Specificities of human CD4+ T cell responses to an inactivated flavivirus vaccine and infection: correlation with structure and epitope prediction.

Tick-borne encephalitis (TBE) virus is endemic in large parts of Europe and Central and Eastern Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to the mosquito-borne yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated whole-virus vaccine can effectively prevent clinical disease. Neutralizing antibodies are directed to the viral envelope protein (E) and an accepted correlate of immunity. However, data on the specificities of CD4(+) T cells that recognize epitopes in the viral structural proteins and thus can provide direct help to the B cells producing E-specific antibodies are lacking. We therefore conducted a study on the CD4(+) T cell response against the virion proteins in vaccinated people in comparison to TBE patients. The data obtained with overlapping peptides in interleukin-2 (IL-2) enzyme-linked immunosorbent spot (ELISpot) assays were analyzed in relation to the three-dimensional structures of the capsid (C) and E proteins as well as to epitope predictions based on major histocompatibility complex (MHC) class II peptide affinities. In the C protein, peptides corresponding to two out of four alpha helices dominated the response in both vaccinees and patients, whereas in the E protein concordance of immunodominance was restricted to peptides of a single domain (domain III). Epitope predictions were much better for C than for E and were especially erroneous for the transmembrane regions. Our data provide evidence for a strong impact of protein structural features that influence peptide processing, contributing to the discrepancies observed between experimentally determined and computer-predicted CD4(+) T cell epitopes. Importance: Tick-borne encephalitis virus is endemic in large parts of Europe and Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated vaccine can effectively prevent disease. Both vaccination and natural infection induce the formation of antibodies to a viral surface protein that neutralize the infectivity of the virus and mediate protection. B lymphocytes synthesizing these antibodies require help from other lymphocytes (helper T cells) which recognize small peptides derived from proteins contained in the viral particle. Which of these peptides dominate immune responses to vaccination and infection, however, was unknown. In our study we demonstrate which parts of the proteins contribute most strongly to the helper T cell response, highlight specific weaknesses of currently available approaches for their prediction, and demonstrate similarities and differences between vaccination and infection.

A deep intronic recurrent CHEK2 variant c.1009-118_1009-87delinsC affects pre-mRNA splicing and contributes to hereditary breast cancer predisposition.

Germline CHEK2 pathogenic variants confer an increased risk of female breast cancer (FBC). Here we describe a recurrent germline intronic variant c.1009-118_1009-87delinsC, which showed a splice acceptor shift in RNA analysis, introducing a premature stop codon (p.Tyr337PhefsTer37). The variant was found in 21/10,204 (0.21%) Czech FBC patients compared to 1/3250 (0.03%) controls (p = 0.04) and in 4/3639 (0.11%) FBC patients from an independent German dataset. In addition, we found this variant in 5/2966 (0.17%) Czech (but none of the 443 German) ovarian cancer patients, three of whom developed early-onset tumors. Based on these observations, we classified this variant as likely pathogenic.

Frequency of Leiden Mutation in Newborns with Birth Weight below 1500 g.

It has been hypothesized that fetal prematurity or Intrauterine Growth Restriction (IUGR) could be related to the presence of factor V of Leiden mutation. This mutation is associated with a higher incidence of pregnancy difficulties that can result in preterm birth. The frequency of Leiden mutation was investigated in the group of newborns with a low birth weight below 1500 g over a six-year period from 2015 to 2020. During this period, 339 newborns were tested, of which 42 newborns with V Leiden mutation (12.4%) were detected. The average of its occurrence frequency in the Czech population was determined as 5.0% based on published studies. In our research, the occurrence of the V Leiden mutation was found significantly higher in newborns under 1500 g. At the same time, we did not demonstrate an increased frequency of births at lower gestational weeks, lower birth weight, or an association with sex in newborns with a positive diagnosis of the Leiden V factor.

Human CD4+ T Helper Cell Responses after Tick-Borne Encephalitis Vaccination and Infection.

Tick-borne encephalitis virus (TBEV) is a human-pathogenic flavivirus that is endemic in large parts of Europe and Asia and causes severe neuroinvasive illness. A formalin-inactivated vaccine induces strong neutralizing antibody responses and confers protection from TBE disease. CD4+ T cell responses are essential for neutralizing antibody production, but data on the functionalities of TBEV-specific CD4+ T cells in response to vaccination or infection are lacking. This study provides a comprehensive analysis of the cytokine patterns of CD4+ T cell responses in 20 humans after TBE vaccination in comparison to those in 18 patients with TBEV infection. Specifically, Th1-specific cytokines (IFN-γ, IL-2, TNF-α), CD40 ligand and the Th1 lineage-specifying transcription factor Tbet were determined upon stimulation with peptides covering the TBEV structural proteins contained in the vaccine (C-capsid, prM/M-membrane and E-envelope). We show that TBEV-specific CD4+ T cell responses are polyfunctional, but the cytokine patterns after vaccination differed from those after infection. TBE vaccine responses were characterized by lower IFN-γ responses and high proportions of TNF-α+IL-2+ cells. In vaccine-induced responses-consistent with the reduced IFN-γ expression patterns-less than 50% of TBEV peptides were detected by IFN-γ+ cells as compared to 96% detected by IL-2+ cells, indicating that the single use of IFN-γ as a read-out strongly underestimates the magnitude and breadth of such responses. The results provide important insights into the functionalities of CD4+ T cells that coordinate vaccine responses and have direct implications for future studies that address epitope specificity and breadth of these responses.

Refined quantitative fluorescent PCR of Y-chromosome DNA sequences mosaics in Turner's syndrome patients--alternative to real-time PCR.

BACKGROUND: Real-time polymerase chain reactions (PCRs) are the most frequently used techniques for gonosomal mosaics quantification. The primary aim of this work is to assess and optimize the refined technique of quantitative fluorescent polymerase chain reaction (RQF PCR) in the quantification of Y-chromosome sequences in gonosomal mosaics. The method was applied to the analysis of Y-chromosome sequences (amelogenin gene, AMELX/Y-loci) in peripheral lymphocytes and gonadal tissues in Y-positive Turner's syndrome (TS) patients. METHODS: RQF PCR was used for molecular quantification, and fluorescent in situ hybridization (FISH) technique was used for comparison. RESULTS: Based on a formulated calibration curve, DNA mosaics from six Y-positive patients and gonads from one patient were deducted. For calculation of rare mosaics, it is possible to take advantage of a new empirical formula. FISH results were comparable to RQF PCR. CONCLUSION: The sensitivity of RQF PCR brings significant progress in the analysis of gonosomal aberrations. RQF PCR also finds applications in prenatal diagnostics of maternal contaminations of amniotic fluid and foetal DNA in maternal blood and analysis of chimerism in patients after bone marrow transplantation. The method is very convenient for determining the number of testis-specific protein, Y-linked (TSPY) gene repetitions.