BACKGROUND: Zoster vaccination is recommended for people with a history of herpes zoster (HZ), but the most effective timing of vaccine administration after zoster illness is unresolved. This prospective observational study compared the immunogenicity and safety of administering HZ vaccine at 6-12 months and 1-5 years after zoster illness. MATERIALS AND METHODS: Blood samples were collected before the administration of live zoster vaccine and 6 weeks after vaccination. Varicella-zoster virus (VZV) IgG concentrations and T-cell responses were assessed by glycoprotein enzyme-linked immunosorbent assay and interferon-γ enzyme-linked immunospot assay (ELISPOT), respectively. RESULTS: The baseline geometric mean value (GMV) of VZV IgG was higher in the 6-12 months group than in the 1-5 years group (245.5 IU/mL vs. 125.9 IU/mL; P = 0.021). However, the GMV increased significantly in both groups (P = 0.002 in the 6-12 months group; P <0.001 in the 1-5 years group). The results of the ELISPOT assay were not significant for differences of the GMV between baseline and 6-week post-vaccination groups, while the GMV increased significantly in both groups (P = 0.001 in the 6-12 months group; P <0.001 in the 1-5 years group). CONCLUSION: The immunogenicity of zoster vaccine may be similar whether administered 6-12 months, or >1 year after zoster illness. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02704572
Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Glycoproteins ; Herpes Zoster Vaccine* ; Herpes Zoster* ; Herpesvirus 3, Human ; Immunoglobulin G ; Observational Study ; Prospective Studies* ; T-Lymphocytes ; Vaccination*

BACKGROUND: Zoster vaccination is recommended for people with a history of herpes zoster (HZ), but the most effective timing of vaccine administration after zoster illness is unresolved. This prospective observational study compared the immunogenicity and safety of administering HZ vaccine at 6-12 months and 1-5 years after zoster illness. MATERIALS AND METHODS: Blood samples were collected before the administration of live zoster vaccine and 6 weeks after vaccination. Varicella-zoster virus (VZV) IgG concentrations and T-cell responses were assessed by glycoprotein enzyme-linked immunosorbent assay and interferon-γ enzyme-linked immunospot assay (ELISPOT), respectively. RESULTS: The baseline geometric mean value (GMV) of VZV IgG was higher in the 6-12 months group than in the 1-5 years group (245.5 IU/mL vs. 125.9 IU/mL; P = 0.021). However, the GMV increased significantly in both groups (P = 0.002 in the 6-12 months group; P <0.001 in the 1-5 years group). The results of the ELISPOT assay were not significant for differences of the GMV between baseline and 6-week post-vaccination groups, while the GMV increased significantly in both groups (P = 0.001 in the 6-12 months group; P <0.001 in the 1-5 years group). CONCLUSION: The immunogenicity of zoster vaccine may be similar whether administered 6-12 months, or >1 year after zoster illness. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02704572
Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Glycoproteins ; Herpes Zoster Vaccine* ; Herpes Zoster* ; Herpesvirus 3, Human ; Immunoglobulin G ; Observational Study ; Prospective Studies* ; T-Lymphocytes ; Vaccination*

The detailed kinetics of the cytomegalovirus (CMV)-specific T cell response in hematopoietic stem cell transplant (HCT) recipients have not yet been fully assessed. We evaluated these kinetics of CMV-specific T cell response and factors associated with high CMV-specific T cell responses 1 year after HCT. In HCT recipients, CMV pp65 and IE1-specific ELISPOT assay were performed before HCT (D0), and at 30 (D30), 90 (D90), 180 (D180), and 360 (D360) days after HCT. Of the 51 HCT recipients with donor-positive (D+)/recipient-positive (R+) serology, 26 (51%) developed CMV infections after HCT. The patterns of post-transplantation reconstitution for CMV-specific T cell response were classified into 4 types: 1) an initial decrease at D30 followed by gradual T cell reconstitution without CMV infection (35%), 2) an initial decrease at D30 followed by gradual T cell reconstitution preceded by CMV infection (35%), 3) failure of gradual or constant T cell reconstitution (26%), and 4) no significant T cell reconstitution (4%). There was no significant difference between ELISPOT counts of D360 and those of D0. High CMV-specific T cell responses at D360 were not associated with high CMV-specific T cell response at D0, CMV infection, ganciclovir therapy, graft versus host disease (GVHD), and immunosuppressant use. In conclusion, there are 4 distinct patterns of reconstitution of the CMV-specific T cell response after HCT. In addition, reconstituted donor-origin CMV-specific T cell responses appeared to be constant until day 360 after HCT, regardless of the level of the pre-transplant CMV-specific T cell response, CMV infection, and immunosuppressant use.
Cytomegalovirus ; Enzyme-Linked Immunospot Assay ; Follow-Up Studies* ; Ganciclovir ; Graft vs Host Disease ; Hematopoietic Stem Cell Transplantation* ; Hematopoietic Stem Cells* ; Kinetics* ; Theophylline

BACKGROUND/AIMS: We evaluated the proposed clinical application of the combined interpretation of host factors and viral factors in two different cytomegalovirus (CMV) co-infection models. METHODS: We prospectively enrolled all human immunodeficiency virus non-infected patients with confirmed Pneumocystitis jirovecii pneumonia (PCP) and those with suspected gastrointestinal CMV disease in a tertiary hospital. All patients underwent CMV interferon-γ releasing assay (IGRA) for CMV (T-track CMV, Lophius Biosciences). We created the 2-axis model with the CMV IGRA results as the x-axis and the results for CMV virus replication as the y-axis, and hypothesized that cases falling in the left upper quadrant (high viral load and low CMV-specific immunity) of the model would be true CMV infections. The CMV IGRA results were concealed from the attending physicians. RESULTS: Of 39 patients with PCP, four (10%) were classified as combined CMV pneumonia, 13 (33%) as bystander activation, and the remaining 22 (56%) as no CMV infection. The data for all four patients with PCP and CMV pneumonia fell in the left upper quadrant of the 2-axis model. Of 24 patients with suspected gastrointestinal CMV disease, 12 (50%) were classified as gastrointestinal CMV disease and the remaining 12 (50%) as bystander activation with no gastrointestinal CMV disease. The data for 11 of the 12 patients (92%) with gastrointestinal CMV disease were located in the left upper quadrant of the 2-axis model. CONCLUSIONS: Cases yielding low CMV IGRA results and high CMV viral replication appear to be true CMV infections. Further studies with large number of cases in different types of CMV disease should be proposed.
Accidental Falls ; Coinfection ; Cytomegalovirus* ; Enzyme-Linked Immunospot Assay ; HIV ; Humans ; Pneumonia ; Prospective Studies ; Tertiary Care Centers ; Viral Load ; Virus Replication

Cytomegalovirus (CMV) is one of the most important opportunistic infections in transplant recipients. Tests for CMV-specific T cell responses have been proposed to change the current risk stratification strategy using CMV assays. We evaluated the usefulness of pre-transplant CMV-specific T cell assays in kidney transplant (KT) candidates for predicting the development of CMV infection after transplantation comparing the results of the overlapping peptides (OLPs)-based enzyme-linked immunospot (ELISPOT) assay and the commercial QuantiFERON-CMV assay. We prospectively enrolled all cases of KT over a 5-month period, except donor CMV-seropositive and recipient seronegative transplants that are at highest risk of CMV infection. All the patients underwent QuantiFERON-CMV, CMV OLPs-based pp65, and immediate-early 1 (IE-1)-specific ELISPOT assays before transplantation. The primary outcome was the incidence of CMV infection at 6 months after transplant. The total of 47 KT recipients consisted of 45 living-donor KTs and 2 deceased-donor KTs. There was no association between positive QuantiFERON-CMV results and CMV infection. However, 10 of 34 patients with phosphoprotein 65 (pp65)- or IE-1-specific ELISPOT results higher than cut-off value developed CMV infections compared with none of 13 patients with results lower than cut-off value developed CMV. The OLPs-based ELISPOT assays are more useful than the QuantiFERON-CMV assay for predicting CMV infection. Patients with higher CMV-specific T cell immunity at baseline appear to be more likely to develop CMV infections after KT, suggesting that the abrupt decline in CMV-specific T cell responses after immunosuppression, or high CMV-specific T cell responses due to frequent CMV activation before KT, may promote CMV infection.
Cytomegalovirus ; Enzyme-Linked Immunospot Assay* ; Humans ; Immunity, Cellular ; Immunosuppression ; Incidence ; Interferon-gamma Release Tests ; Kidney* ; Opportunistic Infections ; Peptides ; Prospective Studies ; Tissue Donors ; Transplant Recipients*

The current cytomegalovirus (CMV) prevention strategies in solid organ transplantation (SOT) recipients have contributed towards overcoming the detrimental effects caused by CMV lytic infection, and improving the long-term success rate of graft survival. Although the quantification of CMV in peripheral blood is the standard method, and an excellent end-point for diagnosing CMV replication and modulating the anti-CMV prevention strategies in SOT recipients, a novel biomarker mimicking the CMV control mechanism is required. CMV-specific immune monitoring can be employed as a basic tool predicting CMV infection or disease after SOT, since uncontrolled CMV replication mostly originates from the impairment of immune responses against CMV under immunosuppressive conditions in SOT recipients. Several studies conducted during the past few decades have indicated the possibility of measuring the CMV-specific cell-mediated immune response in clinical situations. Among several analytical assays, the most advancing standardized tool is the QuantiFERON®-CMV assay. The T-Track® CMV kit that uses the standardized enzyme-linked immunospot assay is also widely employed. In addition to these assays, immunophenotyping and intracellular cytokine analysis using flow cytometry (with fluorescence-labeled monoclonal antibodies or peptide-major histocompatibility complex multimers) needs to be adequately standardized and validated for potential clinical applications.
Antibodies, Monoclonal ; Cytomegalovirus* ; Enzyme-Linked Immunospot Assay ; Flow Cytometry ; Graft Survival ; Immunity, Cellular ; Immunophenotyping ; Major Histocompatibility Complex ; Methods ; Monitoring, Immunologic ; Organ Transplantation* ; Transplants*

BACKGROUND: In March 2013, human infection with avian influenza A (H7N9) virus emerged in China, causing serious public health concerns and raising the possibility of avian-source pandemic influenza. Thus, the development of an effective vaccine for preventing and rapidly controlling avian influenza A (H7N9) virus is needed. In this study, we evaluated the immunogenicity of a synthetic DNA vaccine against H7 HA antigens in mice. MATERIALS AND METHODS: The synthetic consensus H7 HA DNA vaccine (25 or 50 µg) was administered to BALB/c mice at 0, 14, and 28 days by intramuscular injection followed by electroporation. Humoral and cellular immune responses were analyzed in a hemagglutination inhibition test and interferon-gamma enzyme-linked immunospot (ELISpot) assay, respectively. RESULTS: H7 HA-vaccinated mice showed 100% seroprotection and seroconversion rate against H7N9 reassortant influenza virus after both second and third immunizations. The geometric mean titer by the hemagglutination inhibition test increased with an increasing number of immunizations. However, there was no significant difference in geometric titer between the two groups injected with 25 and 50 µg of H7 HA DNA vaccine after two (79.98 vs. 107.65, P = 0.39) and three (159.96 vs. 215.28, P = 0.18) doses. In addition, the ELISpot assay revealed that administration of H7 HA DNA vaccine induced potent interferon-gamma production from mouse splenocytes. CONCLUSIONS: This study demonstrated the humoral and cellular immunogenicity of synthetic consensus H7 HA DNA vaccine in mice. This work demonstrates the potential of the H7 HA DNA vaccine as an efficient tool for the rapid control of emerging influenza A (H7N9) virus.
Animals ; China ; Consensus ; DNA* ; Electroporation ; Enzyme-Linked Immunospot Assay ; Hemagglutination Inhibition Tests ; Humans ; Immunity, Cellular ; Immunization ; Influenza in Birds* ; Influenza, Human ; Injections, Intramuscular ; Interferon-gamma ; Mice* ; Orthomyxoviridae ; Pandemics ; Public Health ; Seroconversion

OBJECTIVETo establish a method of detecting spinal tuberculosis (TB) infection by enzyme-linked immunospot (ELlSPOT) assay and evaluate the value of CFP10/ESAT6 fusion protein for diagnosis of spinal TB.

METHODSSuspected spinal TB patients were prospectively recruited in two hospitals (First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine; Nanfang Hospital, Southern Medical University) from May 2012 to December 2013. Data on clinical characteristics of the patients and conventional laboratory results were collected. Compare and analyze the positive detection rate in spinal TB diagnosis by different methods including ELISPOT detection and conventional detection methods.

RESULTS47 patients with spinal TB had available biopsy or surgical specimens for histopathological examination and 41 specimens had pathological features consistent with a diagnosis of TB infection. Among the spinal TB patients and non-TB disease patients,the overall sensitivity, specificity, positive predictive value, and negative predictive value of the ELISPOT assay in spinal TB diagnosis were 82.7%,87.2%,89.6%, and 79.1%,respectively; the 4 indexes of the PPD skin test were 61.5%, 46.2%, 60.4%, and 47.4%, respectively;those of the antibody detection were 55.8%, 61.5%, 65.9%, and 51.1%. The positive rate of ELISPOT was significantly higher than those of PPD skin test and antibody detection test (82.7% vs. 61.5%, Χ² =5.786, P=0.016; 82.7% vs. 55.8%, Χ² =8.847, P=0.003), but not significantly different from the positive rate of pathological examination (82.7% vs. 87.2%, Χ² =0.396, P=0.529). Moderate agreement was found between pathological examination and the ELISPOT assay (87.2%, Κ=0.498, P=0.001).

CONCLUSIONWith high sensitivity and specificity, the ELISPOT assay using CFP10/ESAT6 fusion protein as antigen is an effective technique for auxiliary diagnosis of spinal TB.

We established an ELISPOT for bovine interferon-gamma (BoIFN-γ), and applied it in the diagnosis of bovine tuberculosis (bTB). Monoclonal antibodies that can bind with native BoIFN-γ were screened as the coating antibody and detecting antibody. After optimization of detecting conditions including coating antibody concentration, cell number, and detecting antibody concentration, the ELISPOT assay was established. Peripheral mononuclear cells (PBMCs) isolated from 30 cows were co-cultured with PPD, and detected with the ELISPOT assay. The optimal conditions of ELISPOT assay were 2.5 μg/mL coating antibody 2G5, 2.5 x 10(5) cells/well, and 1 μg/mL detecting antibody Bio-5E11. In these 30 cows tested both with the ELISPOT assay and the BOVIGAM kit, 11 cows were proved to be positive in ELISOPT assay with the sensitivity of 78.6%, and 12 cows were proved to be negative in ELISOPT assay with the specificity of 75%. The ELISPOT assay for BoIFN-γ could be used to detect bTB efficiently and it might be an alternative method for the diagnosis of bTB.
Animals ; Antibodies, Monoclonal ; Cattle ; Enzyme-Linked Immunospot Assay ; veterinary ; Female ; Interferon-gamma ; isolation & purification ; Sensitivity and Specificity ; Tuberculosis, Bovine ; diagnosis

PURPOSE: To evaluate the diagnostic utility and predictors for determinate results of an enzyme-linked immunospot assay using induced sputum cells (IS ELISPOT) for a rapid diagnosis of pulmonary tuberculosis (TB). MATERIALS AND METHODS: Subjects suspected of pulmonary TB who had either sputum acid fast bacilli smear-negative or not producing sputum spontaneously were prospectively enrolled. ELISPOT assay was performed using cells from induced sputum. RESULTS: A total of 43 subjects, including 25 with TB (TB group) and 18 with non-TB disease (non-TB group) were enrolled. Results of IS ELISPOT were determinate in only 17/43 (39%) subjects, but all of determinate results were consistent with the final diagnosis. Of the 43 sputum samples, 11 (26%) were inadequate to perform IS ELISPOT. Of 32 adequate sputum samples, the proportion of determinate results was significantly higher in the TB group (75%, 15/20) than in the non-TB group (17%, 2/12) (p=0.002). The status of active TB was a unique predictor but smear positivity was not a significant predictor for determinate results. In addition, sensitivity of IS ELISPOT (75%, 9/12) in smear negative TB was higher than that of TB-polymerase chain reaction (25%, 3/12). CONCLUSION: IS ELISPOT showed relatively high diagnostic value and accuracy in the TB group, independent of smear positivity. IS ELISPOT may provide additional diagnostic yield for microbiological tools in the rapid diagnosis of smear-negative TB.
Adult ; Aged ; *Enzyme-Linked Immunospot Assay ; Female ; Humans ; Immunologic Tests/*methods ; Male ; Middle Aged ; Mycobacterium tuberculosis/*isolation & purification ; Predictive Value of Tests ; Prospective Studies ; Reproducibility of Results ; Risk Factors ; Sensitivity and Specificity ; Sputum/*microbiology ; Tuberculosis, Pulmonary/*diagnosis/microbiology