BNT162b2 vaccine induced variant-specific immunity, safety and risk of Omicron breakthrough infection in children aged 5 to 11 years: A cohort study (preprint)

There is little information on BNT162b2 vaccine-induced variant-specific immunogenicity, safety data and dynamics of breakthrough infections in pediatric populations. We addressed these questions using a prospective two dose BNT162b2(10mcg) vaccination cohort study of healthy children 5–11 years in Singapore. Follow up included blood samples at scheduled visits, daily vaccination symptom diary and confirmation of SARS-CoV-2 infection. Surrogate virus neutralization test (sVNT) and spike-specific T cell responses against SARS-CoV-2 variants were performed. The mean age of 127 participants was 8.27 years (SD: 1.95) and 51.2% were males. The median sVNT level against original variant after 1 dose and 2 dose vaccination was 61.4% and 95.1% respectively (p < 0.0001). Neutralizing antibodies against the Omicron variant was the lowest, median 22.4% (IQR:16.5 to 30.8). However, T cell IFN-γ cytokine response against Omicron variant was high and remained so about 4 months after vaccination. Fever rate increased significantly from 4% (dose 1) to 11.5% (dose 2). The risk of Omicron breakthrough infection decreased by 7.8% for every 1% increase in sVNT inhibition level measured after dose 2 vaccination. BNT162b2 vaccines were safe, induced good T cell responses but poor neutralizing antibodies against Omicron in children. Neutralizing antibody levels post-vaccination was predictive of subsequent breakthrough infection.

Engineering Immunosuppressive Drug Resistant Armored (IDRA) SARS-CoV-2 T cells for cell therapy (preprint)

Solid organ transplant (SOT) recipients receive immunosuppressive drugs (ISDs) and are susceptible to develop severe COVID-19. Here, we analyzed the Spike-specific T cell response after 3 doses of mRNA vaccine in a group of SOT patients (n=136) treated with different ISDs. We demonstrated that combination of calcineurin inhibitor (CNI), mycophenolate mofetil (MMF), and prednisone (Pred) treatment regimen displayed strong suppression of mRNA vaccine-induced Spike-specific cellular response. Such defects have clinical consequences since the magnitude of vaccine-induced Spike-specific T cells was directly proportional to the ability of SOT patients to rapidly clear SARS-CoV-2 after breakthrough infection. To then compensate the T cell defects induced by immunosuppressive treatment and to develop an alternative therapeutic strategy for SOT patients, we describe the production of 6 distinct SARS-CoV-2 epitope-specific ISD-resistant T Cell Receptor (TCR)-T cells engineered using mRNA electroporation method with reactivity minimally affected by mutations occurring in Beta, Delta, Gamma, and Omicron variants. This strategy with transient expression characteristics marks an improvement in the immunotherapeutic field and provides an attractive and novel therapeutic possibility for immunosuppressed COVID-19 patients.

Anti-inflammatory SARS-CoV-2 T cell immunity in asymptomatic seronegative Kenyan adults (preprint)

Antibodies are used to estimate prevalence of past infection. However, T cell responses against SARS-CoV-2 may more accurately define prevalence because SARS-CoV-2-specific antibodies wane. In November-December 2021, we studied serological and cellular immune responses in residents of rural Kenya who had not experienced any respiratory symptom nor had contact with COVID-19 cases. Among participants we detected anti-spike antibodies in 41.0% and T cell responses against [≥]2 SARS-CoV-2 proteins in 82.5%, which implies that serosurveys underestimate SARS-CoV-2 prevalence in settings where asymptomatic infections prevail. Distinct from cellular immunity in European and Asian COVID-19 convalescents, strong T cell immunogenicity was observed against viral accessory proteins in these asymptomatic Africans, as well as a higher IL-10/IFN-{gamma} ratio cytokine profile, suggesting that environmental or genetic factors modulate pro-inflammatory responses.

Omicron reactive multi protein specific CD4 T cells defines cellular immune response induced by inactivated virus vaccines (preprint)

Summary Unlike mRNA vaccines based only on the Spike protein, inactivated SARS-CoV-2 vaccines should induce a diversified T cell response recognizing distinct structural proteins. Here we performed a comparative analysis of SARS-CoV-2 specific T cells in healthy individuals following vaccination with inactivated SARS-CoV-2 or mRNA vaccines. Relative to Spike mRNA vaccination, inactivated vaccines elicited a lower magnitude of Spike-specific T cells, but the combined Membrane, Nucleoprotein and Spike-specific T cell response was quantitatively comparable to the sole Spike T cell response induced by mRNA vaccines, and they efficiently tolerate the mutations characterizing the Omicron lineage. However, this multi-protein specific T cell response was not mediated by a coordinated CD4 and CD8 T cell expansion but by selected priming of CD4 T cells. These findings can help in defining the role of CD4 and CD8 T cells in the efficacy of the different vaccines to control severe COVID-19 after Omicron infection.

SARS-CoV-2 infection in vaccinated individuals induces virus-specific nasal resident CD8 and CD4 T cells of broad specificity (preprint)

Rapid recognition of SARS-CoV-2-infected cells by T cells resident in the upper airway might provide an important layer of protection against COVID-19. Whether parenchymal SARS-CoV-2 vaccination or infection induce nasal resident T cells specific for distinct SARS-CoV-2 proteins is unknown. We collected T cells from the nasal secretion of COVID-19 vaccinees, who either experienced SARS-CoV-2 infection after vaccination (n=20) or not (n=15) and analyzed their phenotype, SARS-CoV-2 specificity and function. Nasal-resident IFN-γ producing SARS-CoV-2-specific CD8 and CD4 T cells were detected exclusively in vaccinees who experienced SARS-CoV-2 breakthrough infection. Importantly, the vaccine priming of Spike-specific T cells does not suppress the induction of CD8 and CD4 T cells specific for different SARS-CoV-2 proteins (i.e. NP and NSP-12) that persisted in the nasal cavity up to 3 months after infection. These data highlight the importance of viral nasal challenge in the formation of SARS-CoV-2 specific antiviral immunity at the site of primary infection and further define the immunological features of SARS-CoV-2 hybrid immunity.

Favourable vaccine-induced SARS-CoV-2 specific T cell response profile in patients undergoing immune-modifying therapies (preprint)

Patients undergoing immune-modifying therapies demonstrate a reduced humoral response after COVID-19 vaccination, but we lack a proper evaluation of the impact of such therapies on vaccine-induced T cell responses. Here, we longitudinally characterised humoral and Spike-specific T cell responses in IBD patients who are on antimetabolite therapy (azathioprine or methotrexate), TNF inhibitors and/or other biologic treatment (anti-integrin or anti-p40) after mRNA vaccination. We demonstrated that a Spike-specific T cell response is not only induced in treated IBD patients at levels similar to healthy individuals, but also sustained at higher magnitude, particularly in those treated with TNF inhibitor therapy. Furthermore, the Spike-specific T cell response in these patients is mainly preserved against mutations present in SARS-CoV-2 B.1.1.529 (Omicron) and characterized by a Th1/IL-10 cytokine profile. Thus, despite the humoral response defects, the favourable profile of vaccine-induced T cell responses might still provide a layer of COVID-19 protection to patients under immune-modifying therapies.

Mutations of SARS-CoV-2 variants of concern escaping Spike-specific T cells (preprint)

The amino acid (AA) mutations that characterise the different variants of concern (VOCs), which replaced the ancestral SARS-CoV-2 Wuhan-Hu-1 isolate worldwide, provide biological advantages such as increased infectivity and partial escape from humoral immunity. Here we analysed the impact of these mutations on vaccination- and infection-induced Spike-specific T cells. We confirmed that, in the majority of infected or vaccinated individuals, different mutations present in a single VOC (Delta) or a combined mosaic of more than 30 AA substitutions and deletions found in Alpha, Beta, Gamma, Delta and Omicron VOCs cause modest alteration in the global Spike-specific T cell response. However, distinct numerically dominant Spike-specific CD4 and CD8 T cells preferentially targeted regions affected by AA mutations and do not recognise the mutated peptides. Importantly, some of these mutations, such as N501Y (present in Alpha, Beta, Gamma, and Omicron) and L452R (present in Delta), known to provide biological advantage to SARS-CoV-2 in terms of infectivity also abolished CD8 T cell recognition. Taken together, our data show that while global mRNA vaccine- and infection-induced Spike-specific T cells largely tolerate the diverse mutations present in VOCs, single Spike-specific T cells might contribute to the natural selection of SARS-CoV-2 variants.

Durable T cell responses contrast with faster antibody waning in BNT162b2-vaccinated elderly at 6 month (preprint)

Efficient COVID-19 vaccines have been developed in record time. Here, we present findings from a comprehensive and integrated analysis of multiple compartments of the memory immune response in 312 individuals vaccinated with the BNT162b2 mRNA vaccine. Two vaccine doses induced high antibody and T cell responses in most individuals. However, antibody recognition of the Spike protein of delta variant was less efficient than that of the Wuhan strain. Age stratified analyses identified a group of low antibody responders where individuals ≥ 60 years were overrepresented. Waning of the antibody and cellular responses was observed in 30% of the vaccinees after six months. However, age did not influence the waning of these responses. Taken together, while individuals ≥ 60 years old took longer to acquire vaccine-induced immunity, they develop more sustained acquired immunity at six months post-vaccination. However, the higher proportion of older individuals in the group of antibody low responders and the lower antibody reactivity the Delta variant call for a booster immunization to increase immune responses and protection.

Differential immunogenicity of homologous versus heterologous boost in Ad26.COV2.S vaccine recipients (preprint)

Protection offered by COVID-19 vaccines wanes over time, requiring an evaluation of different boosting strategies to revert such a trend and enhance the quantity and quality of Spike-specific humoral and cellular immune responses. These immunological parameters in homologous or heterologous vaccination boosts have thus far been studied for mRNA and ChAdOx1 nCoV-19 vaccines, but knowledge on individuals who received a single dose of Ad26.COV2.S is lacking. We studied Spike-specific humoral and cellular immunity in Ad26.COV2.S vaccinated individuals (n=55) who were either primed with Ad26.COV2.S only (n=13), or boosted with a homologous (Ad26.COV2.S, n=28) or heterologous (BNT162b2, n=14) second dose. We compared our findings with the results found in individuals vaccinated with a single (n=16) or double (n=44) dose of BNT162b2. We observed that a strategy of heterologous vaccination enhanced the quantity and breadth of both, Spike-specific humoral and cellular immunity in Ad26.COV2.S vaccinated. In contrast, the impact of homologous boost was quantitatively minimal in Ad26.COV2.S vaccinated and Spike-specific antibodies and T cells were narrowly focused to the S1 region. Although a direct association between quantity and quality of immunological parameters and in vivo protection has not been demonstrated, the immunological features of Spike-specific humoral and cellular immune responses support the utilization of a heterologous strategy of vaccine boost in individuals who received Ad26.COV2.S vaccination.

Pre-existing polymerase-specific T cells expand in abortive seronegative SARS-CoV-2 infection (preprint)

Individuals with likely exposure to the highly infectious SARS-CoV-2 do not necessarily develop PCR or antibody positivity, suggesting some may clear sub-clinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1-5. We hypothesised that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-26-12, would expand in vivo to mediate rapid viral control, potentially aborting infection. We studied T cells against the replication transcription complex (RTC) of SARS-CoV-2 since this is transcribed first in the viral life cycle13-15 and should be highly conserved. We measured SARS-CoV-2-reactive T cells in a cohort of intensively monitored healthcare workers (HCW) who remained repeatedly negative by PCR, antibody binding, and neutralisation for SARS-CoV-2 (exposed seronegative, ES). 16-weeks post-recruitment, ES had memory T cells that were stronger and more multispecific than an unexposed pre-pandemic cohort, and more frequently directed against the RTC than the structural protein-dominated responses seen post-detectable infection (matched concurrent cohort). The postulate that HCW with the strongest RTC-specific T cells had an abortive infection was supported by a low-level increase in IFI27 transcript, a robust early innate signature of SARS-CoV-2 infection16. We showed that the RNA-polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and was preferentially targeted by T cells from UK and Singapore pre-pandemic cohorts and from ES. RTC epitope-specific T cells capable of cross-recognising HCoV variants were identified in ES. Longitudinal samples from ES and an additional validation cohort, showed pre-existing RNA-polymerase-specific T cells expanded in vivo following SARS-CoV-2 exposure, becoming enriched in the memory response of those with abortive compared to overt infection. In summary, we provide evidence of abortive seronegative SARS-CoV-2 infection with expansion of cross-reactive RTC-specific T cells, highlighting these highly conserved proteins as targets for future vaccines against endemic and emerging Coronaviridae.

Rapid determination of the wide dynamic range of SARS-CoV-2 Spike T cell responses in whole blood of vaccinated and naturally infected (preprint)

Background: Antibodies and T cells cooperate to control virus infections. The definition of the correlates of protection necessary to manage the COVID-19 pandemic, require both immune parameters but the complexity of traditional tests limits virus-specific T cell measurements. Methods: We test the sensitivity and performance of a simple and rapid SARS-CoV-2 Spike-specific T cell test based on stimulation of whole blood with peptides covering the SARS-CoV-2 Spike protein followed by cytokine (IFN-{gamma}, IL-2) measurement in different cohorts including BNT162b2 vaccinated (n=112; 201 samples), convalescent asymptomatic (n=62; 62 samples) and symptomatic (n=68; 115 samples) COVID-19 patients and SARS-CoV-1 convalescent individuals (n=12; 12 samples). Results: The sensitivity of the rapid cytokine whole blood test equates traditional methods of T cell analysis (ELISPOT, Activation Induced Markers). Utilizing this test we observed that Spike-specific T cells in vaccinated preferentially target the S2 region of Spike and that their mean magnitude is similar between them and SARS-CoV-2 convalescents at 3 months after vaccine or virus priming respectively. However, a wide heterogeneity of Spike-specific T cell magnitude characterizes the individual responses irrespective of the time of analysis. No correlation between neutralizing antibody levels and Spike-specific T cell magnitude were found. Conclusions: Rapid measurement of cytokine production in whole blood after peptide activation revealed a wide dynamic range of Spike-specific T cell response after vaccination that cannot be predicted from neutralizing antibody quantities. Both Spike-specific humoral and cellular immunity should be tested after vaccination to define the correlates of protection necessary to evaluate current vaccine strategies.

Differential Effects of the Second SARS-CoV-2 mRNA Vaccine Dose on T Cell Immunity in Naïve and COVID-19 Recovered Individuals (preprint)

The rapid development and deployment of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naïve individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naïve individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. Here we characterized, SARS-CoV-2 spike-specific humoral and cellular immunity in naïve and previously infected individuals during and after two-doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naïve individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.Funding: Research reported in this publication was supported in part by National Cancer Institute of the NIH (5R01HD102614-02; R01CA249204 and R01CA248984) and ISMMS seed fund to EG. The authors gratefully acknowledge use of the services and facilities of the Tisch Cancer Institute supported by the NCI Cancer Center Support Grant (P30 CA196521). MS was supported by a NCI training grant (T32CA078207). This work was supported by ISMMS seed fund to JO; Instituto de Salud Carlos III, COV20-00668 to RCR; Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181) co-financed by European Development Regional Fund “A way to achieve Europe” to EP; Instituto de Salud Carlos III, Spain (COV20/00170); Government of Cantabria, Spain (2020UIC22-PUB 0019) to MLH; Instituto de Salud Carlos III (PI16CIII/00012) to PP; Fondo Social Europeo e Iniciativa de Empleo Juvenil YEI (Grant PEJ2018-004557-A) to MPE; REDInREN 016/009/009 ISCIII; This project has received funding from the European Union’s Horizon 2020 research and innovation programme VACCELERATE under grant agreement No [101037867] to JO.Conflict of Interest: AB declares the filling of a patent application relating to the use of peptide pools in whole blood for detection of SARS-CoV-2 T cells (pending). The other authors declare no competing interests.Ethical Approval: The study protocols for the collection of clinical specimens from individuals with and without SARS-CoV-2 infection were reviewed and approved by Hospital La Paz, Hospital 12 de Octubre, Hospital Gregorio Marañón, IIS-Fundación Jimenez Díaz, Hospital Universitario Marqués de Valdecilla-IDIVAL and Hospital Puerta de Hierro Clinical Research Ethics Committee (CEIm), and Mount Sinai Hospital Institutional Review Board (IRB).

Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals (preprint)

The rapid development and deployment of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has been questioned. Here we characterized SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during full BNT162b2 vaccination. Our results demonstrate that the second dose increases both the humoral and cellular immunity in naive individuals. On the contrary, the second BNT162b2 vaccine dose results in a reduction of cellular immunity in COVID-19 recovered individuals, which suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.

Early T Cell and Binding Antibody Responses are Associated with COVID-19 RNA Vaccine Efficacy Onset (preprint)

RNA vaccines against Covid-19 have demonstrated ~95% efficacy in Phase III clinical trials. Although complete vaccination consisted of two-doses, the onset of protection for both licensed RNA vaccines was observed as early as 12 days after a single dose. The adaptive immune response that coincides with this onset of protection could represent the necessary elements of immunity against Covid-19. Herein, we tracked the early adaptive immune responses after Covid-19 RNA vaccination, in a cohort of 20 healthcare workers. Our findings suggest that early T cell and binding antibody responses, rather than either receptor-blocking or virus neutralizing activity, induced early protection against Covid-19.Funding: This study was partially funded through a generousdonation from The Hourglass to support Covid-19 research in ViREMiCS. SK receives salary support from the Transition Award, RdA receives funding from the Open Research Fund Young Investigator Award, JGL and EEO receive salary support from the Clinician Scientist Award, and AB receives salary support from the Singapore Translational Research Award, all administered by the National Medical Research Council of Singapore.Conflict of Interest: Duke-NUS Medical School is in partnership with Arcturus Therapeutics to develop a self-replicating RNA vaccine against Covid-19, with EEO as the principal investigator. No monetary or personal benefits are derived from this partnership.Ethical Approval: This study was approved by the SingHealth Centralized Institutional Review Board (CIRB/F2021/2014). Healthcare workers (HCWs) from the Singapore Health Services institutions whowere eligible for Covid-19 vaccination were invited to participate in this study, and written informed consent was obtained.

Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection (preprint)

The efficacy of virus-specific T cells in clearing pathogens involves a fine balance between their antiviral and inflammatory features. SARS-CoV-2-specific T cells in individuals who clear SARS-CoV-2 infection without symptoms or disease could reveal non-pathological yet protective characteristics. We therefore compared the quantity and function of SARS-CoV-2-specific T cells in a cohort of asymptomatic individuals (n=85) with that of symptomatic COVID-19 patients (n=76), at different time points after antibody seroconversion. We quantified T cells reactive to structural proteins (M, NP and Spike) using ELISpot assays, and measured the magnitude of cytokine secretion (IL-2, IFN-{gamma}, IL-4, IL-6, IL-1{beta}, TNF- and IL-10) in whole blood following T cell activation with SARS-CoV-2 peptide pools as a functional readout. Frequencies of T cells specific for the different SARS-CoV-2 proteins in the early phases of recovery were similar between asymptomatic and symptomatic individuals. However, we detected an increased IFN-{gamma} and IL-2 production in asymptomatic compared to symptomatic individuals after activation of SARS-CoV-2-specific T cells in blood. This was associated with a proportional secretion of IL-10 and pro-inflammatory cytokines (IL-6, TNF- and IL-1{beta}) only in asymptomatic infection, while a disproportionate secretion of inflammatory cytokines was triggered by SARS-CoV-2-specific T cell activation in symptomatic individuals. Thus, asymptomatic SARS-CoV-2 infected individuals are not characterized by a weak antiviral immunity; on the contrary, they mount a robust and highly functional virus-specific cellular immune response. Their ability to induce a proportionate production of IL-10 might help to reduce inflammatory events during viral clearance.

Unheeded SARS-CoV-2 protein? Look deep into negative-sense RNA (preprint)

SARS-CoV-2 is a novel ssRNA+ virus from the Coronaviridae family, which has caused the global COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest of RNA viruses, comprising of 26 known protein-coding loci. This study aimed to explore the coding potential of negative-strand RNA intermediate for its potential to contain additional protein coding-loci. Surprisingly, we have found several putative ORFs and one brandt new functional SARS-CoV-2 protein-coding loci and called it Avo1 (Ambient viral ORF1). This sequence is located on negative-sense RNA intermediate and bona fide coding for 81 amino acid residues long protein and contains strong Kozak sequence for translation on eukaryotic ribosomes. In silico translated protein Avo1 has a predominantly alpha-helical structure. The existence of Avo1 gene is supported also by its evolutionarily and structural conservation in RaTG13 bat coronavirus. The nucleotide sequence of Avo1 also contains a unique SREBP2 binding site which is closely related to the so-called cytokine storm in severe COVID-19 patients. Altogether, our results suggest the existence of still undescribed SARS-CoV-2 protein, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.

Additional analyses exploring the hypothesized transdifferentiation of plasmablasts to developing neutrophils in severe COVID-19 (preprint)

We thank Alquicira-Hernandez et al. for their reanalysis of our single-cell transcriptomic dataset profiling peripheral immune responses to severe COVID-19. We agree that careful analysis of single-cell sequencing data is important for generating cogent hypotheses but find several aspects of their criticism of our analysis to be problematic. Here we respond briefly to misunderstandings and inaccuracies in their commentary that may have led to misinformed interpretation of our results.

Early induction of SARS-CoV-2 specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients (preprint)

Virus-specific humoral and cellular immunity act synergistically to protect the host from viral infection. We interrogated the dynamic changes of virological and immunological parameters in 12 patients with symptomatic acute SARS-CoV-2 infection from disease onset to convalescence or death. We quantified SARS-CoV-2 viral RNA in the respiratory tract in parallel with antibodies and circulating T cells specific for various structural (NP, M, ORF3a and spike) and non-structural proteins (ORF7/8, NSP7 and NSP13). We observed that while rapid induction and quantity of humoral responses were associated with increased disease severity, an early induction of SARS-CoV-2 specific T cells was present in patients with mild disease and accelerated viral clearance. These findings provide further support for a protective role of SARS-CoV-2 specific T cells over antibodies during SARS-CoV-2 infection with important implications in vaccine design and immune-monitoring.

Different pattern of pre-existing SARS-COV-2 specific T cell immunity in SARS-recovered and uninfected individuals (preprint)

Memory T cells induced by previous infections can influence the course of new viral infections. Little is known about the pattern of SARS-CoV-2 specific pre-existing memory T cells in human. Here, we first studied T cell responses to structural (nucleocapsid protein, NP) and non-structural (NSP-7 and NSP13 of ORF1) regions of SARS-CoV-2 in convalescent from COVID-19 (n=24). In all of them we demonstrated the presence of CD4 and CD8 T cells recognizing multiple regions of the NP protein. We then show that SARS-recovered patients (n=23), 17 years after the 2003 outbreak, still possess long-lasting memory T cells reactive to SARS-NP, which displayed robust cross-reactivity to SARS-CoV-2 NP. Surprisingly, we observed a differential pattern of SARS-CoV-2 specific T cell immunodominance in individuals with no history of SARS, COVID-19 or contact with SARS/COVID-19 patients (n=18). Half of them (9/18) possess T cells targeting the ORF-1 coded proteins NSP7 and 13, which were rarely detected in COVID-19- and SARS-recovered patients. Epitope characterization of NSP7-specific T cells showed recognition of protein fragments with low homology to "common cold" human coronaviruses but conserved among animal betacoranaviruses. Thus, infection with betacoronaviruses induces strong and long-lasting T cell immunity to the structural protein NP. Understanding how pre-existing ORF-1-specific T cells present in the general population impact susceptibility and pathogenesis of SARS-CoV-2 infection is of paramount importance for the management of the current COVID-19 pandemic.