Early warning system using primary healthcare data in the post-COVID-19-pandemic era: Brazil nationwide case-study (preprint)

BackgroundSyndromic surveillance utilising primary health care (PHC) data is a valuable tool for early outbreak detection, as demonstrated in the potential to identify COVID-19 outbreaks. However, the potential of such an early warning system in the post-COVID-19 era remains largely unexplored. MethodsWe analysed PHC encounter counts due to respiratory complaints registered in the Brazilian database of the Universal Health System between January and July 2023. We applied EARS (variation C1-C2-C3) and EVI to estimate the weekly thresholds. An alarm was determined when the number of encounters exceeded the week-specific threshold. We used data on hospitalisation due to respiratory disease to classify weeks in which the number of cases surpassed predetermined thresholds as anomalies. We compared EARS and EVIs efficacy in anticipating anomalies. FindingsA total of 119 anomalies were identified across 116 immediate regions during the study period. The EARS-C2 presented the highest early alarm rate, with 81/119 (68%) early alarms, and C1 the lowest, with 71 (60%) early alarms. The lowest true positivity was the EARS-C1 118/1354 (8.7%) and the highest EARS-C3 99/856 (11.6%). ConclusionRoutinely collected PHC data can be successfully used to detect respiratory disease outbreaks in Brazil. Syndromic surveillance enhances timeliness in surveillance strategies, albeit with lower specificity. A combined approach with other strategies is essential to strengthen accuracy, offering a proactive and effective public health response against future outbreaks.

Vaccine coverage and effectiveness against laboratory-confirmed symptomatic and severe Covid-19 in indigenous people in Brazil: a cohort study (preprint)

Background Indigenous people have historically suffered devastating impacts from epidemics and continue to have lower access to healthcare and be especially vulnerable to respiratory infectious. We estimated the coverage and effectiveness of Covid-19 vaccines against laboratory-confirmed Covid-19 cases among indigenous people in Brazil.Methods We linked nationwide Covid-19 vaccination data with flu-like surveillance records and studied a cohort of vaccinated indigenous people aged ≥ 5 years between 18th Jan 2021 and 1st Mar 2022. We considered individuals unexposed from the date they received the first dose of vaccine until the 13th day of vaccination, partially vaccinated from the 14th day after the first dose until the 13th day after receiving the second dose, and fully vaccinated onwards. We estimated the Covid-19 vaccination coverage and used Poisson regression to calculate the relative risks (RR) and vaccine effectiveness (VE) of CoronaVac, ChAdOx1, and BNT162b2 against Covid-19 laboratory-confirmed cases incidence, mortality, hospitalisation, and hospital-progression to Intensive Care Unit (ICU) or death. VE was estimated as (1-RR)*100, comparing unexposed to partially or fully vaccinated.Results By 1st Mar 2022, 48·7% (35·0–62·3) of eligible indigenous people vs 74·8% (57·9–91·8) overall Brazilians had been fully vaccinated for Covid-19. VE for the three Covid-19 vaccines combined was 53% (95%CI:44–60%) for symptomatic cases, 53% (95%CI:-56-86%) for mortality and 41% (95%CI:-35-75%) for hospitalisation. Among hospitalised patients, VE was 87% (95%CI:27–98%) for progression to ICU and 96% (95%CI: 90–99%) for death.Conclusions Lower coverage but similar Covid-19 VE among indigenous people than overall Brazilians suggest the need to expand access, timely vaccination, and urgently offer booster doses to achieve a great level of protection among this group.

Prevalence and risk factors for long COVID after mild disease: a longitudinal study with a symptomatic control group. (preprint)

Background There is limited data on the prevalence and risk factors for long COVID, with a shortage of prospective studies with appropriate control groups and adequate sample size. We therefore performed a prospective study to determine the prevalence and risk factors for long COVID. Methods We recruited patients age [≥] 15 years who were clinically suspected of having acute SARS-CoV-2 infection from September 2020 to April 2021. Nasopharyngeal swabs were collected for RT-PCR 3-5 days post symptom onset. Clinical and sociodemographic characteristics were collected using structured questionnaires from persons positive and negative for SARS-COV-2. Follow-up was performed by telephone interview to assess early outcomes and persistent symptoms. For COVID-19 cases, 5D-3L EuroQol questionnaire was used to assess the impact of symptoms on quality of life. Results We followed 814 participants (412 COVD-19 positive and 402 COVID-19 negative persons) of whom the majority (741 / 814) had mild symptoms. Both the COVID-19 positive and the COVID-19 negative groups had similar sociodemographic and clinical characteristics, except for the rate of hospitalization (15.8% vs 1.5%, respectively). One month after disease onset, 122 (29.6%) individuals reported residual symptoms in the COVID-19 positive group or the long COVID group versus 24 (6%) individuals in the COVID-19 negative group. In the long COVID group, fatigue, olfactory disorder, and myalgia were the most frequent symptoms which occurred in the acute phase. Compared to recovered patients, female sex, older age and having > 5 symptoms during the acute phase were risk factors for long COVID. Quality of life was evaluated in 102 out of 122 cases of long COVID with 57 (55.9%) reporting an impact in at least one dimension of the EuroQol 5D-3L questionnaire. Conclusion In this prospective study consisting predominantly of patients with mild disease, the persistence of symptoms after acute disease was highly associated with long COVID-19 (29.6% vs 6% of COVID negative group). The risk factors for long COVID were older age, female sex, and polysymptomatic acute disease.

Vaccine effectiveness of CoronaVac against symptomatic and severe COVID-19 among children in Brazil during the Omicron period (preprint)

Compared to previous variants, the SARS-CoV-2 B.1.1.529 (Omicron) variant has relatively high infection rates, including among children. Even though severe COVID-19 in children is rare, this group is susceptible to the multisystem inflammatory syndrome in Children (MIS-C), long-COVID and downstream effects of COVID-19, including social isolation and education disruption. There is evidence that vaccination with an mRNA vaccine offers protection against infection and severe forms of COVID-19 for children. However, data on the effectiveness of inactivated virus vaccine, the most used platform worldwide, is scarce during the Omicron period. In Brazil, children between 6 to 11 years are eligible to receive the CoronaVac vaccine. Using a national linked database from January 21, 2022, up to April 19, 2022, during the Omicron dominant period in Brazil, we conducted a test-negative design with 194,258 tests to assess CoronaVac effectiveness against infection and severe (hospitalisation or death) outcomes among children aged 6 to 11 years. The estimated VE for symptomatic infection was 35.0% (95% CI 27.7–41.5) at 0–13 days and 41.5% (95% CI: 34.4–47.7) at ≥ 14 days post-second dose. For severe outcomes (hospitalisation or death) VE was 69.2% (95% CI: 11.7–93.6) at 0–13 days and 63.5% (95% CI: 5.8–90.0). Two doses of CoronaVac in children during the Omicron period showed low levels of protection against symptomatic infection, and modest levels against severe illness.

Protection conferred by vaccine plus previous infection (hybrid immunity) with vaccines of three different platforms during the Omicron variant period in Brazil (preprint)

Hybrid immunity (infection plus vaccination) provided high protection against infection and severe disease in the periods of delta and gamma variants of concern. However, the protection of hybrid immunity in the Omicron era remains unknown. We performed a test-negative study using Brazilian national databases between January 01 and March 22, 2022, a period of predominant circulation of the Omicron variant in Brazil. Hybrid immunity offered low protection against infection, with rapid waning, compared to unvaccinated with or without previous infection. For severe illness (hospitalisation or death), the protection, although already high for unvaccinated pre-infected increased regardless of the type of vaccine (Ad26.COV2.S, BNT162b2, ChAdOx-1 or CoronaVac). In conclusion, during the Omicron-dominant period in Brazil, hybrid immunity offered high protection against severe illness and low protection against infection.

SARS-CoV-2 antibody dynamics in blood donors and COVID-19 epidemiology in eight Brazilian state capitals (preprint)

There are large differences in the shape and size of regional SARS-CoV-2 epidemics in Brazil. Here we tested monthly blood donation samples for IgG antibodies from March 2020 to March 2021 in eight of Brazil’s most populous cities. There was large variation in the inferred attack rate adjusted for seroreversion across cities, and seroprevalence was consistently smaller in women and donors older than 55 years. The age-specific infection fatality rate differed between cities and consistently increased with age. The infection hospitalisation rate (IHR) increased significantly during the gamma-dominated second wave in Manaus, suggesting increased morbidity of the Gamma VOC compared to previous variants circulating in Manaus. The higher disease penetrance associated with the health system’s collapse increased the overall IFR by a minimum factor of 2.91 (95% CrI 2.43–3.53). These results demonstrate large heterogeneity in epidemic spread and highlight the utility of blood donor serosurveillance to monitor SARS-CoV-2 epidemics.

Effectiveness of CoronaVac, ChAdOx1, BNT162b2 and Ad26.COV2.S among individuals with prior SARS-CoV-2 infection in Brazil (preprint)

Background. COVID-19 vaccines have proven highly effective among SARS-CoV-2 naive individuals, but their effectiveness in preventing symptomatic infection and severe outcomes among individuals with prior infection is less clear. Methods. Utilizing national COVID-19 notification, hospitalization, and vaccination datasets from Brazil, we performed a case-control study using a test-negative design to assess the effectiveness of four vaccines (CoronaVac, ChAdOx1, Ad26.COV2.S and BNT162b2) among individuals with laboratory-confirmed prior SARS-CoV-2 infection. We matched RT-PCR positive, symptomatic COVID-19 cases with RT-PCR-negative controls presenting with symptomatic illnesses, restricting both groups to tests performed at least 90 days after an initial infection. We used multivariable conditional logistic regression to compare the odds of test positivity, and the odds of hospitalization or death due to COVID-19, according to vaccination status and time since first or second dose of vaccines. Findings. Among individuals with prior SARS-CoV-2 infection, vaccine effectiveness against symptomatic infection [≥] 14 days from vaccine series completion was 39.4% (95% CI 36.1-42.6) for CoronaVac, 56.0% (95% CI 51.4-60.2) for ChAdOx1, 44.0% (95% CI 31.5-54.2) for Ad26.COV2.S, and 64.8% (95% CI 54.9-72.4) for BNT162b2. For the two-dose vaccine series (CoronaVac, ChAdOx1, and BNT162b2), effectiveness against symptomatic infection was significantly greater after the second dose compared with the first dose. Effectiveness against hospitalization or death [≥] 14 days from vaccine series completion was 81.3% (95% CI 75.3-85.8) for CoronaVac, 89.9% (95% CI 83.5-93.8) for ChAdOx1, 57.7% (95% CI -2.6-82.5) for Ad26.COV2.S, and 89.7% (95% CI 54.3-97.7) for BNT162b2.

Effectiveness of the CoronaVac Vaccine in Prevention of Symptomatic and Progression to Severe COVID-19 in Pregnant Women in Brazil (preprint)

Background: The effectiveness of Covid-19 inactivated vaccines in pregnant women is unknown. We estimated vaccine effectiveness (VE) of CoronaVac against symptomatic and severe Covid-19 and in preventing progression from symptomatic to severe Covid-19 in pregnant women in Brazil. Methods: We conducted a test-negative design study in all pregnant women aged 18 to 49 years in Brazil, linking records of negative and positive SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) tests to national vaccination records. We also linked records of test positive cases with notification of severe, hospitalized or fatal Covid-19. Using logistic regression, we estimated adjusted odds and VE against symptomatic Covid-19 by comparing vaccine status in test positive (confirmed cases) to that in subjects with a negative test result. We also calculated the odds/VE against progression by comparing vaccine status in symptomatic cases to that in severe Covid-19 cases. Results: Of 19838 tested pregnant women, 7424 (37.4%) tested positive for Covid-19 and 588 (7.9%) had severe disease. Only 83% of pregnant women who received a first dose of CoronaVac completed the vaccination scheme. A single dose of the CoronaVac vaccine was not effective at preventing symptomatic Covid-19. Effectiveness of two doses of CoronaVac was 41% (95% CI 27.1- 52.2) against symptomatic Covid-19, 85% (95% CI 59.5-94.8) against severe Covid-19 and (75%; 95% CI 27.9- 91.2) in preventing progression to severe Covid-19 among those infected. Conclusion: A complete regimen of CoronaVac in pregnant women was effective in preventing symptomatic Covid-19, and highly effective against severe illness in a setting that combines high disease burden and elevated Covid-19 related maternal deaths.Funding Information: This study is part of the VigiVac Fiocruz program, partially supported by a donation from the "Fazer o bem faz bem" program. EPS is funded by the Wellcome Trust [Grant number 213589/Z/18/Z]. Declaration of Interests: We declare no competing interests. VO, VB, MB, and MB-N are employees from Fiocruz, a federal public institution, which manufactures Vaxzevria in Brazil, through a full technology transfer agreement with AstraZenecaEthics Approval Statement: This study analysed de-identified data and was approved by the National Ethics committee (CONEP) (CAAE registration no. 50199321.9.0000.0040).

The effectiveness of Vaxzevria and CoronaVac vaccines: A nationwide longitudinal retrospective study of 61 million Brazilians (VigiVac-COVID19). (preprint)

BackgroundHigh rates of virus transmission and the presence of variants of concern can affect vaccine effectiveness (VE). Both conditions occur in low-income countries, which primarily use viral vector or inactivated virus vaccine technologies. Such countries conducted few VE analyses, and most lack the power to evaluate effectiveness in subgroups. MethodsThe present retrospective cohort study evaluated the effectiveness of Vaxzevria and CoronaVac vaccines for COVID-19-related infection in 75,919,840 Brazilian vaccinees from January 18 to July 24, 2021. Study outcomes included documented infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19-related hospitalization, ICU admission, and death. We estimated VE using Cox regression adjusted for individual demographic characteristics. ResultsVaccination with Vaxzevria or CoronaVac was effective against SARS-CoV-2 infection and highly effective against hospitalization, ICU admission, and death in individuals up to 79 years. From 80-89 years of age, Vaxzevria led to 89.9%(95CI:87.7-91.7) VE against death versus 67.2%(95CI:63.6-70.5) for CoronaVac. Above 90 years, 65.4%(95CI:46.1-77.8) protection was conferred to Vaxzevria-vaccinated individuals versus 33.6%(95CI:21.9-43.5) in CoronaVac-vaccinated individuals. Furthermore, the post-vaccination daily incidence rate shows a stepwise increase from younger to elder decades of life. ConclusionsVaxzevria demonstrated overall effectiveness against severe COVID-19 up to 89 years and CoronaVac up to 79 years of age. There is a stepwise effectiveness reduction for both vaccines for each decade of life. Our results suggest that individuals aged 80 years or older may benefit from an expedited booster dose. Ongoing evaluations, including any additional vaccines authorized, are crucial to monitoring long-term vaccine effectiveness.

Post-Acute COVID Syndrome, the Aftermath of Mild to Severe COVID-19 in Brazilian Patients (preprint)

ABSTRACT Objective To describe persistent symptoms after acute COVID-19 in different spectrum of disease severity in a population from an upper/middle income country, and identify the main clinical features impacting the quality of life. Design Cross-sectional study. Setting Outpatient clinic from a public post-COVID-19 health center (CPC) at Bahia-Brazil, a state where 80% are black or mixed race. Participants Patients admitted between August 2020 and February 2021 with symptoms at least one month after the onset of COVID-19. Main outcome measures PACS and related disorders such as hospitalization one month or later after disease onset, biochemical dysregulation and reduced quality of life (EQ-5D-5L questionnaire). Results Among 683 individuals assisted at CPC in this period, 602 were recruited. Patients had average of 52 (±14.6) years, 355 (59%) were female, 528 (88%) black/brown. Individuals were classified as mild (39.9%), moderate (27.9%) or severe (32.2%) during acute illness if outpatient, hospitalized non-UCI or UCI, respectively. Most patients reported a polysymptomatic profile, in median eight (IQR=6-9) acute symptoms. The most frequent residual symptoms were dyspnea (66%), fatigue (62%) and chest pain (43%). Women were more affected regardless disease severity at acute stage: presented more residual symptoms [4 (2-6) vs 3 (2-4)] and a higher impact in quality of life. Altered HbA1c [(184/275 (66.9%)], high CRP levels [195/484 (40.3%)] and anemia [143/545 (26.2%)] were the most common abnormalities in laboratory exams. 76 patients presented HbA1c above 6.4% although only 42 referred previous diagnosis of diabetes mellitus. After one month of disease onset, 30 patients required hospitalization, including seven cases with mild acute illness. Hospital admission after acute disease was required on 30 patients, seven (23%) were mild. Quality of life had been affected for 357/404 (88.4%) patients according to EuroQoL (EQ-5D-5L), mainly the domains of anxiety/depression [severe or extreme anxiety for 79/401 (19.7%)] and pain/discomfort [severe or extreme pain for 71/403 (17.6%)]. The median EuroQoL Global Score was 70 [IQR 50-80]. PACS symptoms such as dyspnea, chest pain, and fatigue, was associated with decreased quality of life. Conclusions PACS, such as dyspnea, chest pain and fatigue, occurred after variable degree of disease severity. Among this majority black/mixed-race patients, woman seemed to be more affected. Other consequences included post-acute hospitalization, and abnormal glucose metabolism and reduced quality of life. Summary Box Section 1: What is already known on this topic: ✓ Post-Acute COVID Syndrome (PACS) comprises a set of persistent or new-onset symptoms after illness onset. ✓ As far as we know, there are no studies describing PACS in a population principally black and mixed-race. Additionally, few studies have addressed PACS among outpatients. Section 2: What this study adds: ✓ Similar PACS were reported after mild, moderate and severe illness. Dyspnea, fatigue and chest pain were the most prevalent symptoms in this population presenting majority of black/mixed-race patients. ✓ Women presented more residual symptoms, a higher frequency of myalgia and worse score for mobility, usual activities, anxiety/depression, and pain. ✓ Hospitalization may occur one month or later after mild or moderate/severe acute infection due to respiratory and vascular disorders. Abnormal glucose metabolism was detected in the absence of previous diagnosis of diabetes mellitus.

COVID-19 herd immunity in the Brazilian Amazon (preprint)

The herd immunity threshold is the proportion of a population that must be immune to an infectious disease, either by natural infection or vaccination such that, in the absence of additional preventative measures, new cases decline and the effective reproduction number falls below unity. This fundamental epidemiological parameter is still unknown for the recently-emerged COVID-19, and mathematical models have predicted very divergent results. Population studies using antibody testing to infer total cumulative infections can provide empirical evidence of the level of population immunity in severely affected areas. Here we show that the transmission of SARS-CoV-2 in Manaus, located in the Brazilian Amazon, increased quickly during March and April and declined more slowly from May to September. In June, one month following the epidemic peak, 44% of the population was seropositive for SARS-CoV-2, equating to a cumulative incidence of 52%, after correcting for the false-negative rate of the antibody test. The seroprevalence fell in July and August due to antibody waning. After correcting for this, we estimate a final epidemic size of 66%. Although non-pharmaceutical interventions, plus a change in population behavior, may have helped to limit SARS-CoV-2 transmission in Manaus, the unusually high infection rate suggests that herd immunity played a significant role in determining the size of the epidemic.

Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection (preprint)

The COVID-19 pandemic has revealed that infection with SARS-CoV-2 can result in a wide range of clinical outcomes in humans, from asymptomatic or mild disease to severe disease that can require mechanical ventilation. An incomplete understanding of immune correlates of protection represents a major barrier to the design of vaccines and therapeutic approaches to prevent infection or limit disease. This deficit is largely due to the lack of prospectively collected, pre-infection samples from indiviuals that go on to become infected with SARS-CoV-2. Here, we utilized data from a screen of genetically diverse mice from the Collaborative Cross (CC) infected with SARS-CoV to determine whether circulating baseline T cell signatures are associated with a lack of viral control and severe disease upon infection. SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Further, early control of virus in the lung correlates with an increased abundance of activated CD4 and CD8 T cells and regulatory T cells prior to infections across strains. A basal propensity of T cells to express IFNg and IL17 over TNFa also correlated with early viral control. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. While future studies of human samples prior to infection with SARS-CoV-2 are required, our studies in mice with SARS-CoV serve as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection. Identification of basal immune predictors in humans could allow for identification of individuals at highest risk of severe clinical and virologic outcomes upon infection, who may thus most benefit from available clinical interventions to restrict infection and disease.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry (preprint)

Coronavirus interaction with viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter the host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkeys, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants of susceptibility of ACE2 orthologs to viral entry, we compared koala and mouse ACE2 sequences with human ortholog, and identified the key residues in koala or mouse ACE2 that restrict its viral receptor activity. Humanization of these critical residues could render the capabilities of koala and mouse ACE2 to bind viral spike protein and facilitate the viral entry. Our work identifies the genetic determinant of ACE2 for SARS-CoV-2 susceptibility, and a single mutation could restore the mouse ACE2 receptor activity, providing a potential avenue for the development of mouse model of SARS-CoV-2.

Cryo-EM structure of S-Trimer, a subunit vaccine candidate for COVID-19 (preprint)

Less than a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 22 million people worldwide with a death toll approaching 1 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Angstrom and 2.6 Angstrom respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine.

SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism (preprint)

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus that underlies the current COVID-19 pandemic. SARS-CoV-2 is thought to disable various features of host immunity and cellular defense. The SARS-CoV-2 nonstructural protein 1 (Nsp1) is known to inhibit host protein translation and could be a target for antiviral therapy against COVID-19. However, how SARS-CoV-2 circumvents this translational blockage for the production of its own proteins is an open question. Here, we report a bipartite mechanism of SARS-CoV-2 Nsp1 which operates by: (1) hijacking the host ribosome via direct interaction of its C-terminal domain (CT) with the 40S ribosomal subunit and (2) specifically lifting this inhibition for SARS-CoV-2 via a direct interaction of its N-terminal domain (NT) with the 5 untranslated region (5 UTR) of SARS-CoV-2 mRNA. We show that while Nsp1-CT is sufficient for binding to 40S and inhibition of host protein translation, the 5 UTR of SARS-CoV-2 mRNA removes this inhibition by binding to Nsp1-NT, suggesting that the Nsp1-NT-UTR interaction is incompatible with the Nsp1-CT-40S interaction. Indeed, lengthening the linker between Nsp1-NT and Nsp1-CT of Nsp1 progressively reduced the ability of SARS-CoV-2 5 UTR to escape the translational inhibition, supporting that the incompatibility is likely steric in nature. The short SL1 region of the 5 UTR is required for viral mRNA translation in the presence of Nsp1. Thus, our data provide a comprehensive view on how Nsp1 switches infected cells from host mRNA translation to SARS-CoV-2 mRNA translation, and that Nsp1 and 5 UTR may be targeted for anti-COVID-19 therapeutics.

An immunodominance hierarchy exists in CD8+ T cell responses to HLA-A*02:01-restricted epitopes identified from the non-structural polyprotein 1a of SARS-CoV-2. (preprint)

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-{gamma}-producing CD8+ T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines. ImportanceFor the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.