Characteristics of Norovirus capsid protein-specific CD8+ T-Cell responses in previously infected individuals.

Norovirus (NV) infection causes acute gastroenteritis in children and adults. Upon infection with NV, specific CD8+ T cells, which play an important role in anti-infective immunity, are activated in the host. Owing to the NV's wide genotypic variability, it is challenging to develop vaccines with cross-protective abilities against infection. To aid effective vaccine development, we examined specific CD8+ T-cell responses towards viral-structural protein (VP) epitopes, which enable binding to host susceptibility receptors. We isolated peripheral blood mononuclear cells from 196 participants to screen and identify predominant core peptides towards NV main and small envelope proteins using ex vivo and in vitro intracellular cytokine staining assays. Human leukocyte antigen (HLA) restriction characteristics were detected using next-generation sequencing. Three conservative immunodominant VP-derived CD8+ T-cell epitopes, VP294-102 (TDAARGAIN), VP2153-161 (RGPSNKSSN), and VP1141-148 (FPHIIVDV), were identified and restrictively presented by HLA-Cw * 0102, HLA-Cw * 0702, and HLA-A *1101 alleles, separately. Our findings provide useful insights into the development of future vaccines and treatments for NV infection.

ENTPD1-AS1-miR-144-3p-mediated high expression of COL5A2 correlates with poor prognosis and macrophage infiltration in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a malignant tumor with high morbidity and mortality. Expression of COL5A2 is significantly elevated in GC. Abnormal expression of noncoding RNAs (ncRNAs) have been found in GC, including microRNA (miRNA) and long noncoding RNA (lncRNA). Competing endogenous RNA network plays an important regulatory role in GC. However, its specific regulatory mechanism has not been elucidated. AIM: To gain insight into the ncRNA regulatory mechanism and immune microenvironment related to COL5A2 in GC. METHODS: RNA sequencing data and clinical information from The Cancer Genome Atlas data portal were used to analyze the expressions of COL5A2, miRNA and lncRNA related to the prognosis of GC. Cox regression analysis and Kyoto Encyclopedia of Genes and Genomes analysis were performed to assess the risk factors and relevant function of COL5A2. StarBase was used to predict the interaction of miRNA-lncRNA or miRNA-mRNA in GC. The relationship between COL5A2, miR-144-3p and ENTPD1-AS1 were verified by dual luciferase reporter assay. The association of COL5A2 with immune cell infiltration were analyzed using the Tumor Immune Estimation Resource database and single sample gene set enrichment analysis. The expression of COL5A2 and macrophages in paired GC tissues were detected by immunohistochemical staining. RESULTS: We verified that the upregulation of COL5A2 expression was associated with the prognosis of GC and was an independent risk factor for GC. miR-144-3p was downregulated and correlated with the prognosis of GC. miR-144-3p regulated the expression of COL5A2 through direct interaction with COL5A2. ENTPD1-AS1 was elevated in GC and competitively bound to miR-144-3p, thus inhibiting the expression of miR-144-3p. ENTPD1-AS1 enhanced the expression of COL5A2 through sponging miR-144-3p. Compared to paired normal tissue, COL5A2 expression was upregulated at the protein level, especially in the middle and late stages of GC. The high expression of COL5A2 was positively linked to macrophage infiltration in GC. CONCLUSION: COL5A2 regulated by ENTPD1-AS1-miR-144-3p was associated with poor prognosis and macrophage infiltration in GC. This could be a new biomarker and therapeutic target in GC.

Linear epitopes on the capsid protein of norovirus commonly elicit high antibody response among past-infected individuals.

BACKGROUND: Human norovirus (HuNoV) is the leading cause of acute nonbacterial gastroenteritis globally, and its infection is usually self-limited, so most people become past Norovirus (NoV)-infected individuals. It is known that some antibody responses may play a critical role in preventing viral infection and alleviating disease; however, the characteristics and functions of particular antibody responses in persons with previous infections are not fully understood. Capsid proteins, including VP1 and VP2, are crucial antigenic components of NoV and may regulate antibody immune responses, while epitope-specific antibody responses to capsid proteins have not been comprehensively characterized. METHODS: We prepared purified VP1 and VP2 proteins by ion exchange chromatography and measured serum antigen-specific IgG levels in 398 individuals by ELISA. Overlapping 18-mer peptides covering the full length of VP1 and VP2 were synthesized, and then we identified linear antigenic epitopes from 20 subjects with strong IgG positivity. Subsequently, specific antibody responses to these epitopes were validated in 185 past infected individuals, and the conservation of epitopes was analyzed. Finally, we obtained epitope-specific antiserum by immunizing mice and expressed virus-like particles (VLPs) in an insect expression system for a blockade antibody assay to evaluate the receptor-blocking ability of epitope-specific antibodies. RESULTS: The IgG responses of VP1 were significantly stronger than those of VP2, both of which had high positive rates of over 80%. The overall positive rate of VP1-IgG and/or VP2-IgG was approximately 94%, which may be past NoV-infected individuals. Four linear antigenic B-cell epitopes of capsid proteins were identified, namely, VP1199-216, VP1469-492, VP297-120, and VP2241-264, all of which were conserved. The IgG response rates of the above epitopes in past NoV-infected individuals were 38.92%, 22.16%, 8.11% and 28.11%, respectively. In addition, VP1199-216- and VP1469-492-specific antibodies can partially block the binding of VLPs to the receptor histo-blood group antigen (HBGA). CONCLUSION: This is the first study to describe specific antibody responses of VP2 and to identify its B-cell epitopes. Our findings offer data for a more thorough understanding of norovirus capsid protein-specific IgG responses and could provide useful information for designing and developing vaccines.

UreB immunodominant epitope-specific CD8+ T-cell responses were beneficial in reducing gastric symptoms in Helicobacter pylori-infected individuals.

BACKGROUND AND AIMS: Although Helicobacter pylori is recognized as an extracellular infection bacterium, it can lead to an increase in the number of CD8+ T cells after infection. At present, the characteristics of H. pylori antigen-specific CD8+ T cells and the epitope response have not been elucidated. This study was focused on putative protective antigen UreB to detect specific CD8+ T-cell responses in vitro and screen for predominant response epitopes. METHODS: The PBMCs collected from H. pylori-infected individuals were stimulated by UreB peptide pools in vitro to identify the immunodominant CD8+ T-cell epitopes. Furthermore, their HLA restriction characteristics were detected accordingly by NGS. Finally, the relationship between immunodominant responses and appearance of gastric symptoms after H. pylori infection was conducted. RESULTS: UreB-specific CD8+ T-cell responses were detected in H. pylori-infected individuals. Three of UreB dominant epitopes (A-2 (UreB443-451 : GVKPNMIIK), B-4 (UreB420-428 : SEYVGSVEV), and C-1 (UreB5-13 : SRKEYVSMY)) were firstly identified and mainly presented by HLA-A*1101, HLA-B*4001 and HLA-C*0702 alleles, respectively. C-1 responses were mostly occurred in H. pylori-infected subjects without gastric symptoms and may alleviate the degree of gastric inflammation. CONCLUSIONS: The UreB dominant epitope-specific CD8+ T-cell response was closely related to the gastric symptoms after H. pylori infection, and the C-1 (UreB5-13 ) dominant peptides may be protective epitopes.

Cytotoxin-Associated Gene A-Negative Helicobacter pylori Promotes Gastric Mucosal CX3CR1+CD4+ Effector Memory T Cell Recruitment in Mice.

BACKGROUND: Helicobacter pylori can cause many kinds of gastric disorders, ranging from gastritis to gastric cancer. Cytotoxin-associated gene A (CagA)+ H. pylori is more likely to cause gastric histopathologic damage than CagA- H. pylori. However, the underlying mechanism needs to be further investigated. MATERIALS AND METHODS: Mice were intragastrically administered equal amounts of CagA+ or CagA- H. pylori. Four weeks later, 24 chemokines in stomachs were measured using a mouse chemokine array, and the phenotypes of the recruited gastric CD4+ T cells were analyzed. The migration pathway was evaluated. Finally, the correlation between each pair among the recruited CD4+ T cell sub-population, H. pylori colonization level, and histopathologic damage score were determined by Pearson correlation analysis. RESULTS: The concentration of chemokines, CCL3 and CX3CL1, were significantly elevated in CagA- H. pylori-infected gastric mucosa than in CagA+ H. pylori-infected gastric mucosa. Among them, CX3CL1 secreted by gastric epithelial cells, which was elicited more effectively by CagA- H. pylori than by the CagA+ strain, dramatically promoted mucosal CD4+ T cell migration. The expression of CX3CR1, the only known receptor of CX3CL1, was upregulated on the surface of gastric CD4+ T cells in CagA- H. pylori-infected stomach. In addition, most of the CX3CR1-positive gastric CD4+ T cells were CD44+CD69-CCR7- effector memory T cells (Tem). Pearson correlation analysis showed that the recruited CX3CR1+CD4+ Tem cell population was negatively correlated with H. pylori colonization level and histopathologic damage score. CONCLUSION: CagA- H. pylori promotes gastric mucosal CX3CR1+CD4+ Tem recruitment in mice.

Intranasal immunization with immunodominant epitope peptides derived from HpaA conjugated with CpG adjuvant protected mice against Helicobacter pylori infection.

HpaA is considered to be an effective protective antigen for vaccination against Helicobacter pylori (H. pylori) infection. Oral immunization with HpaA significantly decreases bacterial colonization in H. pylori infected mice. In this study, we investigated whether subcutaneous or intranasal immunization with HpaA could protect against H. pylori infection. Mice immunized subcutaneously with HpaA in Complete Freund's adjuvant, but not mice intranasally immunized with HpaA in CpG adjuvant acquired protection against H. pylori infection. However, intranasal immunization with immunodominant epitope peptides in CpG adjuvant protected mice against H. pylori infection, and immunodominant epitope peptides stimulated stronger Th1 responses and mediated more robust protection against H. pylori infection than subdominant ones. Our results suggest that the length of a candidate antigen is critical for particular vaccination routes, and that immunodominant epitope peptides are promising candidates for protection against H. pylori infection through nasal vaccination.

Immunodominant antigens that induce Th1 and Th17 responses protect mice against Helicobacter pylori infection.

Helicobacter pylori has infected more than half of the world's population, causing gastritis, gastric ulcers, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. The oral recombinant Helicobacter pylori vaccine currently used has made great progress in addressing this problem, however, its efficacy and longevity still need to be improved. Th1 and Th17 cells play essential roles in local protection against Helicobacter pylori in the stomach mucosa. Additionally, protective immunodominant antigens are the preferred for a vaccine. In this work, Helicobacter pylori whole cell lysate was separated into 30 groups based on molecular weight by molecular sieve chromatography. The group best promoting CD4 T cells proliferation was selected and evaluated by immunization. The detail proteins were then analyzed by LC-MS/MS and expressed in Escherichia coli. Eleven proteins were selected and the dominant ones were demonstrated. As a result, three protective immunodominant antigens, inosine 5'-monophosphate dehydrogenase, type II citrate synthase, and urease subunit beta, were selected from Helicobacter pylori whole cell. Two of them (inosine 5'-monophosphate dehydrogenase and type II citrate synthase) were newly identified, and one (urease subunit beta) was confirmed as previously reported. The mixture of the three antigens showed satisfactory protective efficiency, with significant lower H. pylori colonization level (P < 0.001) and stronger Th1 (P < 0.001) and Th17 (P < 0.001) responses than PBS control group. Thus, inosine 5'-monophosphate dehydrogenase, type II citrate synthase, and urease subunit beta are three protective antigens inducing dominant Th1 and Th17 responses to defend against Helicobacter pylori infection.

The influence of adjuvant on UreB protection against Helicobacter pylori through the diversity of CD4+ T-cell epitope repertoire.

Adjuvants are widely used to enhance the effects of vaccines against pathogen infections. Interestingly, different adjuvants and vaccination routes usually induce dissimilar immune responses, and can even have completely opposite effects. The mechanism remains unclear. In this study, urease B subunit (UreB), an antigen of Helicobacter pylori (H. pylori) that can induce protective immune responses, was used as a model to vaccinate mice. We investigated the effects of different adjuvants and routes on consequent T cell epitope-specific targeting and protection against H. pylori infection. Comparison of the protective effects of UreB, administered either subcutaneously (sc) or intranasally (in), with the adjuvants AddaVax (sc), Complete Freund's adjuvant (CFA; sc), or CpG oligonucleotide (CpG; sc or in), indicated that only CFA (sc) and CpG (in) were protective. Protective vaccines induced T cells targeting epitopes that differed from that targeted by control vaccination. Subsequent peptide vaccination demonstrated that only two of the identified epitopes were protective: UreB373-385 and UreB317-329. Overall, we found that both adjuvant and vaccination route affected the T cell response repertoire to antigen epitopes. The data obtained in this study contribute to improved characterization of the relationship between adjuvants, routes of vaccination, and epitope-specific T cell response repertoires.

A 21-35 kDa Mixed Protein Component from Helicobacter pylori Activates Mast Cells Effectively in Chronic Spontaneous Urticaria.

BACKGROUND: Helicobacter pylori (H. pylori) seem to involve in the etiology of chronic spontaneous urticaria (CSU). But studies of the pathogenic mechanism are very little. METHODS: In this study, we detected the serum-specific anti-H. pylori IgG and IgE antibodies in 211 CSU and 137 normal subjects by enzyme-linked immunosorbent assay (ELISA), evaluated the direct activation effects of H. pylori preparations and its protein components on human LAD2 mast cell line in vitro, and analyzed the specific protein ingredients and functions of the most effective H. pylori mixed protein component using liquid chromatography-mass spectrometry and ELISA assay. RESULTS: In CSU patients, the positive rate of anti-H. pylori IgG positive rate was significantly higher than that in normal controls, and the anti-H. pylori IgE levels had no statistical difference between H. pylori-infected patients with and without CSU. Further studies suggested that H. pylori preparations can directly activate human LAD2 mast cell line in a dose-dependent manner and its most powerful protein component was a mixture of 21-35 kDa proteins. Moreover, the 21-35 kDa mixed protein component mainly contained 23 kinds of proteins, which can stimulate the release of histamine, TNF-a, IL-3, IFN-γ, and LTB4 by LAD2 cells in a dose-dependent or time-dependent manner. CONCLUSIONS: A 21-35 kDa mixed protein component should be regarded as the most promising pathogenic factor contributing to the CSU associated with H. pylori infection.