection limited their analysis to the measurement of innate immune cytokines and chemokines in the serum. An early study from Wanzhou, China, compared the immune responses during acute infection of 37 asymptomatic individuals with 37 symptomatic individuals. They reported that B A 0-17 18-39 40-59 60-79 >80 0 20 40 60 80 100 Age in years % of SARS-CoV-2 infected 0-4 5-9 10-16 0 20 40 60 80 100 Age in years % of SARS-CoV-2 infected Asymptomatic cases Mild-moderate cases Severe-critical cases Africa America Europe Asia 0 20 40 60 80 100 Georgraphic region Proportion of asymptomatic SARS-CoV-2 infection (%) C Fig. 3 Proportion of asymptomatic and symptomatic SARS-CoV-2 infection by age and geography. A Proportion of SARS-CoV-2 infected cases with asymptomatic, mild to moderate, and severe to critical symptoms in different age groups. Figure adapted from (Yan et al. Front Med. 2020) [35]. B Proportion of SARS-CoV-2 infected pediatric cases with asymptomatic, mild to moderate, and severe to critical symptoms in different age groups. Figure adapted from (Li et al. Ann Acad Med Singap. 2020) [37]. C Proportion of asymptomatic SARS-CoV-2 infections in different geographic regions. Data from (Chen et al. BMJ Open. 2021) [39] N. Le Bert and T. Samandari 4 Cellular & Molecular Immunology the asymptomatics exhibited lower levels of 18 pro- and anti- in fl ammatory serum cytokines than the symptomatics. Most prominently, the differing molecules were tumor necrosis factor- related apoptosis-inducing ligand (TRAIL), macrophage colony- stimulating factor (M-CSF), growth-regulated oncogene- α (GRO- α ), granulocyte colony-stimulating factor (G-CSF) and IL-6, all of which are known to be pro-in fl ammatory. Interestingly, by comparing asymptomatically infected individuals with healthy controls, they reported signi fi cantly higher levels of stem cell factor (SCF), IL-13, IL-12 p40 and leukemia inhibitory factor (LIF) in the asymptomatic group [55]. In a prospective comparison of U.S. Marine recruits, researchers compared serum proteomic markers among those who either had mild symptoms or asymptomatic seroconversion with anti-SARS- CoV-2 antibodies [56]. They found that while antibody responses and viral shedding were similar in both groups, in fl ammatory chemokines and cytokines including TNF- α , TNF- β , CXCL10 and IL- 8 were higher among the symptomatics, while anti-in fl ammatory analytes known to be involved in tissue repair, such as IL-17C, MMP-10, FGF-19, FGF-21, FGF-23, CXCL5 and CCL-23 were higher in asymptomatic individuals. These differences typically occurred in the fi rst few days after infection. As mentioned above, children have a lower risk of experiencing COVID-19 symptoms compared to adults. Differences in their innate immune system have been suggested as a contributing factor for this distinction [57]. These include children ’ s higher numbers of NK cells and generally reduced levels of pro- in fl ammatory cytokines such as IL-1, IL-2, IL-4, IL-6, IL-10, IFN- γ and TNF- α , a characteristic that would reduce their risk of cytokine storm syndrome. In addition to the reduced pro-in fl ammatory cytokine levels detected in circulation, a strong localized mucosal innate immune response at the site of infection is likely also linked to high rates of asymptomatic infection in young children. An in- depth multi-omics analysis of SARS-CoV-2 infected infants and young children showed profound immune activation in the nose but not in the blood, which was characterized by the in fl ammatory cytokine IFN- α , and T helper (Th) 17 and neutrophil markers IL-17, IL-8, and CXCL1 [58]. This is the opposite of what is observed in adults, who show only minimal changes in nasal cytokine and chemokine levels upon infection. In a review about the immune response to SARS-CoV-2, Agrati et al. assert that several classes of the innate immune response facilitate quick control of SARS-CoV-2 infection: collectins (e.g., mannose-binding lectin), fi colins (e.g., pentraxin 3) and C1q; the early activation of the TLR response; early and potent type 1 IFN; and the absence of autoantibodies against IFNs [59]. THE ANTIBODY RESPONSE IN ASYMPTOMATIC INDIVIDUALS Antibodies that neutralize SARS-CoV-2, whether acquired through vaccination or prior infection, have shown a strong association with protecting against symptomatic and asymptomatic (re-) infections [34, 46, 60]. Additionally, studies demonstrated that monoclonal antibodies targeting SARS-CoV-2 not only signi fi cantly decrease the chances of severe COVID-19 and mortality [61, 62], but also effectively prevent even asymptomatic infections among household contacts [63]. Nevertheless, neutralizing antibodies remain surrogate markers of protection rather than the con fi rmed immune mediator of protection. Initial studies that compared antibody titers between sympto- matic and asymptomatic infection reported contradicting results. One reason might be variation in the time of antibody assessment in relation to viral clearance. Some studies found no difference in antibody titers between individuals who presented with sympto- matic or asymptomatic infection at 4- and 7-months post- resolution [64, 65], while a 180-day longitudinal study of SARS- CoV-2 antibody dynamics associated reduced waning of neutraliz- ing antibodies with disease severity and sustained levels of pro- in fl ammatory molecules [66]. Many studies reported signi fi cantly lower IgG levels and neutralizing antibodies in asymptomatic versus symptomatic infections during the acute and early convalescent phase [55, 67, 68] and a rapid decay [69]. Antibody levels in asymptomatic SARS-CoV-2 infected indivi- duals have most consistently been observed to be lower than in persons with symptoms. This was shown in studies conducted in different regions of the world, such as in Spain, where asymptomatic cases had signi fi cantly reduced neutralizing anti- body titers as compared to those with severe symptoms with sometimes even undetectable levels of neutralizing antibodies [70]. Similar data were reported from coastal Kenya [71], and a study from China showed that asymptomatic cases had signi fi - cantly lower antibody levels relative to symptomatic cases in the acute phase and that 40% of asymptomatic individuals became seronegative as compared to 12.9% of the symptomatic group for anti-nucleoprotein and anti-spike IgG by the early convalescent phase [55]. In infants and very young children with mild symptoms, a recent study found lower titers of spike-binding and neutralizing antibodies during the acute phase of infection than in adults [58]. In contrast to rapidly declining antibody levels during convalescence seen in adults, especially those with asymptomatic infection, the relatively low antibody levels induced in young children, however, persisted for the entire observation period of up to 300 days. Antibodies against SARS-CoV-2 proteins decline over time and if there is no re-infection. With the subsequent evolution of more transmissible variants and the relaxation of non-pharmaceutical interventions in all communities, SARS-CoV-2 has now become an endemic virus throughout the globe; re-infection has become commonplace. Nevertheless, early during the pandemic, it was possible to observe the decline in the antibody response since re- infection after quarantine and isolation was typically instituted. Anti-N antibodies declined more rapidly than anti-S antibodies [72], and a baseline high proportion of virus-speci fi c CD4 + T cells and circulating T follicular helper cells were associated with a slower decline in humoral immunity [73]. A sharp immunological contrast can be drawn between the severely ill and those with mild symptoms. In a study of twelve patients admitted to a Singaporean hospital, we reported that those with mild illness with COVID-19 developed lower antibody levels than those with more severe illness, but those with mild illness had a remarkable early induction of interferon-gamma (IFN- γ )-secreting SARS-CoV-2- speci fi c T cells [74]. Brie fl y, we touch on isotypes (classes) of immune globulins and asymptomatic persons. A study of asymptomatics in Wanzhou, China, showed that the asymptomatics had similar IgM levels as symptomatics but had lower levels of IgG [55]. A study of a Swiss cohort of 431 individuals followed for 6 months post-infection, from Aug 2020 to Jan 2021, before vaccines became available, and while the wildtype strain was still circulating, showed that 17% were asymptomatic. In a subgroup (n = 64) of mostly females between 33 and 68 years old in whom 31% had no symptoms, only 45% developed neutralizing antibody responses that declined rapidly, while 84% had T cell responses that remained more stable [75]. In an adjusted analysis, these researchers showed that the asymptomatics had statistically much lower anti-S IgG and anti-S IgA than symptomatics but no statistical difference in a pooled T cell response or anti-N IgG. Since SARS-CoV-2 infects fi rst the upper airways, Russell and Mestecky proposed in a review the “ primacy ” of the mucosal immune response and speculate that mucosal secretory IgA may explain why most individuals have mild symptoms or no symptoms on exposure to SARS-CoV-2 [76]. They encourage the development of effective mucosal-targeted vaccines and cite three key papers in support of their hypothesis. In the fi rst, higher levels of mucosal anti-RBD and anti-S antibodies (IgM, IgG and IgA) from the upper respiratory tract correlated with reduced N. Le Bert and T. Samandari 5 Cellular & Molecular Immunology SARS-CoV-2 viral load among persons with mild COVID-19 [77]. In the second paper, which recruited a pediatric population, early and intense nasal S1-speci fi c IgA was associated with a rapid decrease in viral load in asymptomatic children as compared to symptomatic children [78]. In the third paper, a research team that developed an oral IgA antibody assay discovered that unvacci- nated children showed evidence of exposure almost exclusively through speci fi c IgA responses in the absence of evidence of symptoms or viral infection [79]. T CELL-MEDIATED IMMUNITY IN ASYMPTOMATICS While neutralizing antibodies can prevent viral infections, the main role of T cells, especially CD8 + T cells, is to detect and clear infected cells. This cellular immune response is important to reduce the spread of the virus within the host and to eliminate it from the body. However, lysis of infected cells can also trigger localized or systemic in fl ammation. Studies of convalescent COVID-19 patients with mild and moderate symptoms quickly established that the vast majority, if not all, mount a multi-speci fi c T cell response comprised of CD4 + and CD8 + T cells [80 – 87]. Though individuals with asymptomatic infections were shown to carry similar viral loads, they clear the infection faster [14, 15], which reduces the time of antigen exposure. Hence questions emerged: Do asymptomatics mount a T cell response? Is it long-lasting? Does it differ in functionality? Does it play a role in the prevention of symptoms? Some early studies suggested, as seen for antibodies [55, 88], that the magnitude of the cellular immune response in an individual correlated with the severity of their disease [64, 87]. Since a large proportion of infections represented as asympto- matic, concerns were raised if this group would develop durable protective immunity so that they would be spared from severe disease upon potential reinfection. The fi rst evidence that SARS-CoV-2-speci fi c T cells can be detected in convalescent individuals with a history of asympto- matic and mild COVID-19 came from a study of Swedish patients recruited in 2020 with symptoms ranging from asymptomatic to severe disease requiring intensive care unit admission. Their data showed that SARS-CoV-2-speci fi c T cells were often detectable and durable in asymptomatic individuals, even in those who remained antibody-seronegative, suggesting that symptoms are not required for the induction of T cell responses [89]. Subsequently, several other studies identi fi ed persons with mild infections who developed low or no antibody responses but had strong T cell responses. In one such study, conducted in Germany among 78 blood donors with mostly mild symptoms of COVID-19 in 2020, 78% of participants did not have detectable antibody responses but had IFN- γ T cell responses against spike and/or membrane at approximately two months after the onset of symptoms [90]. Indeed, Rydyznski Moderbacher and colleagues found that among 39 PCR-positive convalescent patients in San Diego, California, a third of whom had mild disease, the “ strongest associations with low disease severity among acute cases [3- 4 weeks after symptom onset] were IFN- γ -producing CD8 + T cells ” [91]. Evidence that virus-speci fi c T cells expand quickly and stronger in asymptomatic and mild COVID-19 patients came from a single- cell RNA sequencing study. Both effector CD8 + and CD4 + T cell subsets displayed more T-cell receptor clonal expansion in asymptomatic, while enhanced B-cell receptor clonal expansion was seen in those with moderate and severe disease [54]. We performed an analysis of SARS-CoV-2-speci fi c T cell responses during acute infection. This showed that an early and strong T cell response was associated with mild disease, while those with severe COVID-19 mounted a delayed and reduced T cell response during infection [74]. In this longitudinal study, we have seen that the magnitude of the T cell response changes dramatically during the acute and early convalescent phases, with a rapid contraction during the switch from effector to memory T cells. Hence, when comparing T cell responses between symptomatic and asymptomatic individuals, it is important that the time elapsed between viral clearance and the analysis is similar between study participants. However, estimating the time of infection in asymptomatics is far from trivial. Yet, during the initial months of the pandemic, we were able to follow the immune responses of residents from a migrant worker dormitory in Singapore, which allowed us to estimate the time of infection of many asymptomatic individuals [92]. Indeed, in mid-April, the fi rst SARS-CoV-2 infection was con fi rmed in this dormitory, and in June 2020, 478 men were recruited and followed up 2 and 6 weeks later. Throughout the study period, all dormitory residents reported twice daily to a medical post at the dormitory and were tested for temperature, oximeter readings, and other COVID-19-related symptoms. Ser- ological analysis revealed viral spreading before and during the study period, with 27.4% testing seropositive for anti-N and/or neutralizing antibodies at recruitment and nearly 50% of those initially negative seroconverted within the next 6 weeks. Among those who tested seropositive, over 90% remained asymptomatic. By evaluating the period of SARS-CoV-2 antibody seroconversion, we could estimate the time of infection and separate those infected >6 weeks before sampling from those who were likely infected within the last 4 weeks. The immune responses of these asymptomatic individuals were compared with those of hospita- lized COVID-19 patients with mild to severe symptoms. Regardless of antibody persistence, we detected robust T cell frequencies against all viral proteins analyzed (N, M, and S). Interestingly, the magnitude of T cell reactivity and its hierarchy were similar between symptomatic and asymptomatic individuals, an indication that the range of T cell responses was neither augmented nor limited by pathology. Regarding the durability of the response, some patients lost responses to speci fi c antigens after viral clearance, but most asymptomatic individuals did maintain readily detectable T cell responses against multiple antigens. Of note, at 2-3 months post-infection, we found evidence that SARS-CoV-2-speci fi c T cells declined faster in asymptomatic than in symptomatic individuals. This might explain the discrepancy with earlier studies that had compared T cell frequencies several months post-infection and reported a lower T cell response in convalescents with mild or asymptomatic disease [64, 87, 93]. Importantly, our study revealed functional differences of the SARS-CoV-2 speci fi c T cells induced during asymptomatic infec- tion. Despite a similar frequency compared to those with symptoms, upon stimulation, T cells of asymptomatics secreted a nearly log-fold higher level of the antiviral cytokines IFN- γ and IL- 2, accompanied by a low level of IL-10. This highly functional pro fi le, combined with a proportional secretion of anti- in fl ammatory IL-10, might be responsible for the prevention of symptomatic in fl ammation. Notable was the high percentage (93%) of asymptomatic infection in this cohort. This can be partially explained by the fact that workers likely to present with a symptomatic infection (older than 65, having hypertension or diabetes) were moved into other facilities prior to the study. Perhaps more importantly, all subjects in this study were men from either India or Bangladesh, countries that have seen COVID-related fatalities, but at much lower rates as compared with Europe and the US [32]. It is plausible that “ trained immunity ” by live vaccines or more frequent exposure to helminth infections, which have been shown to modify the cytokine pro fi le of T cell responses [94, 95], play a role. Moreover, elevated preexisting and cross-reactive immunity may exist in different parts of the world. A second study by our team that focused on immune responses in asymptomatic infection involved individuals living in rural parts N. Le Bert and T. Samandari 6 Cellular & Molecular Immunology of Kenya [96]. In November 2021, prior to the onset of the omicron wave, we recruited individuals who had not experienced any COVID-19-related symptoms since the beginning of the pandemic, were not in contact with any known case of infection, and who lived in counties with very low reported infection rates. To test for infection prevalence in these asymptomatic study participants, we used serological and T cell assays, which showed positive immune responses in 49% and 81%, respectively. That T cell assays detected a much higher percentage of individuals with a previous SARS-CoV-2 infection or exposure is in line with other studies showing that antibody levels are low and wane more rapidly following asymptomatic infections, while T cell responses can persist for longer. Similar to the migrant workers from Bangladesh and India who showed asymptomatic infection [92], peptide- mediated T cell stimulation triggered IL-10 secretion in addition to the classical Th1 cytokines IFN- γ and IL-2, which suggests this might be a functional pro fi le of T cells in asymptomatics. Interestingly, the hierarchy of T cell responses against various SARS-CoV-2 proteins diffe