Positive ELISPOT Research Articles

Responses to Non-Polymorphic HLA Derived Peptides in a Prospective Study of Renal Transplant Recipients

Introduction: Previous studies have shown that peripheral blood mononuclear cells (PBMCs), isolated from renal transplant patients, at least eighteen months post-transplant produce IFN-g in response to peptides derived from the non-polymorphic a3domain of Class 1 HLA. Further work (non-published) has shown similar results to peptides derived from the non-polymorphic region of HLA class 2. These responses significantly correlated with markers of chronic allograft dysfunction. This may form the basis of a diagnostic assay using synthetically derived peptides to act as antigens. Methods: In this prospective study of 85 renal transplant patients, responses to non-polymorphic HLA derived peptides, were measured pre-transplantation and at time 3 months, 6 months and 12 months post-transplant. Ethical approval was granted from North Staffordshire Research Ethics Committee and in accordance with the declaration of Helsinki. PBMCs were isolated and incubated for 48 hours with media (negative control), 11.4μmol/L of up to 7 15mer peptide mixes, 10μg/ml PPD or 1μg/ml antiCD3 (positive control) in IFN-g ELISPOT plates (Mabtech). Plates were developed according to the manufacturer's instructions and read using AID ELISPOT reader with ELISPOT 4.0 software. Results:Table: [ELISPOT and renal transplant biopsy results]We correlated ELISPOT responses with biopsy confirmed rejection, episodes of infection and change in eGFR and isotopic GFR between 3 and 12 months post-transplant. The frequency of positive ELISPOT responses increased during the course of the first year following transplantation. There was a greater percentage of biopsy confirmed rejection in patients who had a positive ELISPOT response (35%) at 12 months. However, when compared to biopsy confirmed rejection in patients who had an ELISPOT negative response (19%) this failed to reach statistical significance (p=0.17, Chi squared test). The incidence of infections (CMV, viral, bacterial, fungal) was not significantly increased in patients with a positive ELISPOT response at 3, 6 and 12 months. There was a significant reduction in eGFR between 3-12 months in patients with a positive ELISPOT response at 12 months (negative 0.5 ±2.0 ml min -1 vs. positive -0.2 ± 1.6 ml min-1 (p< 0.008)). This was confirmed by change in GFR calculated by isotopic GFRs at 3 months and 12 months, (negative 6.00 ± 1.410 ml min -1 vs. positive 0.13 ±0.91 (p=0.026). Conclusion: These results support our hypothesis that responses to non-polymorphic HLA derived peptides develop during the course of renal transplant follow up. This may have value as a diagnostic and prognostic tool for assessing cellular immunity in a renal transplant population.

HIV-1-Specific Enzyme-Linked Immunosorbent Spot Assay Responses in HIV-1-Exposed Uninfected Partners in Discordant Relationships Compared to Those in Low-Risk Controls

A number of studies of highly exposed HIV-1-seronegative individuals (HESN) have found HIV-1-specific cellular responses. However, there is limited evidence that responses prevent infection or are linked to HIV-1 exposure. Peripheral blood mononuclear cells (PBMC) were isolated from HESN in HIV-1-discordant relationships and low-risk controls in Nairobi, Kenya. HIV-1-specific responses were detected using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays stimulated by peptide pools spanning the subtype A HIV-1 genome. The HIV-1 incidence in this HESN cohort was 1.5 per 100 person years. Positive ELISpot responses were found in 34 (10%) of 331 HESN and 14 (13%) of 107 low-risk controls (odds ratio [OR] = 0.76; P = 0.476). The median immunodominant response was 18.9 spot-forming units (SFU)/10(6) peripheral blood mononuclear cells (PBMC). Among HESN, increasing age (OR = 1.24 per 5 years; P = 0.026) and longer cohabitation with the HIV-1-infected partner (OR = 5.88 per 5 years; P = 0.003) were associated with responses. These factors were not associated with responses in controls. Other exposure indicators, including the partner's HIV-1 load (OR = 0.99 per log(10) copy/ml; P = 0.974) and CD4 count (OR = 1.09 per 100 cells/μl; P = 0.238), were not associated with responses in HESN. HIV-1-specific cellular responses may be less relevant to resistance to infection among HESN who are using risk reduction strategies that decrease their direct viral exposure.

Predictive Value of Interferon-γ ELISPOT Assay in HIV 1-Infected Patients in an Intermediate Tuberculosis-Endemic Area

The tuberculin skin test for diagnosing latent tuberculosis (TB) has some limitations for HIV-infected patients, especially in BCG vaccinated countries. The objective of this study was to identify the incidence rate of new TB cases among HIV-infected patients in an intermediate TB-endemic area and to examine its correlation with the ELISPOT assay. We prospectively followed up 124 patients with HIV-1 infection to monitor development of active TB disease after performing an ELISPOT assay (T-SPOT. TB test, Oxford Immunotec, Ltd., Abingdon, UK). A total of 120 patients were followed for a median of 947 days; four patients with active TB at enrollment were excluded. Eleven patients developed active TB during 238 person-years, giving a high incidence rate of 4621/100,000 person-years. Patients with positive ELISPOT responses had a higher TB incidence rate than those with negative ELISPOT responses; however this was not statistically significant [20% (6/30) vs. 6.02% (5/83), p=0.052]. A Cox regression analysis showed that the independent risk factors associated with progression of TB were low CD4(+) T cell counts, previous history of TB treatment, and positive ELISPOT results. Advanced HIV-infected patients who showed a positive TB ELISPOT assay had a higher rate of progression to TB in the intermediate TB-endemic area.

Interferon-gamma release assays in the detection of latent tuberculosis infection in patients with inflammatory arthritis scheduled for anti-tumour necrosis factor treatment

Biological agents, particularly anti-Tumour Necrosis Factor (TNF)-α agents, have emerged as an effective treatment in patients with chronic inflammatory diseases. An association between anti-TNF-α antibodies and reactivation of latent tuberculosis infection (LTBI) has been established. Appropriate screening for TB infection has become mandatory before starting a treatment based on TNF-α inhibition. The objective was to determine the usefulness of IFN-γ release assays in diagnosing LTBI in patients with inflammatory rheumatic diseases scheduled for anti-TNF-α treatment. The study included 53 individuals with inflammatory rheumatism. All patients had a TST, a chest radiograph, QuantiFERON Gold In-Tube (QFN-G-IT) and T-SPOT.TB. To investigate the influence of non-tuberculous mycobacteria (NTM) infections on non-BCG-vaccinated patients, with a positive TST result and both negative IFN-γ assays, we performed an ex vivo ELISPOT, stimulating the cells separately with NTM sensitins. TST was positive in 7 cases, T-SPOT.TB in 11 and QFN-G-IT in 9 cases. Agreement between TST and T-SPOT.TB and QFN-G-IT was 77.35% (κ = 0.33 and κ = 0.40, respectively), and between both in vitro tests, it was 83.01% (κ = 0.57). Of the three patients with positive TST and negative T-SPOT.TB and QFN-G-IT, one positive ELISPOT result was obtained after stimulation with NTM sensitins. Positive TST, T-SPOT.TB and QFN-G-IT results were not affected by the immunosuppressive therapies. IFN-γ release assays are useful methods for avoiding TST false-positive results, but in those patients with a high risk of developing active TB and in the absence of predictive value studies in this specific kind of population for knowing how safe is the use of IGRAs alone, the combined use of TST and IFN-γ tests should be recommended in order to increase the overall number of LTBI diagnoses.

A Pilot Trial of WT1 Peptide-Loaded Allogeneic Dendritic Cell Vaccine and Donor Lymphocyte Infusion for WT1-Expressing Hematologic Malignancies and Post-Transplant Relapse

Abstract 3055 Background:Treatment of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) remains challenging. The Wilms' tumor 1 (WT1) gene product is a tumor-associated antigen that is expressed in acute leukemia and other hematologic malignancies with limited expression in normal tissues. This pilot trial incorporates antigen-specific immunotherapy and allogeneic adoptive cell transfer for pediatric and adult patients with relapsed hematologic malignancies after alloHSCT. The primary objectives are to assess safety and feasibility of a peptide-loaded donor-derived dendritic cell (DC) vaccine and donor lymphocyte infusion (DLI) designed to enhance the graft-vs -leukemia effect. Secondary objectives are to determine if immunologic and clinical responses to WT1 specific peptides can be generated by this novel vaccine strategy after alloHSCT. Design:HLA-A2+ patients with WT1-expressing hematologic malignancies that have relapsed after alloHSCT are eligible. Donor-derived DC vaccines are given every 2 weeks for 6 doses and DLI every 4 weeks for 3 doses. Peripheral blood monocyte-derived DCs are loaded with a combination of three HLA-A2 binding WT1 peptides (WT1 37–45; WT1 126–134; WT1 187–195) linked to the 11-mer HIV TAT protein transduction domain peptide (47–57) designed to enhance antigen presentation. The DCs are generated from donor peripheral blood monocytes that are separated from DLI collected by apheresis. Monocytes are incubated with GM-CSF and IL-4 followed by maturation with LPS and IFN-g. WT1 expression is assessed using immunohistochemistry and/or quantative RT-PCR. Study endpoints included toxicity, feasibility, and antigen-specific immune and clinical responses. Results:4 patients, aged 9–19 years have been treated to date, 3 with acute lymphoblastic leukemia (ALL) and one with Hodgkin lymphoma (HL). Donors were matched siblings in 3 cases and a 10/10 HLA matched father in one case. Vaccines were successfully produced from all donors. All patients tolerated vaccine and DLI administrations well. The most common adverse events were mild, reversible pain and pruritus at vaccine administration and delayed type hypersensitivity (DTH) skin test sites. One patient developed Grade I skin GVHD that did not require treatment. Immune responses were observed in all 3 patients with ALL. ELISPOT was considered positive when it was at least two times greater than and had at least 10 spots more than background. 3 of 4 (75%) patients had positive ELISPOT responses to WT1 peptides. 3 of 4 (75%) patients had positive DTH responses to the keyhole limpet hemocyanin (KLH) control and 2 of 4 (50%) to the WT1 peptides. Median overall survival was 12 months. 1 patient remains in remission 12 months after initiation of therapy and 3 have died of disease. All donors tolerated apheresis well and there were no complications from donor procedures. Conclusions:This novel allogeneic immunotherapy regimen is feasible, well-tolerated and can induce immune responses in the allogeneic setting. Accrual is ongoing.Table:Summary of DTH and ELISPOT ResponsesPatient #DiagnosisKLH DTH ResponseWT1 DTH ResponseWT1 ELISPOT Response1ALL+++2HL---3ALL+-+4ALL+++ Disclosures:Off Label Use: This is a pilot clinical trial of an investigational cancer vaccine.

Breast Milk HIV-1 RNA Levels and Female Sex Are Associated With HIV-1-Specific CD8+ T-Cell Responses in HIV-1-Exposed, Uninfected Infants in Kenya

Although evidence supports a relationship between human immunodeficiency virus (HIV)-1 exposure and HIV-1-specific CD8(+) T cell responses, studies have not demonstrated a direct association between the quantity of HIV-1 to which a person is exposed and the presence or absence of a response. From 1999 to 2005, maternal HIV-1 RNA levels were measured in blood, cervical secretions, and breast milk at delivery and 1 month after delivery. HIV-1-specific interferon (IFN)-γ Elispot assays were conducted to determine infant CD8(+) T-cell responses at 3 months of age. Among 161 infants tested with Elispot assays, 23 (14%) had positive results. Mothers whose infants had a positive assay had higher breast milk HIV-1 RNA levels at month 1 compared with mothers whose infants had negative Elispot assays (3.1 vs 2.5 log(10) copies/mL; P = .017). Female infants were also more likely to have positive Elispot assays than male infants (P = .046), and in multivariate analyses, both female sex and high breast milk HIV-1 levels remained important predictors of a positive response (P = .022 and P = .015, respectively). Exposure to breast milk HIV-1 and sex were associated with development of HIV-1-specific CD8(+) T-cell responses in infants. These data support a role for mucosal exposure via the oral route in induction of systemic HIV-1-specific cellular immunity.

Adenovirus-5-Vectored P. falciparum Vaccine Expressing CSP and AMA1. Part B: Safety, Immunogenicity and Protective Efficacy of the CSP Component

BackgroundA protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.Methodology/Principal FindingsNMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.SignificanceThe NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.Trial Registration ClinicalTrials.gov NCT00392015

Safety and immunogenicity of a modified pox vector-based HIV/AIDS vaccine candidate expressing Env, Gag, Pol and Nef proteins of HIV-1 subtype B (MVA-B) in healthy HIV-1-uninfected volunteers: A phase I clinical trial (RISVAC02)

To investigate the safety and immunogenicity of a modified vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B), a phase-I, doubled-blind placebo-controlled trial was performed. 30 HIV-uninfected volunteers at low risk of HIV-1 infection were randomly allocated to receive 3 intramuscular injections (1×10(8)pfu/dose) of MVA-B (n=24) or placebo (n=6) at weeks 0, 4 and 16. All volunteers were followed 48 weeks. Primary end-points were adverse events and immunogenicity. A total of 169 adverse events were reported, 164 of grade 1-2, and 5 of grade 3 (none related to vaccination). Overall 75% of the volunteers showed positive ELISPOT responses at any time point. The magnitude (median) of the total responses induced was 288SFC/10(6)PBMC at week 18. Antibody responses against Env were observed in 95% and 72% of vaccinees at week 18 and 48, respectively. HIV-1 neutralizing antibodies were detected in 33% of volunteers. MVA-B was safe, well tolerated and elicited strong and durable T-cell and antibody responses in 75% and 95% of volunteers, respectively. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Clinical Trials.gov identifier: NCT00679497.

A Prospective Longitudinal Study Evaluating the Usefulness of a T-Cell-Based Assay for Latent Tuberculosis Infection in Kidney Transplant Recipients

We evaluated whether ELISPOT assay can predict tuberculosis (TB) development in kidney-transplantation (KT) recipients with a negative tuberculin skin test (TST). All adult patients admitted to a KT institute between June 2008 and December 2009 were enrolled; TB development after KT was observed between June 2008 and December 2010. Isoniazid (INH) was given to those patients with positive TST or clinical risk factors for latent TB infection (LTBI). ELISPOT assay was performed on all patients, and TB development after KT was observed by a researcher blinded to the results of ELISPOT. A total of 312 KT recipients including 242 (78%) living-donor KT were enrolled. Of the 312 patients, 40 (13%) had positive TST or clinical risk factors for LTBI and received INH; none developed TB after KT. Of the remaining 272 patients, 4 (6%) of 71 with positive ELISPOT assay developed TB after KT, whereas none of the 201 patients with negative (n = 171) or indeterminate ELISPOTs (n = 30) developed TB after KT (rate difference between positive and negative/indeterminate ELISPOT, 3.3 per 100 person-years [95% CI 1.4-5.1, p<0.001]). Positive ELISPOT results predict subsequent development of TB in KT recipients in whom LTBI cannot be detected by TST or who lack clinical risk factors for LTBI.

Successful treatment with infliximab despite positive tuberculosis ELISpot results in a patient with pityriasis rubra pilaris taking prophylactic isoniazid

Clinical and Experimental DermatologyVolume 36, Issue 7 p. 808-809 Viewpoints in dermatology • Correspondence Successful treatment with infliximab despite positive tuberculosis ELISpot results in a patient with pityriasis rubra pilaris taking prophylactic isoniazid M. Zirbs, M. Zirbs Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this authorE. Kigitsidou, E. Kigitsidou Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this authorF. Seifert, F. Seifert Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this authorJ. Ring, J. Ring Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this authorK. Brockow, K. Brockow Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this author M. Zirbs, M. Zirbs Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this authorE. Kigitsidou, E. Kigitsidou Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this authorF. Seifert, F. Seifert Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this authorJ. Ring, J. Ring Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this authorK. Brockow, K. Brockow Department of Dermatology and Allergy Clinic and Polyclinic for Dermatology and Allergology, Klinikum Rechts der Isar, Technische Universitaet Muenchen, Biedersteinerstraβe 29, Muenchen, GermanyE-mail: michael.zirbs@lrz.tu-muenchen.deSearch for more papers by this author First published: 20 April 2011 https://doi.org/10.1111/j.1365-2230.2011.04074.xCitations: 10 Conflict of interest: none declared. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume36, Issue7October 2011Pages 808-809 RelatedInformation

146 Strong and Broad Immunogenicity of a Multigene, Multiclade HIV-1 DNA Prime MVA Boost Vaccine Regimen Among Healthy Tanzanian Volunteers

A phase I trial (HIVIS01/02) of a multigene, multiclade HIV-1 DNA prime heterologous MVA boost vaccine regimen among healthy volunteers in Sweden showed that the vaccines were safe and highly immunogenic (J Inf Dis 2008;198:1482-90). A phase I/II trial (HIVIS03) using the same vaccine constructs has subsequently been conducted in Dar es Salaam, Tanzania. Sixty HIV-uninfected volunteers randomised to three groups of 20 received HIV-DNA vaccine at 1 mg intradermally (i.d) or 3.8 mg intramuscularly (i.m.) or placebo using a needle-free device. The DNA plasmids containing HIV-1 gp160 subtypes A,B,C, revB, gag A,B and RtmutB were given at months 0, 1 and 3. The volunteers were boosted i.m. with 108 pfu of MVA containing HIV-1 env, gag, pol of CRF01A_E or placebo at months 9 and 21.Two to four weeks after the second HIV-MVA boost, 28 (97%) of 29 vaccinees had positive IFN-gamma ELISpot responses, 27 (93%) to Gag and 23 (79%) to Env peptides. The i.d. primed vaccinees showed higher immune responses to Env compared to the i.m. primed vaccinees. Intracellular cytokine staining for Gag-specific IFN-gamma/IL-2 production 4 weeks after the second HIV-MVA boost showed both CD8 and CD4 T-cell responses. All of 25 vaccinees had lymphoproliferative responses of a similar high magnitude to AT-2-treated HIV antigens from 3 different subtypes (donated by J Lifson, NCI, USA). After the second HIV-MVA boost, 26/29 (90 %) of the vaccinees developed binding antibodies to gp160. In conclusion, this HIV-DNA prime MVA boost vaccine regimen induced strong and broad immune responses in Tanzanian volunteers.

Diagnostic usefulness of enzyme-linked immunospot assay for interferon-gamma for tuberculosis in cancer patients

The interferon-γ enzyme-linked immunospot assay (ELISPOT) has been demonstrated to be useful in the diagnosis of active tuberculosis (TB). In this study we aimed to evaluate the diagnostic performance of the ELISPOT assay in cancer patients with suspected pulmonary TB. Eighty-one cancer patients with suspected pulmonary TB were prospectively enrolled from April 2007 to December 2008, to investigate the diagnostic sensitivity and specificity of the ELISPOT assay. Of the 38 patients with TB, 33 (86.8%) had positive ELISPOT results. Of the 43 patients without TB, the results of the ELISPOT assay were negative in 35 (81.3%) patients. The overall sensitivity was 86.8%, specificity 81.3%, positive predictive value 80.5% and negative predictive value 87.5%. No significant difference was noted for the diagnostic performance of the ELISPOT assay for diagnosing TB between solid cancer and haematological cancer patients. In addition, a quantitative study did not show that TB patients with solid cancers have a better response than haematological cancer patients as measured by spot-forming cells per 10(6) peripheral blood mononuclear cells after exposure to early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10). In conclusion, the ELISPOT assay could be a useful supplementary tool for the diagnosis of pulmonary TB among cancer patients, irrespective of cancer type.

Reversion and conversion of Mycobacterium tuberculosis IFN-γ ELISpot results during anti-tuberculous treatment in HIV-infected children

BackgroundRecent interest has focused on the potential use of serial interferon gamma (IFN-γ) release assay (IGRA) measurements to assess the response to anti-tuberculous (TB) treatment. The kinetics of IFN-γ responses to Mycobacterium tuberculosis (MTB) antigens in HIV-infected children during treatment have not however been previously investigated.MethodsIFN-γ responses to the MTB antigens, ESAT-6, CFP-10 and PPD were measured by an enzyme-linked immunospot assay (IFN-γ ELISpot) at presentation and at one, two and six months after starting anti-tuberculous treatment in HIV-infected children with definite or probable TB. Responses at different time points were compared using a Mann-Whitney U test with paired data analysed using the Wilcoxon signed rank test. A Fisher's exact or Chi-squared test was used to compare proportions when test results were analysed as dichotomous outcomes.ResultsOf 102 children with suspected TB, 22 (21%) had definite TB and 24 (23%) probable TB. At least one follow up IFN-γ ELISpot assay result was available for 31 (67%) of the 46 children. In children with definite or probable TB in whom the IFN-γ ELISpot assay result was positive at presentation, anti-tuberculous treatment was accompanied by a significant decrease in both the magnitude of the IFN-γ response to individual or combined MTB-specific antigens (ESAT-6 median 110 SFCs/106 PBMC (IQR 65-305) at presentation vs. 15 (10-115) at six months, p = 0.04; CFP-10 177 (48-508) vs. 20 (5-165), p = 0.004, ESAT-6 or CFP-10 median 250 SFCs/106 PBMC (IQR 94-508) vs. 25 (10-165), p = 0.004) and in the proportion of children with a positive IFN-γ ELISpot assay (Fisher's exact test: ESAT-6 15/0 vs 5/11, p = 0.0002, CFP-10 22/0 vs 8/17, p = 0.0001, ESAT-6 or CFP-10 22/0 vs. 9/17, p= 0.002). However almost half of the children had a positive IFN-γ ELISpot assay after six months of anti-tuberculous treatment. In addition, there was conversion of the IFN-γ ELISpot assay result during anti-tuberculous therapy in six of 12 children in whom the initial IFN-γ ELISpot assay was negative.ConclusionsIn HIV-infected children with definite or probable TB, anti-tuberculosis treatment is accompanied by a reduction in the magnitude of the IFN-γ ELISpot response to MTB-antigens. However, serial IFN-γ ELISpot measurements appear to have limited clinical utility in assessing a successful response to anti-tuberculous treatment in HIV infected children.

Evaluation of the Prognostic Value of IFN-γ Release Assay and Tuberculin Skin Test in Household Contacts of Infectious Tuberculosis Cases in Senegal

BackgroundChemoprophylaxis of contacts of infectious tuberculosis (TB) cases is recommended for TB control, particularly in endemic countries, but is hampered by the difficulty to diagnose latent TB infection (LTBI), classically assessed through response to the Tuberculin Skin Test (TST). Interferon-gamma release assays (IGRA) are proposed new tools to diagnose LTBI, but there are limited data on their ability to predict the development of active TB disease. To address this, we investigated the response to TST and IGRA in household contacts of infectious TB cases in a TB high-burden country and the potential correlation with development of TB.Methodology/Principal FindingsProspective household contacts study conducted in two health centres in Dakar, Senegal. A total of 2679 household contacts of 206 newly detected smear and/or culture positive index TB cases aged 18 years or greater were identified A TST was performed in each contact and an ESAT6/CFP10 ELISPOT assay performed in a random sample of those. Contacts were followed-up for 24 months. TB was diagnosed in 52 contacts, an incidence rate of 9.27/1000 person-years. In univariable analysis, the presence of positive TST (≥10 mm) and ELISPOT (>32 SFC/million PBMC) responses at baseline were associated with active TB during follow-up: Rate Ratio [RR] = 2.32 (95%CI:1.12–4.84) and RR = 2.09 (95%CI:0.83–5.31), respectively. After adjustment for age, sex and proximity to index case, adjusted RRs were 1.51 (95%CI:0.71–3.19) and 1.98 (95%CI:0.77–5.09), respectively. Restricting analysis to the 40 microbiologically confirmed cases, the adjusted RR for positive ELISPOT was 3.61 (95%CI:1.03–12.65). The median ELISPOT response in contacts who developed TB was 5-fold greater than in those who did not develop TB (p = 0.02).Conclusions/SignificanceTST and IGRAs are markers of a contact of the immune system with tubercle bacilli. In a TB endemic area, a high ELISPOT response may reflect increased bacterial replication that may subsequently be associated with development of TB disease and may have a prognostic value. Further longitudinal data are needed to assess whether IGRAs are reliable markers to be used for targeting chemoprophylaxis.

Evaluating the non-tuberculous mycobacteria effect in the tuberculosis infection diagnosis

The aim of the present study was to determine the role of previous non-tuberculous mycobacteria sensitisation in children as a factor of discordant results between tuberculin skin test (TST) and an in vitro T-cell based assay (T-SPOT.TB; Oxford Immunotec, Oxford, UK). We enrolled 21 non-bacille Calmette-Guérin-vaccinated paediatric patients for suspicious of latent tuberculosis infection (LTBI). These patients yielded a positive TST and a negative T-SPOT.TB. Cells were stimulated with Mycobacterium avium sensitin (having cross-reaction with Mycobacterium intracellulare and Mycobacterium scrofulaceum) and the presence of reactive T-cells was determined by an ex vivo ELISPOT. From the 21 patients, in 10 cases (47.6%), we obtained a positive ELISPOT result after stimulation with M. avium sensitin, in six (28.6%) cases, the result was negative and in the remaining five (23.8%) cases, the result was indeterminate. In conclusion, previous non-tuberculous mycobacteria sensitisation induces false-positive results in the TST for diagnosing LTBI and the use of gamma-interferon tests could avoid unnecessary chemoprophylaxis treatment among a child population.

High Numbers of Interferon-.GAMMA.-Producing T Cells and Low Titers of Anti-Tuberculous Glycolipid Antibody in Individuals with Latent Tuberculosis

Latent tuberculosis infection (LTBI) is defined as an infection with Mycobacterium tuberculosis (MTB) without clinical, bacteriological, or radiological findings, and its early diagnosis is essential for eradication of tuberculosis. To identify LTBI, we measured the numbers of interferon-gamma producing T cells, based on the ELISPOT assay, and the antibody titers in the sera to tuberculous glycolipid antigen (TBGL-Ab). Seventeen culture-confirmed TB patients, 13 controls from TB endemic areas (EC) and 13 controls from TB non-endemic areas (NEC) were enrolled. Peripheral blood mononuclear cells (2.5 x 10(5) per well) were cultured on plates precoated with antibody against interferon-gamma. ELISPOT response was defined as positive when the MTB-specific antigen-containing wells showed at least 6 spots and twice numbers of spots than negative control wells. ELISPOT responses were positive in 15 (88%), 8 (62%) and 4 (31%) subjects of TB, EC and NEC groups, respectively. The ELISPOT data differ between TB and NEC groups (p < 0.01) but not between TB and EC groups. In contrast, TBGL-Ab titers were elevated (> 2.0 U/ml) in 12 TB patients (71%), but only in one subject (8%) each from EC and NEC groups. These results indicate the high prevalence of LTBI in EC. In conclusion, LTBI is associated with positive ELISPOT assay and the low titer of TBGL-Ab, while positive results both in ELISPOT and TBGL-Ab assays indicate active TB. The low titer of TBGL-Ab is a helpful marker to identify LTBI in ELISPOT-positive individuals in TB endemic areas.

RAPID DIAGNOSIS OF ACTIVE PULMONARY TUBERCULOSIS IN THE ELDERLY USING ENZYME‐LINKED IMMUNOSPOT ASSAY FOR INTERFERON‐GAMMA

RAPID DIAGNOSIS OF ACTIVE PULMONARY TUBERCULOSIS IN THE ELDERLY USING ENZYME‐LINKED IMMUNOSPOT ASSAY FOR INTERFERON‐GAMMA

Despite Absence of Measurable Immune Responses against Adenovirus in the First 100 Days After Unrelated Umbilical Cord Blood Transplant in Vitro amplification Strategies Can Unveil Hexon and Penton-Specific Immunity.

Abstract 4640Unrelated cord blood transplantation (UCBT) has become a life saving modality of hematopoietic cell transplantation (HCT) for those cancer patients who lack HLA-matched sibling donors. However, success after UCBT is limited by the high incidence of opportunistic infections (OI), most of which are viral and typically detected < 3 months after UCBT. A minority of UCBT patients with evidence of viral infection/reactivation post-transplant actually resolve their disease without antiviral therapy. Since these infections are largely controlled by T-cell immunity this suggests that in vivo T-cell priming may occur despite immunosuppressive drugs and functional T-cell deficits and protective antiviral immunity can be attained once a threshold number of antiviral T-cells have been achieved. To determine whether this was the case we collected PBMCs from 10 UCBT patients who underwent myeloablative regimens and were diagnosed with adenovirus infection (defined as positive PCR at least twice and/or viral culture in blood and/or stool and/or nasopharynx). Samples were collected a median of 88 days (range 37-209 days) post-transplant at first indication of Adv positivity (blood PCR + in 5/10 patients at a mean of ∼110,000 copies/ml, median of 350, range 100-1×106 copies/ml, 4 patients positive in stool only, one patient + in respiratory tract only). The median age of patients was 4.6 years (range 2.0-16.6y). Eight (8) out of ten (10) patients had grade 2-4 acute GVHD prior to the studies, while the other two patients had grade I.At baseline we were consistently unable to detect T-cells with reactivity against the immunodominant adenoviral virion proteins hexon and penton by IFN-g ELIspot [hexon - median 1.75 spot forming cells (SFC)/2×105 PBMC (range 1-12.5), penton – median 1 SFC)/2×105 PBMC (range 0.5-1)], even though the patients had evidence of active adenovirus infections. However, in 5/10 patients adenovirus-specific T cells could be amplified by a single in vitro stimulation with donor-derived mononuclear cells transduced with a recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber (MOI 200 virus particles (vp) per cell) and culture in the presence of IL4 (1000u/ml). The median hexon-specific T cell frequency in the reactivated cytotoxic T lymphocyte (CTL) lines was 62.5 SFC/2×105 (range 34.5-299.5), and in one patient hexon epitope mapping revealed reactivity against at least four distinct peptides. Penton was also recognized by the CTL lines with a median T-cell frequency of 30 SFC/2×105 (range 26.5-164), with no recognition of control pepmixes. In contrast we were unable to expand adenovirus-reactive T cells from the remaining five patients. Two of three patients with positive ELIspot results from the CTL cultures against both hexon and penton are alive and adenovirus negative without any antiviral agents used at a median of 446 days post-UCBT (range 422-470). Four (4) patients died despite receiving antiviral treatment with Cidofovir. Two of these patients had no detectable antiviral immunity, while the others had only restricted or weak reactivity (reactivity against only one antigen or reactivity below the median SFC values of the responder cohort).To our knowledge this is the first study that analyzes the anti-adenoviral immune response in UCBT recipients with active infection in the early post-transplant period. It appears that in the first 100 days after UCBT, during a time of active immunosuppression, adenovirus-specific T-cells in peripheral blood are consistently (10/10 patients) below the level of detection of conventional IFN-g ELIspot assays. However, dormant immune responses can be amplified in vitro using a single stimulation in the presence of IL4. Furthermore, detection of functional CTL against hexon and penton proteins may be a useful marker to distinguish between those with effective antiviral T cell immunity and those at risk of developing progressive disease. Disclosures:No relevant conflicts of interest to declare.

Vaccination of Melanoma Patients With Melan-A/Mart-1 Peptide and Klebsiella Outer Membrane Protein P40 as an Adjuvant

Toll-like receptor ligands are potentially useful adjuvants for the development of clinical T cell vaccination. Here we investigated the novel Toll-like receptor2 ligand P40, the outer membrane protein A derived from Klebsiella pneumoniae. Seventeen human leukocyte antigen-A*0201 positive stage III/IV melanoma patients were vaccinated with P40 and Melan-A/Mart-1 peptide subcutaneously in monthly intervals. Adverse reactions were mild-to-moderate. Fourteen patients received at least 8 vaccinations and were thus evaluable for clinical tumor and immune responses. Seven patients experienced progressive disease, whereas 2 patients had stable disease throughout the trial period, 1 of them with regression of multiple skin metastases. The remaining 5 patients had no measurable disease. Melan-A/Mart-1 specific CD8 T cells were analyzed ex vivo, with positive results in 6 of 14 evaluable patients. Increased percentages of T cells were found in three patients, memory/effector T cell differentiation in 4 patients, and a positive interferon-gamma Elispot assay in 1 patient. Antibody responses to P40 were observed in all patients. We conclude that vaccination with peptide and P40 was feasible and induced ex vivo detectable tumor antigen specific T cell responses in 6 of 14 patients with advanced melanoma.

Bronchoalveolar Lavage Enzyme-linked Immunospot for a Rapid Diagnosis of Tuberculosis

The rapid diagnosis of pulmonary tuberculosis (TB) is difficult when acid fast bacilli (AFB) cannot be detected in sputum smears. Following a proof of principle study, we examined in routine clinical practice whether individuals with sputum AFB smear-negative TB can be discriminated from those with latent TB infection by local immunodiagnosis with a Mycobacterium tuberculosis-specific enzyme-linked immunospot (ELISpot) assay. Subjects suspected of having active TB who were unable to produce sputum or with AFB-negative sputum smears were prospectively enrolled at Tuberculosis Network European Trialsgroup centers in Europe. ELISpot with early-secretory-antigenic-target-6 and culture-filtrate-protein-10 peptides was performed on peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage mononuclear cells (BALMCs). M. tuberculosis-specific nucleic acid amplification (NAAT) was performed on bronchoalveolar lavage fluid. Seventy-one of 347 (20.4%) patients had active TB. Out of 276 patients who had an alternative diagnosis, 127 (46.0%) were considered to be latently infected with M. tuberculosis by a positive PBMC ELISpot result. The sensitivity and specificity of BALMC ELISpot for the diagnosis of active pulmonary TB were 91 and 80%, respectively. The BALMC ELISpot (diagnostic odds ratio [OR], 40.4) was superior to PBMC ELISpot (OR, 10.0), tuberculin skin test (OR, 7.8), and M. tuberculosis specific NAAT (OR, 12.4) to diagnose sputum AFB smear-negative TB. In contrast to PBMC ELISpot and tuberculin skin test, the BALMC ELISpot was not influenced by previous history of TB. Bronchoalveolar lavage ELISpot is an important advancement to rapidly distinguish sputum AFB smear-negative TB from latent TB infection in routine clinical practice.