Dissecting the dynamics of SARS-CoV-2 reinfections in blood donors with pauci- or asymptomatic COVID-19 disease course at initial infection.

BACKGROUND: Understanding the dynamics of SARS-CoV-2 reinfections is crucial for public health policy, vaccine development, and long-term disease management. However, data on reinfections in the general population remains scarce. OBJECTIVES: This study aimed to investigate SARS-CoV-2 antibody dynamics among Austrian blood donors, representing healthy adults, over two years following primary infection and to evaluate the reinfection risk. METHODS: 117,895 blood donations were analysed for SARS-CoV-2 total anti-N levels from June 2020 to December 2023. We examined anti-N and anti-S antibody dynamics and in vitro functionality in 230 study participants at five defined times during 24 months, assessing associations with demographics, vaccination status, and reinfection awareness. RESULTS: The seroprevalence of SARS-CoV-2 infection-derived anti-N antibodies increased over time, reaching 90% by February 2023 and remaining at that level since then. According to serological screenings, we found an 88% reinfection rate, which is in contrast to participants' reports indicating a reinfection rate of 59%. Our data further reveal that about 26% of reinfections went completely unnoticed. Antibody dynamics were independent of age, sex, and ABO blood group. Interestingly, individuals with multiple reinfections reported symptoms more frequently during their primary infection. Our results further show that vaccination modestly affected reinfection risk and disease course. CONCLUSION: SARS-CoV-2 reinfections were uncommon until the end of 2021 but became common with the advent of Omicron. This study highlights the underestimation of reinfection rates in healthy adults and underscores the need for continued surveillance, which is an important support for public health policies and intervention strategies.

Epstein-Barr virus reactivation is not causative for post-COVID-19-syndrome in individuals with asymptomatic or mild SARS-CoV-2 disease course.

PURPOSE: Post-COVID-19-Syndrome (PCS) frequently occurs after an infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, the understanding of causative mechanisms is still limited. Aim of this study was to determine the PCS rate among SARS-CoV-2 seropositive blood donors as representatives of supposedly healthy adults, who had experienced an asymptomatic or mild COVID-19 disease course, and to examine whether Epstein-Barr virus (EBV) is reactivated in individuals reporting PCS. METHODS: The PCS rate was determined using questionnaires that included questions about infection and persistent symptoms. Pre-pandemic blood samples and samples collected at regular, pre-defined times after a SARS-CoV-2 infection were analysed for neopterin, a marker for antiviral immune responses, by an enzyme-linked immunosorbent assay (ELISA). Additionally, we determined the rate of SARS-CoV-2 anti-N total antibodies using an electrochemiluminescence immunoassay (ECLIA). Furthermore, quantitative real-time polymerase chain reaction (qPCR) to detect EBV DNA and ECLIA screening for EBV viral capsid-antigen (VCA) IgM, IgG and EBV nuclear antigen 1 (EBNA) IgG were performed. RESULTS: Our data reveal that 18% of all infections result in PCS, with symptoms lasting for up to one year. In individuals reporting PCS, no elevated levels of neopterin were detected, indicating no persisting pro-inflammatory, antiviral immune response. SARS-CoV-2 antibody levels were declining in all participants in comparable manner over time, pointing to a successful virus clearance. In individuals with PCS, no EBV DNA could be detected. Furthermore, no differences in EBV specific antibody levels could be shown in PCS groups compared to non-PCS groups. CONCLUSION: Our data suggest that PCS in per se healthy, immunocompetent adults cannot be ascribed to a reactivation of EBV.

Three rounds of a national external quality assessment reveal a link between disharmonic anti-SARS-CoV-2 antibody quantifications and the infection stage.

OBJECTIVES: The WHO's standardized measuring unit, "binding antibody units per milliliter (BAU/mL)," should allow the harmonization of quantitative results by different commercial Anti-SARS-CoV-2 immunoassays. However, multiple studies demonstrate inter-assay discrepancies. The antigenic changes of the Omicron variant affect the performance of Spike-specific immunoassays. This study evaluated the variation of quantitative Anti-SARS-CoV-2-Spike antibody measurements among 46, 50, and 44 laboratories in three rounds of a national external quality assessment (EQA) prior to and after the emergence of the Omicron variant in a diagnostic near-to-real-life setting. METHODS: We analyzed results reported by the EQA participant laboratories from single and sequential samples from SARS-CoV-2 convalescent, acutely infected, and vaccinated individuals, including samples obtained after primary and breakthrough infections with the Omicron variant. RESULTS: The three immunoassays most commonly used by the participants displayed a low intra-assay and inter-laboratory variation with excellent reproducibility using identical samples sent to the participants in duplicates. In contrast, the inter-assay variation was very high with all samples. Notably, the ratios of BAU/mL levels quantified by different immunoassays were not equal among all samples but differed between vaccination, past, and acute infection, including primary infection with the Omicron variant. The antibody kinetics measured in vaccinated individuals strongly depended on the applied immunoassay. CONCLUSIONS: Measured BAU/mL levels are only inter-changeable among different laboratories when the same assay was used for their assessment. Highly variable ratios of BAU/mL quantifications among different immunoassays and infection stages argue against the usage of universal inter-assay conversion factors.

Comprehensive Comparison of Seven SARS-CoV-2-Specific Surrogate Virus Neutralization and Anti-Spike IgG Antibody Assays Using a Live-Virus Neutralization Assay as a Reference.

Neutralizing antibodies (nAbs) are considered a valuable marker for measuring humoral immunity against SARS-CoV-2. However, live-virus neutralization tests (NTs) require high-biosafety-level laboratories and are time-consuming. Therefore, surrogate virus neutralization tests (sVNTs) have been widely applied, but unlike most anti-spike (S) antibody assays, NTs and sVNTs are not harmonized, requiring further evaluation and comparative analyses. This study compared seven commercial sVNTs and anti-S-antibody assays with a live-virus NT as a reference, using a panel of 720 single and longitudinal serum samples from 666 convalescent patients after SARS-CoV-2 infection. The sensitivity of these assays for detecting antibodies ranged from 48 to 94% after PCR-confirmed infection and from 56% to 100% relative to positivity in the in-house live-virus NT. Furthermore, we performed receiver operating characteristic (ROC) curve analyses to determine which immunoassays were most suitable for assessing nAb titers exceeding a specific cutoff (NT titer, ≥80) and found that the NeutraLISA and the cPass assays reached the highest area under the curve (AUC), exceeding 0.91. In addition, when the assays were compared for their correlation with nAb kinetics over time in a set of longitudinal samples, the extent of the measured decrease of nAbs after infection varied widely among the evaluated immunoassays. Finally, in vaccinated convalescent patients, high titers of nAbs exceeded the upper limit of the evaluated assays' quantification ranges. Based on data from this study, we conclude that commercial immunoassays are acceptable substitutes for live-virus NTs, particularly when additional adapted cutoffs are employed to detect nAbs beyond a specific threshold titer. IMPORTANCE While the measurement of neutralizing antibodies is considered a valuable tool in assessing protection against SARS-CoV-2, neutralization tests employ live-virus isolates and cell culture, requiring advanced laboratory biosafety levels. Including a large sample panel (over 700 samples), this study provides adapted cutoff values calculated for seven commercial immunoassays (including four surrogate neutralization assays and a protein-based microarray) that robustly correlate with specific titers of neutralizing antibodies.

When the available blood supply mismatches the needs of the patient.

BACKGROUND: Substantial regional differences in the genetic patterns related to blood group have been observed across different continents. This diversity means that the blood supply, as an essential part of patient care, is increasingly impacted by global migration. Consequently, the Austrian blood donor population does not match the immigrant patient population. This mismatch is likely to result in the formation of alloantibodies to red cell antigens in the chronically transfused. Subsequently, major difficulties in providing compatible blood emerge. MATERIAL AND METHODS: The study included patients of African origin (n=290) and Caucasians who represent the Austrian donor population (n=1,017). Genetic typing was performed for up to 69 blood group polymorphisms with a multiplex sequence specific primer-PCR including high frequency antigens and antigens for which antisera are not commercially available. By assessing differences in antigen frequencies between the two populations, and using these data for prophylactic matching, we aim to develop tools to increase the quality of patient care. RESULTS: Results indicate various and significant differences (p<0.0001) in antigen frequencies between African patients and the European donor population, especially in the MNS, Duffy, Knops and Rhesus systems. DISCUSSION: Our data highlight the importance of matching the donor population to the demographics of the patient population. In addition, it underlines the need to recruit donors of African origin and to focus on the upcoming challenges, such as malaria semi-immunity and a significantly higher rate of infectious disease in this population. It is also recommended to apply extended genetic typing to detect rare blood types, and (cryo)storage of rare blood in national and international rare blood banks. Co-operation with regional blood banks should also be encouraged.

Storage of packed red blood cells impairs an inherent coagulation property of erythrocytes.

Storage of packed red blood cells is associated with changes in erythrocytes that over time increasingly impair cellular function and potentially contribute to adverse effects associated with blood transfusion. Exposure of phosphatidylserine at the outer membrane leaflet of erythrocytes and shedding of microvesicles (MVs) during packed red blood cell storage are alterations assumed to increase the risk of prothrombotic events in recipients. Here, we used rotational thromboelastometry to study the coagulation process in blood samples with erythrocytes from stored PRBCs reconstituted with freshly prepared platelet-rich plasma. We explored the influence of following effects on the coagulation process: 1) PRBC storage duration, 2) differences between erythrocytes from stored PRBCs compared to freshly drawn erythrocytes, and 3) the contribution of added MVs. Interestingly, despite of a higher fraction of PS-positive cells, erythrocytes from PRBCs stored for 6 weeks revealed longer clotting times than samples with erythrocytes stored for 2 or 4 weeks. Further, clotting times and clot formation times were considerably increased in samples reconstituted with erythrocytes from stored PRBCs as compared to fresh erythrocytes. Moreover, MVs added to reconstituted samples elicited only comparably small and ambiguous effects on coagulation. Thus, this study provides no evidence for an amplified clotting process from prolonged storage of PRBCs but on the contrary implicates a loss of function, which may be of clinical significance in massive transfusion. Our observations add to the increasing body of evidence viewing erythrocytes as active players in the clotting process.

SARS-CoV-2 IgG Levels Allow Predicting the Optimal Time Span of Convalescent Plasma Donor Suitability.

Convalescent plasma (CP) has been in use for the treatment of numerous infectious diseases for more than a century, recently also for coronavirus disease 2019 (COVID-19). A major challenge for this treatment is identifying suitable donors with sufficient levels of functional antibodies and to determine the optimal time span for CP donation. In this retrospective study, we analyzed 189 CP donations of 66 donors regarding anti-SARS-CoV-2 anti-S IgG antibody levels. We found a significant correlation between the semi-quantitative SARS-CoV-2 IgG ratio values and in vitro antibody functionality. A time-to-event analysis allowed us to predict the optimal time span of COVID-19 CP donor suitability. We found that high IgG ratio values, which significantly correlate with high in vitro antibody functionality, were suitable for CP donation for a median of 134 days after the first CP donation. Donors with lower IgG ratios were suitable for a median of 53 days. Our data support plasma collection centers to determine optimal points in time for CP donation by means of widely used semi-quantitative laboratory IgG ratio values.

Sample Buffer Containing Guanidine-Hydrochloride Combines Biological Safety and RNA Preservation for SARS-CoV-2 Molecular Diagnostics.

The COVID-19 pandemic has elicited the need to analyse and store large amounts of infectious samples for laboratory diagnostics. Therefore, there has been a demand for sample storage buffers that effectively inactivate infectious viral particles while simultaneously preserving the viral RNA. Here, we present a storage buffer containing guanidine-hydrochloride that fulfils both requirements. Its ability to preserve RNA stability was confirmed by RT-qPCR, and virus-inactivating properties were tested by tissue culture infectious dose assay. Our data revealed that RNA from samples diluted in this storage buffer was efficiently preserved. Spiking samples with RNase A resulted in RNAse concentrations up to 100 ng/mL being efficiently inhibited, whereas spiking samples with infectious SARS-CoV-2 particles demonstrated rapid virus inactivation. In addition, our buffer demonstrated good compatibility with several commercially available RNA extraction platforms. The presented guanidine-hydrochloride-based storage buffer efficiently inactivates infectious SARS-CoV-2 particles and supports viral RNA stability, leading to a reduced infection risk during sample analysis and an increased period for follow-up analysis, such as sequencing for virus variants. Because the presented buffer is uncomplicated to manufacture and compatible with a variety of commercially available test systems, its application can support and improve SARS-CoV-2 laboratory diagnostics worldwide.

Persistence of Naturally Acquired and Functional SARS-CoV-2 Antibodies in Blood Donors One Year after Infection.

The developmental course of antibodies produced after a SARS-CoV-2 infection has been insufficiently investigated so far. Therefore, the aim of this study was to investigate the dynamics of SARS-CoV-2 antibody levels against the viral nucleocapsid- and spike-protein among Austrian blood donors as a representative group of a supposedly healthy population within the first year after a SARS-CoV-2 infection. The impact of age, sex, vaccination status, AB0-blood group and awareness about the infection was evaluated. Our study shows that the level of anti-N antibodies is declining, while anti-S antibody levels remain stable. Antibodies detected were functional in vitro. Age, sex and blood group do not influence antibody dynamics. However, blood group AB shows significantly lower antibody levels and in vitro functionality compared to other blood groups. Our data reveal that one out of five individuals was not aware of a previous SARS-CoV-2 infection and that the disease course neither affects the level of antibody production nor the in vitro functionality. We also found that 14% of participants show persisting COVID-19-related symptoms for up to nine months. Our results provide valuable insights into the dynamics of the immune response after a SARS-CoV-2 infection in a representative cohort of adult blood donors in Central Europe.

Characterization of the novel alleles: HLA-A*01:305, HLA-B*58:120, HLA-C*04:428Q and HLA-C*05:01:56.

Four new HLA class I alleles were characterized by next-generation sequencing and haplotypes determined.

Seroprevalence of anti-SARS-CoV-2 total antibody is higher in younger Austrian blood donors.

PURPOSE: Frequently the infection with coronavirus 2 (SARS-CoV-2) can be asymptomatic or provoke only mild symptoms. These cases often remain unnoticed, so it is difficult to estimate the actual numbers of infections. Aim of this study was to determine the seroprevalence of anti-SARS-CoV-2 total antibody in Austrian blood donors. METHODS: 20,228 blood donors aged between 18 and 72 years resident in four Austrian federal states were screened for anti-SARS-CoV-2 total antibody between 5th of June and 4th of December 2020. To evaluate the impact of sex, age, AB0-blood group and donation period on the anti-SARS-CoV-2 seroprevalence, multiple logistic regression was done. RESULTS: Our data reveal an anti-SARS-CoV-2 seroprevalence of 2.5% overall, significantly depending on the time point of blood donation: after the first Austrian lockdown the seroprevalence was lower compared to the following months, when the rate was constantly rising. While younger blood donors showed significantly higher seroprevalence, no differences were found concerning sex or AB0 blood group. CONCLUSION: Broad testing strategies are required to better determine the number of SARS-CoV-2 infections. Screening blood donors as a representative group for the adult population could be a valid tool to determine the number of recorded and unrecorded cases of SARS-CoV-2 infection.

Leukocyte-Reactive Antibodies in Female Blood Donors: The Austrian Experience.

INTRODUCTION: Antibody-mediated transfusion-related acute lung injury (TRALI) is caused by antibodies against human leukocyte antigens (HLAs) or human neutrophil antigens (HNAs), and is one of the most serious complications associated with transfusion medicine. Prevention strategies like testing allo-exposed female blood donors have not yet been introduced nationwide in Austria. To assess the need and feasibility of routine leukocyte antibody testing, the prevalence of leukocyte-reactive antibodies in an Austrian female donor population was been determined using classical cell-based methods which were compared with a high-throughput bead-based method. METHODS: Sera from 1,022 female blood donors were screened using a granulocyte aggregation test (GAT) and a white blood cell immunofluorescence test (WIFT) after retesting and specification of positive samples by granulocyte immunofluorescence test (GIFT) and monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA). Potential HLA reactivities were confirmed using the microbeads assay LabScreenTM Mixed. The results in 142 donor sera and 38 well-defined reference sera were investigated by the microbeads assay LabScreenTM Multi and compared with classical cell-based methods. RESULTS: Reactivity with either granulocytes and/or lymphocytes was detected in 79 sera (7.7%), with the majority being HLA-specific. Antibodies against HNA were obtained in 7 samples (0.7%). The aggregating potential of the detected antibodies was observed in 9 cases (0.9%). Most of the leukocyte-reactive antibodies occurred at a donor age of between 35 and 59 years (n = 61). LabScreen Multi showed good agreement (κ = 0.767) for HNA antibody detection by cell-based assays, but double/multiple specificities (100% of 7 anti-HNA-1b sera) as well as false-negative results (40% of 15 HNA-3-specific sera) occurred. DISCUSSION: Leukocyte-reactive antibody screening is advised in Austrian female donors for safe blood transfusion, including single-donor convalescent plasma treatment of COVID-19 that may be implemented soon. For the introduction of LabScreen Multi, the combination with GAT should be considered to ensure correct anti-HNA-3a detection.

Jknull alleles in two patients with anti-Jk3.

BACKGROUND: As of publication, a total of 41 null alleles have been acknowledged by the International Society of Blood Transfusion (ISBT) to cause the rare Jknull phenotype, but none have been discovered in Austria thus far. MATERIALS AND METHODS: Two patients with anti-Jk3 were serologically identified by a positive antibody screening and typed as Jk(a-b-). The initial genotyping using an SSP-PCR method for the common 838A/G polymorphism indicated a JK*02/02, or JK*01/02 genotype, respectively. To find the disruptive mutations, Sanger sequencing was performed and results were compared to the reference sequence. The patient's antibodies were characterized with a monocyte monolayer assay (MMA) for their potential clinical significance. RESULTS: Three novel null-mutations of the SLC14A1 gene were found in two patients. Patient 1 was homozygous for a 10bp deletion in exon 4 (c.157_166del on JK*02). Testing of her family members revealed Mendelian inheritance of the deletional allele. The other patient was compound heterozygous for two mutations: one allele carrying a single base deletion in exon 4 (c.267delC on JK*01) and the other a splice site mutation in intron 3 (c.152-1g>a on JK*02). The MMA results suggest high clinical significance of the anti-Jk3 in both patients. DISCUSSION: The detected mutations led to Jknull phenotypes and are the first description of JKnull alleles in the Austrian population.

Characterization of 100 sequential SARS-CoV-2 convalescent plasma donations.

BACKGROUND: Transfusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent plasma is a promising treatment for severe coronavirus disease 2019 (COVID-19) cases, with success of the intervention based on neutralizing antibody content. Measurement by serologic correlates without biocontainment needs as well as an understanding of donor characteristics that may allow for targeting of more potent donors would greatly facilitate effective collection. STUDY DESIGN AND METHODS: One hundred convalescent plasma units were characterized for functionally active SARS-CoV-2 neutralizing antibodies, as well as for SARS-CoV-2 binding antibodies, with the intention to establish a correlation between the functionally more relevant neutralization assay and the more accessible enzyme-linked immunosorbent assay (ELISA). Donor demographics such as COVID-19 severity, age, and sex were correlated with antibody titers. RESULTS: A mean neutralization titer 50% of 230 (range, <8-1765) was seen for the 100 convalescent plasma units, with highly significant (P < .0001) yet quantitatively limited (R2 = 0.2830) correlation with results of the ELISA. Exclusion of units with particularly high titers (>500) from analysis improved correlation (R2 = 0.5386). A tendency of higher-titer plasma units from donors with increased disease severity, of advanced age, and of male sex was seen, yet the functional relevance of this difference is questionable. CONCLUSION: The ELISA-based correlation to neutralization titer enabled a threshold proposal that could be used to eliminate lower-titer units from the clinical supply for COVID-19 treatment. Disease severity may be associated with the development of higher titers of neutralizing antibodies, although larger case numbers will be needed for additional confirmation.

Association of ABO blood group with bleeding severity in patients with bleeding of unknown cause.

Blood group O has been associated with an increased bleeding tendency due to lower von Willebrand factor (VWF) and factor VIII (FVIII) levels. We explored whether blood group O is independently associated with bleeding severity in patients with mild-to-moderate bleeding of unknown cause (BUC) in the Vienna Bleeding Biobank cohort. Bleeding severity was recorded with the Vicenza bleeding score (BS). Blood group O was overrepresented in 422 patients with BUC compared with its presence in 23 145 healthy blood donors (47.2% vs 37.6%; odds ratio, 1.48; 95% confidence interval [CI], 1.22-1.79). The BS and the number of bleeding symptoms were significantly higher in patients with blood group O than in patients with non-O after adjustment for VWF and FVIII levels and sex (least-square [LS] means of BSs: 6.2; 95% CI, 5.8-6.6 vs 5.3; 4.9-5.7; and of number of symptoms: LS, 3.5; 95% CI, 3.2-3.7 vs 3.0; 2.8-3.2, respectively). Oral mucosal bleeding was more frequent in those with blood group O than in those with other blood types (group non-O; 26.1% vs 14.3%), independent of sex and VWF and FVIII levels, whereas other bleeding symptoms did not differ. Patients with blood group O had increased clot density in comparison with those with blood group non-O, as determined by rotational thromboelastometry and turbidimetric measurement of plasma clot formation. There were no differences in thrombin generation, clot lysis, or platelet function. Our data indicate that blood group O is a risk factor for increased bleeding and bleeding severity in patients with BUC, independent of VWF and FVIII levels.

Two Prevalent ∼100-kb GYPB Deletions Causative of the GPB-Deficient Blood Group MNS Phenotype S-s-U- in Black Africans.

The U antigen (MNS5) is one of 49 antigens belonging to the MNS blood group system (ISBT002) carried on glycophorins A (GPA) and B (GPB). U is present on the red blood cells in almost all Europeans and Asians but absent in approximately 1.0% of Black Africans. U negativity coincides with negativity for S (MNS3) and s (MNS4) on GPB, thus be called S-s-U-, and is thought to arise from homozygous deletion of GYPB. Little is known about the molecular background of these deletions. Bioinformatic analysis of the 1000 Genomes Project data revealed several candidate regions with apparent deletions in GYPB. Highly specific Gap-PCRs, only resulting in positive amplification from DNAs with deletions present, allowed for the exact genetic localization of 3 different breakpoints; 110.24- and 103.26-kb deletions were proven to be the most frequent in Black Americans and Africans. Among 157 CEPH DNAs, deletions in 6 out of 8 African ethnicities were present. Allele frequencies of the deletions within African ethnicities varied greatly and reached a cumulative 23.3% among the Mbuti Pygmy people from the Congo. Similar observations were made for U+var alleles, known to cause strongly reduced GPB expression. The 110- and 103-kb deletional GYPB haplotypes were found to represent the most prevalent hereditary factors causative of the MNS blood group phenotype S-s-U-. Respective GYPB deletions are now accessible by molecular detection of homo- and hemizygous transmission.

Quantification of SARS-CoV-2 antibodies with eight commercially available immunoassays.

Since the emergence of SARS-CoV-2 numerous antibody assays have become available, demonstrating different performance characteristics. This study focused on a quantitative correlation between different commercial assays and a neutralization test (NT). Comparative data is needed as a basis for the production of convalescent plasma and potential interpretations COVID-19 immunity. Sera of 100 SARS-CoV-2 convalescent plasma donors were collected and SARS-CoV-2 antibodies were characterized using three different IgG-ELISAs (EUROIMMUN IgG and NCP-IgG ELISA, Wantai ELISA), two CLIA (Elecsys, LIAISON) and two lateral flow tests (MEDsan IgM/IgG-Rapid-Test, Wantai Rapid Test) and subsequently correlated to neutralization titers. The Wantai ELISA and the Elecsys provide the highest sensitivities in this sample (98 and 95 percent respectively). Titers with the best overall quantitative correlation to the NT titer were obtained with the Euroimmun IgG ELISA assay (Rho=0.759) and the Wantai ELISA assay (Rho=0.729). An infection without fever and negative or weakly positive reactions in the Wantai Rapid test were negative predictive factors for NT titers >1:200 (negative predictive value of 92 % and 92 % respectively, combination of both 100 %). The Wantai ELISA titer could be a suitable substitute for NT. An adequate pooling strategy of plasma units additionally could compensate deviations of individual antibody titers.

CD4 T Cell Determinants in West Nile Virus Disease and Asymptomatic Infection.

West Nile (WN) virus infection of humans is frequently asymptomatic, but can also lead to WN fever or neuroinvasive disease. CD4 T cells and B cells are critical in the defense against WN virus, and neutralizing antibodies, which are directed against the viral glycoprotein E, are an accepted correlate of protection. For the efficient production of these antibodies, B cells interact directly with CD4 helper T cells that recognize peptides from E or the two other structural proteins (capsid-C and membrane-prM/M) of the virus. However, the specific protein sites yielding such helper epitopes remain unknown. Here, we explored the CD4 T cell response in humans after WN virus infection using a comprehensive library of overlapping peptides covering all three structural proteins. By measuring T cell responses in 29 individuals with either WN virus disease or asymptomatic infection, we showed that CD4 T cells focus on peptides in specific structural elements of C and at the exposed surface of the pre- and postfusion forms of the E protein. Our data indicate that these immunodominant epitopes are recognized in the context of multiple different HLA molecules. Furthermore, we observed that immunodominant antigen regions are structurally conserved and similarly targeted in other mosquito-borne flaviviruses, including dengue, yellow fever, and Zika viruses. Together, these findings indicate a strong impact of virion protein structure on epitope selection and antigenicity, which is an important issue to consider in future vaccine design.