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.

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.

Immunodominant SARS-CoV-2-specific CD4+ and CD8+ T-cell responses elicited by inactivated vaccines in healthy adults.

Safety profiles and humoral responses to inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have been previously assessed, but cellular immune responses to inactivated SARS-CoV-2 vaccines remain understudied. Here, we report the comprehensive characteristics of SARS-CoV-2-specific CD4+ and CD8+ T-cell responses elicited by the BBIBP-CorV vaccine. A total of 295 healthy adults were recruited, and SARS-CoV-2-specific T-cell responses were detected after stimulation with overlapping peptide pools spanning the entire length of the envelope (E), membrane (M), nucleocapsid (N), and spike (S) proteins. Robust and durable CD4+ (p < 0.0001) and CD8+ (p < 0.0001) T-cell responses specific to SARS-CoV-2 were detected following the third vaccination, with an increase in specific CD8+ T-cells, compared to CD4+ T-cells. Cytokine profiles showed that interferon gamma and tumor necrosis factor-α were predominantly expressed with the negligible expression of interleukin (IL)-4 and IL-10, indicating a Th1- or Tc1-biased response. Compared to E and M proteins, N and S activated a higher proportion of specific T-cells with broader functions. The predominant frequency of the N antigen (49/89) was highest for CD4+ T-cell immunity. Furthermore, N19-36 and N391-408 were identified to contain dominant CD8+ and CD4+ T-cell epitopes, respectively. In addition, N19-36 -specific CD8+ T-cells were mainly effector memory CD45RA cells, whereas N391-408 -specific CD4+ T-cells were mainly effector memory cells. Therefore, this study reports comprehensive features of T-cell immunity induced by the inactivated SARS-CoV-2 vaccine BBIBP-CorV and proposes highly conserved candidate peptides which may be beneficial in vaccine optimization.

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.

The BBIBP-CorV inactivated COVID-19 vaccine induces robust and persistent humoral responses to SARS-CoV-2 nucleocapsid, besides spike protein in healthy adults.

Vaccination is one of the best ways to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic. Among the various SARS-CoV-2 vaccines approved for use, the BBIBP-CorV inactivated vaccine has been widely used in 93 countries. In order to understand deeply the protective mechanism of inactivated vaccine, which retains all antigenic components of live virus, the analysis of humoral responses triggered by multiple proteins is necessary. In this research, antibody responses were generated with 6 selected recombinant proteins and 68 overlapping peptides that completely covered SARS-CoV-2 nucleocapsid (N) protein in 254 healthy volunteers vaccinated with BBIBP-CorV. As a result, antibody responses to the receptor binding domain (RBD), N, and non-structural protein 8 (NSP8) were induced following immunization by BBIBP-CorV. The antibody responses detected in donors after the 2nd dose vaccination can be maintained for about 6 months. Moreover, specific antibody levels can be restored after the boosting vaccination measured by ELISA. Furthermore, the level of SARS-CoV-2 specific IgG response is independent of age and gender. Moreover, N391-408 was identified as a dominant peptide after vaccination of BBIBP-CorV through peptide screening. Understanding the overview of humoral reactivity of the vaccine will contribute to further research on the protective mechanism of the SARS-CoV-2 inactivated vaccine and provide potential biomarkers for the related application of inactivated vaccine.

Development and validation of an efficient nomogram for risk assessment of norovirus infection in pediatric patients.

This study aimed to establish a predictive model and nomogram based on routine laboratory blood indicators and clinical symptoms, subsequently providing a rapid risk assessment of norovirus (NoV) infection in children. This retrospective study enrolled 307 pediatric patients with symptoms of acute gastroenteritis and detected NoV using real-time quantitative polymerase chain reaction. Significant indicators selected by multivariate logistic regression, including routine blood tests and consultation symptoms, were used to develop the nomogram. We divided the sample into training and internal validation sets and performed external validation of the final model. Furthermore, we evaluated the clinical performance using the Akaike information criterion (AIC), area under the curve (AUC), calibration curve, decision curve analysis (DCA), sensitivity, specificity, concordance rate, positive predictive value, and negative predictive value. Overall, 153 cases were NoV-PCR-positive, and 154 were negative. The multivariate logistic regression included five predictors of NoV infection, including symptoms of vomiting, upper respiratory tract infection, and indicators of white blood cells, lymphocyte absolute counts, and platelet counts. The nomogram showed a significant predictive value with overall internal set diagnosis, with an AUC of 0.827 (95% confidence interval (CI): 0.785-0.868), and 0.812 (95% CI: 0.755-0.869) with 0.799 (95% CI: 0.705-0.894) in the training and internal validation sets, respectively. Nevertheless, the AUC in the external validation set was higher (0.915; 95% CI: 0.862-0.968). This nomogram is a useful tool for risk assessment for NoV infection. Moreover, the evaluated indicators are accessible, substantially reducing the time for laboratory testing.

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.

Fecal calprotectin concentrations in cancer patients with Clostridium difficile infection.

Fecal calprotectin (fCPT) has been used as a surrogate marker for assessment of intestinal inflammation. We explore the utility of fCPT values as a diagnostic aid in cancer patients with suspected Clostridium difficile infection (CDI). A total of 232 stool specimens submitted for GeneXpert C. difficile PCR testing were included in the study. All specimens were tested for fCPT and toxin/GDH antigens. Clinical severity of CDI cases was determined by the IDSA/SHEA criteria. Significant differences of median fCPT values between CDI (n = 117, Median 183.6 µg/g) and non-CDI (n = 115, 145.6 µg/g, p = 0.006) patients were seen. In CDI patents, significantly lower fCPT values were found in patients with mild to moderate (n = 95, 182.1 µg/g) than those with severe and severe to complicated (n = 22, 218.5 µg/g, p = 0.014) scores, and among those that were toxin positive (n = 24, 200.2 µg/g) vs. toxin negative (n = 86, 182.8 µg/g, p = 0.044). Despite this overall trend, wide variations in fCPT values were found in all categories examined. A logistic regression analysis revealed that the fCPT values correlated independently with the severity of clinical manifestations (OR = 2.021, 95%CI = 1.132-3.608); however, it did not correlate with other clinical outcomes. Our study findings show that high fecal calprotectin levels correlate with toxin-positive and clinically severe CDI; however, wide variations in individual measurements preclude establishment of reliable cut-offs for routine diagnostic use in cancer patients.

Multicenter Evaluation of the ePlex Respiratory Pathogen Panel for the Detection of Viral and Bacterial Respiratory Tract Pathogens in Nasopharyngeal Swabs.

The performance of the new ePlex Respiratory Pathogen (RP) panel (GenMark Diagnostics) for the simultaneous detection of 19 viruses (influenza A virus; influenza A H1 virus; influenza A 2009 H1 virus; influenza A H3 virus; influenza B virus; adenovirus; coronaviruses [HKU1, OC43, NL63, and 229E]; human rhinovirus/enterovirus; human metapneumovirus; parainfluenza viruses 1, 2, 3, and 4; and respiratory syncytial virus [RSV] [RSV subtype A and RSV subtype B]) and 2 bacteria (Mycoplasma pneumoniae and Chlamydia pneumoniae) was evaluated. Prospectively and retrospectively collected nasopharyngeal swab (NPS) specimens (n = 2,908) were evaluated by using the ePlex RP panel, with the bioMérieux/BioFire FilmArray Respiratory Panel (BioFire RP) as the comparator method. Discordance analysis was performed by using target-specific PCRs and bidirectional sequencing. The reproducibility of the assay was evaluated by using reproducibility panels comprised of 6 pathogens. The overall agreement between the ePlex RP and BioFire RP results was >95% for all targets. Positive percent agreement with the BioFire RP result for viruses ranged from 85.1% (95% confidence interval [CI], 80.2% to 88.9%) to 95.1% (95% CI, 89.0% to 97.9%), while negative percent agreement values ranged from 99.5% (95% CI, 99.1% to 99.7%) to 99.8% (95% CI, 99.5% to 99.9%). Additional testing of discordant targets (12%; 349/2,908) confirmed the results of ePlex RP for 38% (131/349) of samples tested. Reproducibility was 100% for all targets tested, with the exception of adenovirus, for which reproducibilities were 91.6% at low virus concentrations and 100% at moderate virus concentrations. The ePlex RP panel offers a new, rapid, and sensitive "sample-to-answer" multiplex panel for the detection of the most common viral and bacterial respiratory pathogens.

Norovirus Loads in Stool Specimens of Cancer Patients with Norovirus Gastroenteritis.

In immunocompromised patients with norovirus (NoV) gastroenteritis, the relationship between fecal NoV load and clinical complications has not been examined. In this study, a validated real-time quantitative PCR assay was used to determine viral loads for NoV genogroup I and II (GI and GII) in NoV-positive stool specimens of cancer patients. A total of 234 specimens from 152 patients were positive for NoV, including 201 of GII and 33 of GI. Geometric mean of logarithmic copies per gram of stool (w/w) of NoV-GII were 9.03 ± 1.71 (means ± SD), which was significantly higher than that of NoV-GI [7.87 ± 1.49; odd ratio (OR), 3.22; 95% CI, 1.33-7.76; P = 0.009]. Among 152 patients with gastroenteritis, the fecal NoV geometric mean of logarithmic copy was correlated with mild (n = 85; 7.97 ± 1.55), moderate (n = 23; 9.09 ± 1.38), and severe (n = 44; 10.39 ± 0.91) episodes of severity by modified Vesikari scoring system, respectively. Multivariate analysis revealed that high level of NoV load was correlated with GII infections (OR, 4.13; 95% CI, 1.62-10.55; P = 0.003) and associated with development of severe clinical symptom (OR, 5.53; 95% CI, 2.00-7.24; P = 0.001) at the time of diagnosis. Infection with GII strains was more common than GI infection in cancer patients with viral gastroenteritis.

Laboratory Diagnosis of Central Nervous System Infection.

Central nervous system (CNS) infections are potentially life threatening if not diagnosed and treated early. The initial clinical presentations of many CNS infections are non-specific, making a definitive etiologic diagnosis challenging. Nucleic acid in vitro amplification-based molecular methods are increasingly being applied for routine microbial detection. These methods are a vast improvement over conventional techniques with the advantage of rapid turnaround and higher sensitivity and specificity. Additionally, molecular methods performed on cerebrospinal fluid samples are considered the new gold standard for diagnosis of CNS infection caused by pathogens, which are otherwise difficult to detect. Commercial diagnostic platforms offer various monoplex and multiplex PCR assays for convenient testing of targets that cause similar clinical illness. Pan-omic molecular platforms possess potential for use in this area. Although molecular methods are predicted to be widely used in diagnosing and monitoring CNS infections, results generated by these methods need to be carefully interpreted in combination with clinical findings. This review summarizes the currently available armamentarium of molecular assays for diagnosis of central nervous system infections, their application, and future approaches.

[Relationship between M-Protein of Multiple Myeloma and False Positive Syphilis Serological Results].

BACKGROUND: The false positive in conventional syphilis serological test was found in patients with multiple myeloma (MM). OBJECTIVE: To investigate the relationship between the M-protein of patients with MM and the false positive in conventional syphilis serologic test. METHODS: The M-protein of 68 MM cases was typed with immunofixation electrophoresis and 68 cases of MM were screened with non-specific and specific syphilis serologic tests, then the samples with syphilic serological positive were chosen and confirmed with immonobloting test, finally the relationship between M protein of MM and the false positive of syphilis serological test were analysed. RESULTS: Four out of 68 cases showed the positive in syphilis serological test and further were confimed to be false positive by immunoblotting test, the false positive rate was nearly 6%. The M-protein of MM patients in our hospital mostly possessed IgG, κ type, followed by IgA, κ type, light chain κ type. In general, κ : λ = 2.4 : 1. Among samples of 4 cases with syphilis serological positive 2 cases were of IgG and κ type, 1 case was of IgG, λ type, another 1 case was IgA, κ type. CONCLUSION: The M-protein of IgG and IgA types in MM patients results in syphilis serological false positive reaction. The clinicians and laboratorial technicians should pay a great attention to screen the MM patients for the false positive syphilis serological test so as to avoid the misdiagnosis and subsequent embarassment.

[Preparation and selection of the monoclonal antibody used for kits to detect BNP32 which was applied to clinical research].

AIM: To prepare the monoclonal antibody (mAb) used for kits to detect the BNP32 antigen by means of double-antibody sandwich ELISA assay. Comparasion differences on detection BNP between double-antibody sandwich ELISA and Roche ECL. METHODS: BALB/c mice were immunized with purified recombinant BNP32 protein and by routine hybridoma technique, Then comparasion differences on detection BNP between double-antibody sandwich ELISA and Roche ECL. RESULTS: 16 hybridoma cell lines secreting potent mAb against BNP32 were obtained. The subtype of these 16 mAb were found to be IgG1, IgG2a and IgM, then their cross-blocking properties were analyzed, when they reacted with the BNP32 protein in direct ELISA in order to offer the valuable data for selecting feasible pair of mAb in the detection of the BNP32 antigen. All the mAbs were used for the detection of BNP32 by double-antibody sandwich ELISA. In addition, the mAbs were purified and HRP-labelled in advance. Finally, we had successfully screened a pair of mAbs, which exhibited a sensitivity of 20 ng/L for the detection of BNP32 antigen. The two detection methods have very good consistency (kappa=0.828>0.75)between double-antibody sandwich ELISA and Roche ECL. There was no statistics significance on differences between these two methods(P>0.05). CONCLUSION: It is evident that this pair of mAb shows excellent detection of BNP32. The double-antibody sandwich ELISA can be good apply to detect BNP level in clinical congestive heart failure patients.

[Preparation and application of anti-serum against mouse Foxp3].

AIM: To prepare the high titer and specific anti-serum against mouse Foxp3 and then use them to identify Foxp3 expression in normal mouse tissues. METHODS: The Foxp3 fragment was amplified by polymerase chain reactions(PCR) and cloned into the prokaryotic expression vector pGEX-6p-2. The recombinant plasmid pGEX-6p-2/Foxp3 was transformed into E.coli BL21(DE3) to be expressed. The expressed fusion protein GST-Foxp3 was analyzed by SDS-PAGE and Western blot. The polyclonal clanti-serum was obtained by immunizing New Zealand rabbits with the purified fusion protein as antigen. The Foxp3 expression in normal mouse tissues was detected by Western blot with the anti-serum. RESULTS: The expressed products were analyzed by SDS-PAGE and Western blot. The results showed that the molecular weight of the expressed protein was about 45 000. The titer of the anti-serum was above 1:12 800. We observed that the anti-serum could recognize Foxp3 protein expressed in NIH3T3 cells by immunoblotting. Western blot results showed that the Foxp3 protein was expressed highly in spleen, thymus and lymph nodes, less expressed in stomach, but not expressed in skeletal muscle and adipose tissue. CONCLUSION: High titer antiserum against mouse Foxp3 is produced and can be used to identify the Foxp3 expression in normal mouse tissues.