Enzyme-linked Immunospot Responses Research Articles

Human Antibody Responses Following Vaccinia Immunization Using Protein Microarrays and Correlation With Cell-Mediated Immunity and Antibody-Dependent Cellular Cytotoxicity Responses.

There are limited data regarding immunological correlates of protection for the modified vaccinia Ankara (MVA) smallpox vaccine. A total of 523 vaccinia-naive subjects were randomized to receive 2 vaccine doses, as lyophilized MVA given subcutaneously, liquid MVA given subcutaneously (liquid-SC group), or liquid MVA given intradermally (liquid-ID group) 28 days apart. For a subset of subjects, antibody-dependent cellular cytotoxicity (ADCC), interferon-γ release enzyme-linked immunospot (ELISPOT), and protein microarray antibody-binding assays were conducted. Protein microarray responses were assessed for correlations with plaque reduction neutralization titer (PRNT), enzyme-linked immunosorbent assay, ADCC, and ELISPOT results. MVA elicited significant microarray antibody responses to 15 of 224 antigens, mostly virion membrane proteins, at day 28 or 42, particularly WR113/D8L and WR101H3L. In the liquid-SC group, responses to 9 antigens, including WR113/D8L and WR101/H3L, correlated with PRNT results. Three were correlated in the liquid-ID group. No significant correlations were observed with ELISPOT responses. In the liquid-ID group, WR052/F13L, a membrane glycoprotein, correlated with ADCC responses. MVA elicited antibodies to 15 vaccinia strain antigens representing virion membrane. Antibody responses to 2 proteins strongly increased and significantly correlated with increases in PRNT. Responses to these proteins are potential correlates of protection and may serve as immunogens for future vaccine development. NCT00914732.

Whole blood interleukin-2 release test to detect and characterize rare circulating gluten-specific T cell responses in coeliac disease.

Whole blood cytokine release assays (CRA) assessing cellular immunity to gluten could simplify the diagnosis and monitoring of coeliac disease (CD). We aimed to determine the effectiveness of electrochemiluminescence CRA to detect responses to immunodominant gliadin peptides. HLA-DQ2·5+ CD adults (cohort 1, n=6; cohort 2, n=12) and unaffected controls (cohort 3, n=9) were enrolled. Cohort 1 had 3-day gluten challenge (GC). Blood was collected at baseline, and for cohort 1 also at 3h, 6h and 6days after commencing 3-day GC. Gliadin peptide-stimulated proliferation, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and 14- and 3-plex electrochemiluminescence CRA were performed. Poisson distribution analysis was used to estimate responding cell frequencies. In cohort 1, interleukin (IL)-2 dominated the gliadin peptide-stimulated cytokine release profile in whole blood. GC caused systemic IL-2 release acutely and increased gliadin peptide-stimulated IFN-γ ELISPOT and whole blood CRA responses. Whole blood CRA after GC was dominated by IL-2, but also included IFN-γ, C-X-C motif chemokine ligand 10/IFN-γ-induced protein 10 (CXCL10/IP-10), CXCL9/monokine induced by IFN-γ (MIG), IL-10, chemokine (C-C motif) ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP-1α), TNF-α and IL-8/CXCL8. In cohorts 2 and 3, gliadin peptide-stimulated whole blood IL-2 release was 100% specific and 92% sensitive for CD patients on a gluten-free diet; the estimated frequency of cells in CD blood secreting IL-2 to α-gliadin peptide was 0·5 to 11per ml. Whole blood IL-2 release successfully mapped human leucocyte antigen (HLA)-DQ2·5-restricted epitopes in an α-gliadin peptide library using CD blood before and after GC. Whole blood IL-2 release assay using electrochemiluminescence is a sensitive test for rare gliadin-specific T cells in CD, and could aid in monitoring and diagnosis. Larger studies and validation with tetramer-based assays are warranted.

Cellular and humoral cytomegalovirus immunity changes in one-year combined prophylaxis after lung transplantation: suggestions from and for clinical practice.

Background:Immune responses, both cellular and humoral, against cytomegalovirus (CMV) are used to predict CMV manifestations in solid organ recipients. The aim of this study is to evaluate CMV enzyme-linked immunospot (ELISPOT) assay and serology during CMV infections, their concordance and variations after lung transplantation (LTx).Methods:We retrospectively analysed in one year the follow-up data of 43 patients receiving combined CMV prophylaxis with antiviral agents and CMV-specific immunoglobulin G (IgG). CMV infections were investigated by using molecular analyses on both 167 bronchoalveolar lavage and biopsy specimens and 1134 blood samples. Cellular CMV immunity was assessed with specific ELISPOT whereas the humoral one was assessed by quantifying specific immunoglobulins.Results:At the first month after LTx the majority of patients were ELISPOT responders (52.3%) and 30.9% were non-responders. ELISPOT responders had a lower incidence of CMV viremia (p = 0.047), whereas neither effects on CMV pulmonary asymptomatic infection nor on acute rejection were observed. Responders had a higher CMV IgG titre (p < 0.0001) in particular at the first month after LTx (p = 0.0001). Concordance among CMV ELISPOT assay and IgG levels was moderate (Cohen’s K 0.524), with an agreement of 89.8%. All ELISPOT responders maintained their status and almost all non-responders became responders during follow-up (92.3%); the percentage of IgG seropositive subjects increased from 74.4% at the first month of follow-up to 97.4% after 1 year.Conclusions:Despite a moderate concordance with serology, ELISPOT response predicted a lower incidence of CMV viremia in LTx patients; no effects were reported on pulmonary clinical manifestations nor on acute rejection. The ELISPOT response as well as serology changed during the follow-up, not only after first CMV contact. The reviews of this paper are available via the supplemental material section.

Plasma cell and serum antibody responses to influenza vaccine in preterm and full-term infants.

Preterm (PT) infants are at greater risk for severe influenza infection and experience decrements in long-term antibody responses to vaccines. This may related to defects in antibody secreting cell (ASC) generation. To investigate the relationships among the frequencies of influenza-specific antibody secreting cells, ASC numbers and subsets, and antibody responses to influenza vaccines (IV) among PT and full-term (FT) infants. We enrolled 11 former PT (≤32weeks' gestation, ≤1500 g' birth weight) and 11FT infants, 6-17months of age, receiving their first influenza immunizations. Infants received two doses of inactivated trivalent (T)IV or quadrivalent (Q)IV during the 2012-2013 and 2013-2014 influenza seasons, respectively, at 0 and 28days, and blood was drawn at 0, 10, 35, and 56days and 9months. Vaccine-specific antibody was measured by hemagglutination inhibition (HAI) at 0 and 56days and 9months, vaccine-specific ASC numbers by enzyme linked immunospot (ELISPOT) at 10 and 35days, and ASC subsets by flow cytometry at 0, 10 and 35days. PT infants had post-vaccine HAI titers to all 4 vaccine strains at least equal to FT infants at 56days and 9months after beginning immunization. Influenza-specific ASC ELISPOT responses at 35days were higher among PT than FT infants (median 100 v. 30 per 106 PBMC, p=0.04). ASC numbers at 35days were positively correlated with serum HAI titers at 56days (ρ=0.50-0.80). There were no statistical differences between PT and FT infants in the frequency of five ASC subsets and no specific ASC subset correlated with durability of serum antibody titers. Influenza-specific ASC numbers in both FT and PT infants correlated with peak antibody titers, but ASC subsets did not correlate with durability of antibody response.

Human Cytomegalovirus-Specific Memory CD4+ T-Cell Response and Its Correlation With Virus Transmission to the Fetus in Pregnant Women With Primary Infection.

Primary human cytomegalovirus (HCMV) infection during pregnancy is the major cause of congenital viral sequelae. The HCMV-specific T-cell response may have a role in the prevention of virus transmission to the fetus. HCMV-specific memory T cells were investigated in the second month after primary infection onset in 44 pregnant women (15 transmitting the infection to the fetus) and 8 pregnant women with remote infection. Peripheral blood mononuclear cells were stimulated for 12 days with peptide pools of HCMV proteins IE-1, IE-2, and pp65, and subsequently restimulated for 24 hours with the same peptide pools in a cultured enzyme-linked immunospot (ELISPOT) assay. In pregnant women with primary infection, the cultured ELISPOT assay detected a higher T-cell response to pp65 than to IE-1 or IE-2, whereas in remote infection pp65-, IE-1-, and IE-2-specific T cells were detected at comparable levels. During primary infection, the cultured ELISPOT response was mainly mediated by CD4+ T cells, and was lower than in remote infection. Strikingly, the cultured ELISPOT response to pp65 (but not to IE-1 or IE-2) was significantly higher in nontransmitting mothers. To detect other factors potentially associated with nontransmission, different serological parameters were analyzed. Only immunoglobulin G avidity index was higher in nontransmitting mothers, who showed also a lower DNAemia level. These 2 parameters remained associated with congenital infection in multivariate analysis. Determination of HCMV-specific T cells by cultured ELISPOT, in pregnant women with primary HCMV infection, in association with avidity index and DNAemia may help to assess the risk of HCMV fetal transmission.

Surveillance Study of Influenza Occurrence and Immunity in a Wisconsin Cohort During the 2009 Pandemic.

BackgroundAntibody and T-cell immunity to conserved influenza virus antigens can protect animals against infection with diverse influenza strains. Although immunity against conserved antigens occurs in humans, whether such responses provide cross-protection in humans and could be harnessed as the basis for universal influenza vaccines is controversial. The 2009 pandemic provided an opportunity to investigate whether pre-existing cross-reactive immunity affected susceptibility to infection.MethodsIn 2009, we banked sera and peripheral blood mononuclear cells (PBMC) from blood donors, then monitored them for pandemic influenza infection (pH1N1) by polymerase chain reaction or seroconversion. Antibodies to hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix 2 (M2), and HA-pseudotypes were measured in sera. T-cell inteferon-γ enzyme-linked immunospot responses were measured in PBMC.ResultsThere were 13 infections in 117 evaluable donors. Pre-existing T-cell reactivity to pH1N1 was substantial (of 153 donors tested, 146 had >100 spot-forming cells/106 cells). Antibodies reactive with pH1N1 were common: anti-NP (all donors) and anti-M2 (44% of donors). Pseudotype-neutralizing antibodies to H1 were detected, but not to highly conserved HA epitopes. Unexpectedly, donors with symptomatic pH1N1 infection had sharp rises in HA pseudotype-neutralizing antibodies, not only pH1N1 but also against multiple seasonal H1s. In addition, an exploratory study of a T-cell marker (response to NP418-426) identified probable infection missed by standard criteria.ConclusionsAlthough the number of infections was inadequate for conclusions about mechanisms of protection, this study documents the wide variety of pre-existing, cross-reactive, humoral and cellular immune responses to pandemic influenza virus antigens in humans. These responses can be compared with results of other studies and explored in universal influenza vaccine studies.

Human papillomavirus 16 (HPV16) and HPV52 E6-specific immunity in HIV-infected adults on combination antiretroviral therapy.

Human papillomavirus (HPV)-associated cancers disproportionately affect those infected with HIV despite effective combination antiretroviral therapy (cART). The primary aim of this study was to quantify HPV16 and HPV52 E6-specific interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) T-cell responses, a correlate of protective immunity, in the first year following cART initiation and subsequently in those patients with suboptimal (sIR) and optimal (oIR) immune reconstitution. Ninety-four HIV-infected patients were recruited to the study; a longitudinal cohort of patients recruited just prior to commencing cART and followed up for 48 weeks (n = 27), and a cross-sectional cohort (n = 67) consisting of patients with sIR (CD4 T-cell count < 350 cells/μL) and oIR (CD4 T-cell count > 500 cells/μL) after a minimum of 2 years on cART. Controls (n = 29) consisted of HIV-negative individuals. IFN-γ ELISPOT responses against HPV16 and HPV52 E6 were correlated to clinical characteristics, anal and oral HPV carriage, T-cell maturational subsets, markers of activation, senescence and T-regulatory cells. HPV16 and HPV52 E6-specific T-cell responses were detected in only one of 27 patients (3.7%) during the initial phase of immune recovery. After at least 2 years of cART, those who achieved oIR had significantly higher E6-specific responses (9 of 34; 26.5%) compared with those with sIR (2 of 32; 6.3%) (P = 0.029). Apart from higher CD4 T-cell counts and lower CD4 T-cell activation, no other immunological correlates were associated with the detection of HPV16 and HPV52 E6-specific responses. HPV16 and HPV52 E6-specific IFN-γ T-cell responses, a correlate of protective immunity, were detected more frequently among HIV-infected patients who achieved optimal immune recovery on cART (26.5%) compared with those with suboptimal recovery (6.3%).

Memory T cells specific for HBV enumerated by a peptide-based cultured enzyme-linked immunospot assay in healthy HBV-vaccinated subjects

Hepatitis B vaccine is the most effective strategy to control hepatitis B virus (HBV) infection and disease. It is considered that an anti-HBs (antibodies against HBV surface antigen) titer >10 mIU/ml, measured shortly after a complete vaccination schedule, provides protection against infection. Approximately 4-10% of healthy individuals fail to respond to 3-dose vaccination. Long-term HBV-specific memory T-cell response has not been fully investigated, mainly due to the lack of a suitable assay. We quantified HBV-specific expandable memory T cells by using a cultured IFN-γ enzyme-linked immunospot (ELISPOT) assay. Cultured ELISPOT response to an overlapping peptide pool representing the complete L (large) HBV envelope polypeptide was evaluated in 41 healthy subjects vaccinated 15-20 y earlier and 5 unvaccinated. Plasma samples were tested for anti-HBs. Vaccinated subjects had significantly higher HBV-specific T-cellular response than unvaccinated (p = 0.0002). HBV-specific T-cell response was mainly mediated by CD4+ T cells. No concordance was found between cultured ELISPOT and anti-HBs data in vaccinated subjects. Thirty-one (76%) vaccinated subjects were responders (anti-HBs >10 mIU/ml). Nineteen (46%) vaccinated subjects were considered to be responders in cultured ELISPOT. Twenty-two (54%) vaccinated subjects were considered non-responders in cultured ELISPOT; 5 of them (23%) were also humoral non-responders. About 12% of healthy HBV-vaccinated subjects are both humoral and cellular non-responders. Although the prognostic value of this assay has not been established in terms of predictability for susceptibility to de-novo HBV infection, ELISPOT data suggest that these subjects may be at risk for HBV infection and disease, especially health care workers.

Memory T cells specific for HBV enumerated by a peptide-based cultured enzyme-linked immunospot assay in healthy HBV-vaccinated subjects

ABSTRACTHepatitis B vaccine is the most effective strategy to control hepatitis B virus (HBV) infection and disease. It is considered that an anti-HBs (antibodies against HBV surface antigen) titer >10 mIU/ml, measured shortly after a complete vaccination schedule, provides protection against infection. Approximately 4–10% of healthy individuals fail to respond to 3-dose vaccination. Long-term HBV-specific memory T-cell response has not been fully investigated, mainly due to the lack of a suitable assay. We quantified HBV-specific expandable memory T cells by using a cultured IFN-γ enzyme-linked immunospot (ELISPOT) assay. Cultured ELISPOT response to an overlapping peptide pool representing the complete L (large) HBV envelope polypeptide was evaluated in 41 healthy subjects vaccinated 15–20 y earlier and 5 unvaccinated. Plasma samples were tested for anti-HBs. Vaccinated subjects had significantly higher HBV-specific T-cellular response than unvaccinated (p = 0.0002). HBV-specific T-cell response was mainly mediated by CD4+ T cells. No concordance was found between cultured ELISPOT and anti-HBs data in vaccinated subjects. Thirty-one (76%) vaccinated subjects were responders (anti-HBs >10 mIU/ml). Nineteen (46%) vaccinated subjects were considered to be responders in cultured ELISPOT. Twenty-two (54%) vaccinated subjects were considered non-responders in cultured ELISPOT; 5 of them (23%) were also humoral non-responders. About 12% of healthy HBV-vaccinated subjects are both humoral and cellular non-responders. Although the prognostic value of this assay has not been established in terms of predictability for susceptibility to de-novo HBV infection, ELISPOT data suggest that these subjects may be at risk for HBV infection and disease, especially health care workers.

A phase 1a, first-in-human, randomized study of a respiratory syncytial virus F protein vaccine with and without a toll-like receptor-4 agonist and stable emulsion adjuvant

BackgroundRespiratory syncytial virus (RSV) causes significant illness in older adults resulting in substantial health and economic impact. A successful vaccine would reduce morbidity in this growing segment of the population. MethodsIn this double-blind phase 1 study, subjects 60 years of age and older were enrolled by cohort and randomized to receive vaccines containing escalating doses (20, 50, or 80μg) of soluble RSV fusion protein (sF) alone or adjuvanted with 2.5μg of glucopyranosyl lipid A, a toll-like receptor-4 agonist, in 2% stable emulsion (GLA-SE). Each cohort included 20 vaccine and 4 placebo recipients. Immune responses were evaluated using assays for RSV microneutralizing, anti-F IgG, and palivizumab competitive antibodies and for F-specific interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) responses. ResultsThe inclusion of adjuvant increased local reactogenicity, with the majority of subjects who received sF and adjuvant reporting low-grade injection site pain or tenderness. At all doses, the safety profile was acceptable for further development. Immune responses were antigen dose-dependent, and the inclusion of adjuvant increased both humoral and cellular immune responses, with responses statistically higher than for placebo recipients in all 4 assays. At the highest dosage level with adjuvant, half of the subjects had a ≥3-fold rise from day 0 in RSV neutralizing antibody titers, and all had a ≥3-fold rise in antibody levels by anti-F IgG and palivizumab competitive antibody assays on day 29. For the day 8 IFNγ ELISPOT assay, 74% of subjects in the highest dosing cohort had a ≥3-fold rise from baseline. ConclusionsThe safety and immunogenicity results from this study support inclusion of the GLA-SE adjuvant in this RSV vaccine for older adults and also support assessment of the efficacy of the vaccine in a larger clinical trial. Clinicaltrials.gov NCT02115815.

Utility of the Enzyme-Linked Immunospot Interferon-γ–Release Assay to Predict the Risk of Cytomegalovirus Infection in Hematopoietic Cell Transplant Recipients

The ability to distinguish allogeneic hematopoietic cell transplant (allo-HCT) recipients at risk for cytomegalovirus (CMV) reactivation from those who are not is central for optimal CMV management strategies. Interferon γ (IFN-γ) produced by CMV-challenged T cells may serve as an immune marker differentiating these 2 populations. We prospectively monitored 63 CMV-seropositive allo-HCT recipients with a CMV-specific enzyme-linked immunospot (ELISPOT) assay and for CMV infection from the period before transplantation to day 100 after transplantation. Assay results above certain thresholds (50 spots per 250 000 cells for immediate early 1 or 100 spots per 250 000 cells for phosphoprotein 65) identified patients who were protected against CMV infection as long as they had no graft-versus-host disease and/or were not receiving systemic corticosteroids. Based on the multivariable Cox proportional hazards regression model, the only significant factor for preventing CMV reactivation was a CMV-specific ELISPOT response above the determined thresholds (adjusted hazard ratio, 0.21; 95% confidence interval, .05–.97; P = .046). Use of this assay as an additional tool for managing allo-HCT recipients at risk for CMV reactivation needs further validation in future studies. Application of this new approach may reduce the duration and intensity of CMV monitoring and the duration of prophylaxis or treatment with antiviral agents in those who have achieved CMV-specific immune reconstitution.

Immunization of HIV-1-Infected Persons With Autologous Dendritic Cells Transfected With mRNA Encoding HIV-1 Gag and Nef: Results of a Randomized, Placebo-Controlled Clinical Trial.

Background:HIV-1 eradication may require reactivation of latent virus along with stimulation of HIV-1-specific immune responses to clear infected cells. Immunization with autologous dendritic cells (DCs) transfected with viral mRNA is a promising strategy for eliciting HIV-1-specific immune responses. We performed a randomized controlled clinical trial to evaluate the immunogenicity of this approach in HIV-1-infected persons on antiretroviral therapy.Methods:Fifteen participants were randomized 2:1 to receive intradermal immunization with HIV-1 Gag- and Nef-transfected DCs (vaccine) or mock-transfected DCs (placebo) at weeks 0, 2, 6, and 10. All participants also received DCs pulsed with keyhole limpet hemocyanin (KLH) to assess whether responses to a neo-antigen could be induced.Results:After immunization, there were no differences in interferon-gamma enzyme-linked immunospot responses to HIV-1 Gag or Nef in the vaccine or placebo group. CD4 proliferative responses to KLH increased 2.4-fold (P = 0.026) and CD8 proliferative responses to KLH increased 2.5-fold (P = 0.053) after vaccination. There were increases in CD4 proliferative responses to HIV-1 Gag (2.5-fold vs. baseline, 3.4-fold vs. placebo, P = 0.054) and HIV-1 Nef (2.3-fold vs. baseline, 6.3-fold vs. placebo, P = 0.009) among vaccine recipients, but these responses were short-lived.Conclusion:Immunization with DCs transfected with mRNA encoding HIV-1 Gag and Nef did not induce significant interferon-gamma enzyme-linked immunospot responses. There were increases in proliferative responses to HIV-1 antigens and to a neo-antigen, KLH, but the effects were transient. Dendritic cell vaccination should be optimized to elicit stronger and long-lasting immune responses for this strategy to be effective as an HIV-1 therapeutic vaccine.

Targeting CD138−/CD20+ Clonogenic Myeloma Precursor Cells Decreases These Cells and Induces Transferable Antimyeloma Immunity

This phase Ib clinical trial evaluated whether pretargeting of CD20+ clonogenic myeloma precursor cells (CMPCs) with anti-CD3 × anti-CD20 bispecific antibody-armed T cells (BATs) before autologous stem cell transplantation (SCT) in patients with standard-risk and high-risk multiple myeloma would induce antimyeloma immunity that could be detected and boosted after SCT. All 12 patients enrolled in this study received 2 BATs infusions before SCT, and 4 patients received a booster infusion of BATs after SCT. Pretargeting CD138−/CD20+ CMPCs with BATs before SCT was safe and reduced levels of CMPCs by up to 58% in the postinfusion bone marrow in patients who remained in remission. Four of 5 patients who remained in remission had a >5-fold increase in IFN-γ enzyme-linked immunospot responses. SOX2 antibody increased after BATs infusions and persisted after SCT. The median anti-SOX2 level at 3 months after SCT was 28.1 ng/mL (range, 4.6 to 256 ng/mL) in patients who relapsed and 46 ng/mL (range, 28.3 to 73.3 ng/mL) in patients who remained in remission. The immune correlates suggest that infusions of targeted T cells given before SCT were able to reduce CMPC levels and induced cellular and humoral antimyeloma immunity that could be transferred and boosted after SCT.

A novel recombinant BCG-expressing pro-apoptotic protein BAX enhances Th1 protective immune responses in mice

One-third of the world's population is infected with Mycobacterium tuberculosis (MTB). The protective efficacy of bacille Calmette Guérin (BCG) vaccine against tuberculosis (TB) in adults is highly controversial even though the BCG vaccine has been available for more than 90 years. Because BCG is effective against infantile tuberculosis meningitis and miliary tuberculosis in young children and provides cost-effective prevention from tuberculosis for developing countries, it would be desirable to modify the existing BCG vaccine to provide more comprehensive protection. In our study, we constructed a novel recombinant BCG strain expressing pro-apoptotic BAX (rBCG::BAX) and demonstrated that it significantly induced the apoptosis of macrophages infected with rBCG::BAX both in vitro and in vivo. In addition, it significantly enhanced Ag85B-specific IFN-γ enzyme-linked immunospot responses, IFN-γ secretion, IL-2 secretion and the ratio of Ag85B-specific IgG2b/IgG1, and it significantly decreased Ag85B-specific IL-4. Furthermore, it presumably facilitated antigen presentation by inducing a significant up-regulation in the expression of MHC-II and B7.1 (CD80) co-stimulatory molecules on macrophages. In conclusion, these results suggest that the rBCG::BAX strain elicited predominantly a Th1 protective immune responses and might be a potential tuberculosis vaccine candidate for further study.

Maternal plasma and breastmilk viral loads are associated with HIV-1-specific cellular immune responses among HIV-1-exposed, uninfected infants in Kenya.

Infants exposed to maternal HIV-1 provide an opportunity to assess correlates of HIV-1-specific interferon (IFN)-γ responses and may be informative in the development of HIV-1 vaccines. HIV-1-infected women with CD4 counts 200-500 cells/mm(3) were randomized to short-course zidovudine/nevirapine (ZDV/NVP) or highly active anti-retroviral therapy (HAART) between 2003 and 2005. Maternal plasma and breastmilk HIV-1 RNA and DNA were quantified during the first 6-12 months postpartum. HIV-1 gag peptide-stimulated enzyme-linked immunospot (ELISPOT) assays were conducted in HIV-1-exposed, uninfected infants (EU), and correlates were determined using regression and generalized estimating equations. Among 47 EU infants, 21 (45%) had ≥1 positive ELISPOT result during follow-up. Infants had a median response magnitude of 177 HIV-1-specific spot-forming units (SFU)/106 peripheral blood mononuclear cells (PBMC) [interquartile range (IQR)=117-287] directed against 2 (IQR = 1-3) gag peptide pools. The prevalence and magnitude of responses did not differ by maternal anti-retroviral (ARV) randomization arm. Maternal plasma HIV-1 RNA levels during pregnancy (P=0.009) and breastmilk HIV-1 DNA levels at 1 month (P=0.02) were associated with a higher magnitude of infant HIV-1-specific ELISPOT responses at 1 month postpartum. During follow-up, concurrent breastmilk HIV-1 RNA and DNA (cell-free virus and cell-associated virus, respectively) each were associated positively with magnitude of infant HIV-1-specific responses (P=0.01). Our data demonstrate the importance of antigenic exposure on the induction of infant HIV-1-specific cellular immune responses in the absence of infection.

ROBUST INDUCTION OF TYPE-1 CD8+ T-CELL RESPONSES IN WHO GRADE II LOW-GRADE GLIOMA PATIENTS RECEIVING PEPTIDE-BASED VACCINES IN COMBINATION WITH POLY-ICLC

BACKGROUND: WHO grade II low-grade gliomas (LGGs) with high risk factors for recurrence are mostly lethal despite current treatments, and novel approaches are needed. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in human leukocyte antigen (HLA)-A2+ adults with high-risk LGGs in the following three cohorts: 1) newly diagnosed patients without prior radiation therapy (RT); 2) newly diagnosed patients with prior RT, and 3) recurrent patients. METHODS: GAAs were interleukin-13 receptor (IL-13R)α2, EphA2, Wilms Tumor (WT)1, and Survivin, and synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for 8 courses with intramuscular injections of Toll-like receptor 3 agonist Polyinosinic-Polycytidylic Acid Stabilized by Lysine and Carboxymethylcellulose (poly-ICLC), followed by q12-week booster vaccines. Primary endpoints were safety and CD8+ T-cell responses against vaccine-targeted GAAs. RESULTS: Cohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity has been encountered except for one case with Common Terminology Criteria for Adverse Events (CTCAE) Grade 3 fever (Cohort 1). Enzyme-linked Immuno-SPOT (ELISPOT) assays demonstrated robust and sustained interferon (IFN)-γ responses against at least 3 of the 4 GAA epitopes in 10 and 4 cases of Cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFN-γ ELISPOT responses than Cohort 3 patients, suggesting newly diagnosed patients have superior vaccine-responsiveness to recurrent patients. IFN-γ ELISPOT response levels in this study is significantly higher than those observed in our previous phase I/II study in high-grade glioma patients (Okada et al. JCO 2011). Furthermore, IFN-γ ELISPOT response levels were significantly higher than those for IL-5, indicating effective type-1 skewing by the current regimen. Median progression-free survival (PFS) periods are 21 months (Cohort 1; since diagnosis; range 10-42) and 12 months (Cohort 3; since the 1st vaccine; range 3-26). The only patient with large astrocytoma in Cohort 2 has been progression-free for over 58 months since diagnosis. There was a positive trend between IFN-γ ELISPOT responses and progression-free survival (PFS) in Cohort 3 patients (P = 0.08 by The Cox proportional hazards model). CONCLUSIONS: The current regimen is well tolerated and induces robust GAA-specific responses in WHO grade II LGG patients. These results suggest these patients may be suitable populations for vaccine therapy and warrant further evaluations of this approach. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Normalizing ELISPOT responses to T-cell counts: A novel approach for quantification of HCMV-specific CD4+ and CD8+ T-cell responses in kidney transplant recipients

BackgroundHuman cytomegalovirus (HCMV) is the most common opportunistic virus infection in solid organ transplant recipients. The analysis of HCMV-specific T-cell immunity after organ transplant is of relevant clinical interest. ObjectivesTo analyze HCMV-specific CD4+ and CD8+ T-cell responses in healthy subjects and kidney transplant recipients (KTR). Study designHCMV-specific T-cell responses were evaluated by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phosphoprotein 65 (pp65) (for stimulation of both CD4+ and CD8+ T-cell responses) and a pool of 34 short peptides (8–12 amino acids in length, for stimulation of CD8+ T-cell responses). ELISPOT results were normalized to T-cell subset counts and their correlations with a reported dendritic cell (DC)-based assay, which simultaneously quantifies HCMV-specific CD4+ and CD8+ T-cell responses, were analyzed. ResultsHCMV-seropositive KTR showed higher ELISPOT responses compared to HCMV-seropositive healthy subjects. IE-1 and pp65 ELISPOT responses were mediated mainly by CD8+ T-cells and, to a lesser extent, CD4+ T cells; IE-2 peptides appear to stimulate CD56+ cells (natural killer cells). In HCMV-seropositive healthy subjects, ELISPOT results (expressed either as net spots/million cells or normalized to the corresponding T-cell count) significantly correlated with the DC assay. However, in HMCV-seropositive KTR, only normalized ELISPOT responses to overlapping 15-mer peptide pools significantly correlated with DC-assay responses. ConclusionsThe normalized ELISPOT represents a novel and simple approach for quantifying and monitoring HCMV-specific CD4+ and CD8+ T-cell responses in KTR.

Brucella melitensis T Cell Epitope Recognition in Humans with Brucellosis in Peru

Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4(+) T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4(+) T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen.

Inhibition of Mycobacterial GrowthIn Vitrofollowing Primary but Not Secondary Vaccination with Mycobacterium bovis BCG

Despite the widespread use of the Mycobacterium bovis BCG vaccine, there are more than 9 million new cases of tuberculosis (TB) every year, and there is an urgent need for better TB vaccines. TB vaccine candidates are selected for evaluation based in part on the detection of an antigen-specific gamma interferon (IFN-γ) response. The measurement of mycobacterial growth in blood specimens obtained from subjects immunized with investigational TB vaccines may be a better in vitro correlate of in vivo vaccine efficacy. We performed a clinical study with 30 United Kingdom adults who were followed for 6 months to evaluate the abilities of both a whole-blood- and a novel peripheral blood mononuclear cell (PBMC)-based mycobacterial growth inhibition assay to measure a response to primary vaccination and revaccination with BCG. Using cryopreserved PBMCs, we observed a significant improvement in mycobacterial growth inhibition following primary vaccination but no improvement in growth inhibition following revaccination with BCG (P < 0.05). Mycobacterial growth inhibition following primary BCG vaccination was not correlated with purified protein derivative (PPD) antigen-specific IFN-γ enzyme-linked immunospot (ELISPOT) responses. We demonstrate that a mycobacterial growth inhibition assay can detect improved capacity to control growth following primary immunization, but not revaccination, with BCG. This is the first study to demonstrate that an in vitro growth inhibition assay can identify a difference in vaccine responses by comparing both primary and secondary BCG vaccinations, suggesting that in vitro growth inhibition assays may serve as better surrogates of clinical efficacy than the assays currently used for the assessment of candidate TB vaccines.

Detection of Epstein–Barr virus‐specific memory CD4+T cells using a peptide‐based cultured enzyme‐linked immunospot assay

Approaches to evaluate T-cell responses to Epstein-Barr virus (EBV) include enzyme-linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon-γ secretion upon antigen stimulation. However, evaluation of expandable EBV-specific memory T cells in an ELISPOT format has not been described previously. We quantified EBV-specific T-cell precursors with high proliferative capacity by using a peptide-based cultured interferon-γ ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15-mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV-specific T-cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T-cell responses quantified by cultured ELISPOT were mainly mediated by CD4+ T cells and a marked pattern of immunodominance to latent-phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV-specific T-cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung-transplanted patient with EBV-associated diseases. Analysis of T-cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV-related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.