Interferon-gamma Enzyme-linked Immunosorbent Spot Research Articles

Utility of the Interferon-Gamma Enzyme-Linked Immunosorbent Spot Assay to Predict Risk of Cytomegalovirus Infection in Kidney Transplant Recipients.

Non-specific interferon-gamma (IFN-γ) enzyme-linked immunosorbent (ELISpot) responses after solid organ transplant (SOT) and their relationship with cytomegalovirus (CMV) reactivation have hardly been investigated. Adult kidney transplant (KT) recipients underwent measurement of IFN-γ-producing T cells using the ELISpot assay before and 1 month after transplantation. Data for CMV infection episodes were collected. Risk factors for post-transplant CMV infection, based on IFN-γ responses, were analyzed using a Cox proportional hazards model. A total of 93 KT recipients were enrolled in the study and 84 evaluable participants remained at 1 month post KT. Thirty-three (39%) recipients developed subsequent CMV infection within 6 months post-transplant. At 1-month post-transplant, IFN-γ-producing T cells with <250 spot-forming units (SFUs)/2.5 × 105 peripheral blood mononuclear cells (PBMCs) were significantly associated with CMV infection (HR 3.1, 95% CI 1.4-7.1, p = 0.007). On multivariable analysis, posttransplant IFN-γ-producing T cells with <250 SFUs/2.5 × 105 PBMCs remained independently associated with CMV infection (HR 3.1, 95% CI 1.2-7.8, p = 0.019). Conclusions: KT recipients with low IFN-γ-producing T cells measured by the ELISpot assay are more likely to develop CMV infection after transplantation. Therefore, measurement of nonspecific cell-mediated immunity ELISpot responses could potentially stratify recipients at risk of CMV infection (Thai Clinical Trials Registry, TCTR20210216004).

Survivin (BIRC5) Peptide Vaccine in the 4T1 Murine Mammary Tumor Model: A Potential Neoadjuvant T Cell Immunotherapy for Triple Negative Breast Cancer: A Preliminary Study

A triple negative breast cancer model using the murine 4T1 tumor cell line was used to explore the efficacy of an adjuvanted survivin peptide microparticle vaccine using tumor growth as the outcome metric. We first performed tumor cell dose titration studies to determine a tumor cell dose that resulted in sufficient tumor takes but allowed multiple serial measurements of tumor volumes, yet with minimal morbidity/mortality within the study period. Later, in a second cohort of mice, the survivin peptide microparticle vaccine was administered via intraperitoneal injection at the study start with a second dose given 14 days later. An orthotopic injection of 4T1 cells into the mammary tissue was performed on the same day as the administration of the second vaccine dose. The mice were followed for up to 41 days with subcutaneous measurements of tumor volume made every 3-4 days. Vaccination with survivin peptides was associated with a peptide antigen-specific gamma interferon enzyme-linked immunosorbent spot response in the murine splenocyte population but was absent from the control microparticle group. At the end of the study, we found that vaccination with adjuvanted survivin peptide microparticles resulted in statistically significant slower primary tumor growth rates in BALB/c mice challenged with 4T1 cells relative to the control peptideless vaccination group. These studies suggest that T cell immunotherapy specifically targeting survivin might be an applicable neoadjuvant immunotherapy therapy for triple negative breast cancer. More preclinical studies and clinical trials are needed to explore this concept further.

Long-Term CD4+ T-Cell and Immunoglobulin G Immune Responses in Oncology Workers following COVID-19 Vaccination: An Interim Analysis of a Prospective Cohort Study.

We conducted a prospective study to evaluate immune responses to SARS-CoV-2 in oncology workers in which we collected blood and clinical data every 6 months. Spike-specific CD4+ T-cells and immunoglobulin G responses were measured using interferon-gamma enzyme-linked immunosorbent spot and enzyme-linked immunosorbent assay, respectively. Sixty (81%) vaccinated and 14 (19%) unvaccinated individuals were enrolled. CD4+ T-cell responses of those individuals currently naturally infected were comparable to those who were 6 months from receiving their last dose of the vaccine; both responses were significantly higher than among those who were unvaccinated. Unvaccinated participants who became vaccinated while in the study showed a significant increase in both types of spike-specific immune responses. Previously vaccinated individuals who received a third dose (booster) showed a similar response to the spike protein. However, this response decreases as soon as 3 months but does not dip below the established response following two doses. Response to variants of concern B.1.617.2 (Delta) and B.1.1.529 (Omicron) also increased, with the Omicron variant having a significantly lower response when compared to Delta and the wild type. We conclude that antibody and T-cell responses increase in oncology workers after serial vaccination but can wane over time.

Immunological characteristics of MAV/06 strain of varicella-zoster virus vaccine in an animal model

BackgroundVaricella-zoster virus (VZV) is a pathogen that causes chickenpox and shingles in humans. Different types of the varicella vaccines derived from the Oka and MAV/06 strains are commercially available worldwide. Although the MAV/06 vaccine was introduced in 1990s, little was known about immunological characteristics.ResultsHere, we evaluated B and T cell immune response in animals inoculated with the Oka and MAV/06 vaccines as well as a new formulation of the MAV/06 vaccine. A variety of test methods were applied to evaluate T and B cell immune response. Plaque reduction neutralization test (PRNT) and fluorescent antibody to membrane antigen (FAMA) assay were conducted to measure the MAV/06 vaccine-induced antibody activity against various VZVs. Glycoprotein enzyme-linked immunosorbent assay (gpELISA) was used to compare the degree of the antibody responses induced by the two available commercial VZV vaccines and the MAV/06 vaccine. Interferon-gamma enzyme-linked immunosorbent spot (IFN-γ ELISpot) assays and cytokine bead array (CBA) assays were conducted to investigate T cell immune responses. Antibodies induced by MAV/06 vaccination showed immunogenicity against a variety of varicella-zoster virus and cross-reactivity among the virus clades.ConclusionsIt is indicating the similarity of the antibody responses induced by commercial varicella vaccines and the MAV/06 vaccine. Moreover, VZV-specific T cell immune response from MAV/06 vaccination was increased via Th1 cell response. MAV/06 varicella vaccine induced both humoral and cellular immune response via Th1 cell mediated response.

Antigen Specific Humoral and Cellular Immunity Following SARS-CoV-2 Vaccination in ANCA-Associated Vasculitis Patients Receiving B-Cell Depleting Therapy.

Humoral vaccine responses are known to be suboptimal in patients receiving B-cell targeted therapy, and little is known about vaccine induced T-cell immunity in these patients. In this study, we characterized humoral and cellular antigen-specific anti-SARS-CoV2 responses following COVID-19 vaccination in patients with ANCA-associated vasculitis (AAV) receiving anti-CD20 therapy, who were either B-cell depleted, or B-cell recovered at the time of vaccination and in normal control subjects. SARS-CoV-2 anti-spike (S) and anti-nucleocapsid (NC) antibodies were measured using electrochemiluminescence immunoassays, while SARS-CoV-2 specific T-cell responses to S glycoprotein subunits 1 (S1) and 2 (S2) and receptor binding domain peptide pools were measured using interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assays. In total, 26 recently vaccinated subjects were studied. Despite the lack of a measurable humoral immune response, B-cell depleted patients mounted a similar vaccine induced antigen-specific T-cell response compared to B-cell recovered patients and normal controls. Our data indicate that to assure a humoral response in patients receiving anti-CD20 therapy, SARS-CoV-2 vaccination should ideally be delayed until B-cell recovery (CD-20 positive B-cells > 10/μl). Nevertheless, SARS-CoV-2 vaccination elicits robust, potentially protective cellular immune responses in these subjects. Further research to characterize the durability and protective effect of vaccine-induced anti-SARS-CoV-2 specific T-cell immunity are needed.

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

ABSTRACTAfrican swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs.IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates.

Humoral and Cellular Immunogenicity of Zoster Vaccine within One Year after Herpes Zoster

BackgroundHerpes zoster vaccination is recommended to patients with a prior history of herpes zoster to prevent reactivation. However, the appropriate timing of vaccination is controversial. We compared immunogenicity of vaccine according to timing of vaccination after zoster illness.MethodsIn this prospective observational study, subjects were stratified into two groups by the vaccination timing since their zoster illness: 6–12 months (within-1 year group) vs. 1–5 years (after-1 year group). Blood samples were collected before and 6 weeks after vaccination of zoster vaccine live. Varicella-zoster virus (VZV)-specific IgG concentrations were measured by enzyme-linked immunosorbent assay. Interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assays were performed to assess VZV specific T-cell responses.ResultsA total of 59 patients (18 in the within-1 year group and 41 in the after-1 year group) were enrolled. Ages were not significantly different between groups. The baseline geometric mean titer (GMT) of VZV IgG was higher in the within-1 year group than in the after-1 year group (245.8 IU/mL vs. 124.9 IU/mL; P = 0.040). The geometric mean fold-rise (GMFR) of VZV IgG was lower in the within-1 year group than in the after-1 year group (1.42 vs. 2.46; P = 0.002). The GMT of spot forming cell (SFC) counts by ELISPOT at baseline and 6 weeks after vaccination were not significantly different between groups. The GMFRs of SFCs were also comparable.ConclusionZoster vaccination within 1 year after zoster illness may have disadvantage in the aspect of humoral immune response (ClinicalTrials.gov number, NCT02704572).Disclosures All authors: No reported disclosures.

A Novel Non-Replication-Competent Cytomegalovirus Capsid Mutant Vaccine Strategy Is Effective in Reducing Congenital Infection.

Congenital cytomegalovirus (CMV) infection is a leading cause of mental retardation and deafness in newborns. The guinea pig is the only small animal model for congenital CMV infection. A novel CMV vaccine was investigated as an intervention strategy against congenital guinea pig cytomegalovirus (GPCMV) infection. In this disabled infectious single-cycle (DISC) vaccine strategy, a GPCMV mutant virus was used that lacked the ability to express an essential capsid gene (the UL85 homolog GP85) except when grown on a complementing cell line. In vaccinated animals, the GP85 mutant virus (GP85 DISC) induced an antibody response to important glycoprotein complexes considered neutralizing target antigens (gB, gH/gL/gO, and gM/gN). The vaccine also generated a T cell response to the pp65 homolog (GP83), determined via a newly established guinea pig gamma interferon enzyme-linked immunosorbent spot assay. In a congenital infection protection study, GP85 DISC-vaccinated animals and a nonvaccinated control group were challenged during pregnancy with wild-type GPCMV (10(5) PFU). The pregnant animals carried the pups to term, and viral loads in target organs of pups were analyzed. Based on live pup births in the vaccinated and control groups (94.1% versus 63.6%), the vaccine was successful in reducing mortality (P = 0.0002). Additionally, pups from the vaccinated group had reduced CMV transmission, with 23.5% infected target organs versus 75.9% in the control group. Overall, these preliminary studies indicate that a DISC CMV vaccine strategy has the ability to induce an immune response similar to that of natural virus infection but has the increased safety of a non-replication-competent virus, which makes this approach attractive as a CMV vaccine strategy. Congenital CMV infection is a leading cause of mental retardation and deafness in newborns. An effective vaccine against CMV remains an elusive goal despite over 50 years of CMV research. The guinea pig, with a placenta structure similar to that in humans, is the only small animal model for congenital CMV infection and recapitulates disease symptoms (e.g., deafness) in newborn pups. In this report, a novel vaccine strategy against congenital guinea pig cytomegalovirus (GPCMV) infection was developed, characterized, and tested for efficacy. This disabled infectious single-cycle (DISC) vaccine strategy induced a neutralizing antibody or a T cell response to important target antigens. In a congenital infection protection study, animals were protected against CMV in comparison to the nonvaccinated group (52% reduction of transmission). This novel vaccine was more effective than previously tested gB-based vaccines and most other strategies involving live virus vaccines. Overall, the DISC vaccine is a safe and promising approach against congenital CMV infection.

Variability of the IFN-γ ELISpot assay in the context of proficiency testing and bridging studies

Assays that assess cellular mediated immune responses performed under Good Clinical Laboratory Practice (GCLP) guidelines are required to provide specific and reproducible results. Defined validation procedures are required to establish the Standard Operating Procedure (SOP), include pass and fail criteria, as well as implement positivity criteria. However, little to no guidance is provided on how to perform longitudinal assessment of the key reagents utilized in the assay. Through the External Quality Assurance Program Oversight Laboratory (EQAPOL), an Interferon-gamma (IFN-γ) Enzyme-linked immunosorbent spot (ELISpot) assay proficiency testing program is administered. A limit of acceptable within site variability was estimated after six rounds of proficiency testing (PT). Previously, a PT send-out specific within site variability limit was calculated based on the dispersion (variance/mean) of the nine replicate wells of data. Now an overall ‘dispersion limit’ for the ELISpot PT program within site variability has been calculated as a dispersion of 3.3. The utility of this metric was assessed using a control sample to calculate the within (precision) and between (accuracy) experiment variability to determine if the dispersion limit could be applied to bridging studies (studies that assess lot-to-lot variations of key reagents) for comparing the accuracy of results with new lots to results with old lots. Finally, simulations were conducted to explore how this dispersion limit could provide guidance in the number of replicate wells needed for within and between experiment variability and the appropriate donor reactivity (number of antigen-specific cells) to be used for the evaluation of new reagents. Our bridging study simulations indicate using a minimum of six replicate wells of a control donor sample with reactivity of at least 150 spot forming cells per well is optimal. To determine significant lot-to-lot variations use the 3.3 dispersion limit for between and within experiment variability.

Updated Results of a Phase 1/2a, Dose Escalation Study of Pvx-410 Multi-Peptide Cancer Vaccine in Patients with Smoldering Multiple Myeloma (SMM)

Updated Results of a Phase 1/2a, Dose Escalation Study of Pvx-410 Multi-Peptide Cancer Vaccine in Patients with Smoldering Multiple Myeloma (SMM)

Interferon Gamma ELISPOT Testing as a Risk-Stratifying Biomarker for Kidney Transplant Injury: Results From the CTOT-01 Multicenter Study.

Previous studies suggest that quantifying donor-reactive memory T cells prior to kidney transplantation by interferon gamma enzyme-linked immunosorbent spot assay (IFNγELISPOT) can assist in assessing risk of posttransplant allograft injury. Herein, we report an analysis of IFNγELISPOT results from the multicenter, Clinical Trials in Organ Transplantation-01 observational study of primary kidney transplant recipients treated with heterogeneous immunosuppression. Within the subset of 176 subjects with available IFNγELISPOT results, pretransplant IFNγELISPOT positivity surprisingly did not correlate with either the incidence of acute rejection (AR) or estimated glomerular filtration rate (eGFR) at 6- or 12-month. These unanticipated results prompted us to examine potential effect modifiers, including the use of T cell-depleting, rabbit anti-thymocyte globulin (ATG). Within the no-ATG subset, IFNγELISPOT(neg) subjects had higher 6- and 12-month eGFRs than IFNγELISPOT(pos) subjects, independent of biopsy-proven AR, peak PRA, human leukocyte antigen mismatches, African-American race, donor source, and recipient age or gender. In contrast, IFNγELISPOT status did not correlate with posttransplant eGFR in subjects given ATG. Our data confirm an association between pretransplant IFNγELISPOT positivity and lower posttransplant eGFR, but only in patients who do not receive ATG induction. Controlled studies are needed to test the hypothesis that ATG induction is preferentially beneficial to transplant candidates with high frequencies of donor-reactive memory T cells.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multiantigen" vaccine that elicits robust CMI. Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

Initial Results of a Phase 1/2a, Dose Escalation Study of PVX-410 Multi-Peptide Cancer Vaccine in Patients with Smoldering Multiple Myeloma (SMM)

Introduction: The standard of treatment for SMM is watchful waiting. PVX-410 (OncoPep, Inc.) is being developed for the treatment of SMM to stop progression to active MM.Methods: PVX-410 consists of 4 HLA-A2 restricted, synthetic 9-mer peptides from unique regions of 3 multiple myeloma (MM)-associated antigens (XBP1 US184-192; XBP1 SP367-375; CD138260-268; and CS1239-247) emulsified in Montanide® ISA-720 VG (Seppic, Inc.). Adults with SMM at high risk of progression to active MM (ie, ≥2 of the following risk factors: serum monoclonal [M]-protein ≥3 g/dL; bone marrow clonal plasma cells >10%; and/or abnormal serum free light chain ratio [0.26-1.65]) and HLA-A2+were eligible. The primary objective of this study phase was to determine the tolerability of PVX-410 as a single agent. Immune response to PVX-410 and change in M protein were also assessed. This ongoing study is similarly evaluating PVX-410 co-administered with lenalidomide (Celgene Corp.).Patients received 6 doses of PVX-410 subcutaneously plus 0.5 mL (1 mg) Hiltonol®(poly-ICLC, Oncovir, Inc.) intramuscularly over 10 weeks. Patients initially received PVX-410 0.4 mg (3 patients, 0.1 mg/peptide / 0.4 mg total dose; low-dose) with escalation to 0.8 mg (9 patients, 0.2 mg/peptide / 0.8 mg total dose; target-dose). Patients were followed for 12 months post-treatment. Blood for immune response evaluation is collected at Week 0 (Baseline; pre-dose), 2, 4, and 8 during treatment and at Months 1, 3, 6, 9, and 12 post-treatment; disease response is assessed at the same timepoints, except Weeks 0 and 2.Immune response to PVX-410 was determined by interferon-gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) and multimer assays. Samples were tested for IFN-γ production in response to peptide stimulation using ELISPOT and analyzed according to the distribution free sampling (DFR) method (Moodie et al, 2010). A positive (+) response was defined as 1) DFR+ at the timepoint (p-value ≤0.05); and 2) >2-fold increase from Baseline. A result also was considered + if the Baseline value was negative and the post-dose value was DFR+. For determination of peptide-specific memory CD8+ T cell responses, a flow cytometry-based major histocompatibility complex (MHC) multimer assay was performed using tetramers specific for HLA-A2 and each of the 4 peptides. A + response was defined as a value ≥2 X the negative control; ≥Baseline; or, if the negative control or Baseline value was 0.0 or 0.1, the patient result was ≥0.03.Results: Twelve patients were enrolled (3 low-dose; 9 target-dose). Ten subjects were male and all were white. Median age was 56 years. Median time since diagnosis was 0.8 years (range 0.3 to 7.3 years); time since diagnosis was <1 year for 8 patients. All patients had at least 2, and 6 had all 3 high-risk factors. Results are available for all 12 patients through at least 1 month post-treatment.All 12 patients had a positive immune response to at least 1 peptide, as measured by IFN-γ ELISPOT: XBP1 US184-192 (9/12); XBP1 SP367-375 (8/12); CD138260-268 (5/12); and CS1239-247(2/9). DFR-positivity to 1, 2, 3, and all 4 peptides was seen in 5, 3, 3, and 1 patient, respectively.The multimer assay was performed for 9 patients, of whom 7 had vaccine-induced, peptide-specific memory CD8+ T cells: XBP1 US184-192 (7/9); XBP1 SP367-375 (4/9); CD138260-268 (2/9); and CS1239-247(1/9).Five subjects, 2 of 3 in the low-dose and 3 of 9 in the target-dose cohorts, experienced progression to active disease within 9 months post-treatment. Seven patients had stable disease at last follow-up (median follow up of 9 months).PVX-410 was well-tolerated. All adverse events (AEs) were ≤Grade 2 and non-serious. Study vaccine-related AEs generally occurred within the first 2 days post-dose and consisted of systemic symptoms and local reactions commonly seen with vaccines (eg, fever, chills, fatigue, nausea, and other flu-like symptoms / localized erythema, induration, pain, rash, and pruritus).Conclusions: Six doses of PVX-410 were well tolerated in 12 patients with SMM. All 12 patients showed an immune response to the vaccine, with 4 having an immune response to ≥3 of 4 peptides. Based on these promising findings, PVX-410 is being investigated in combination with 3 cycles of fixed-dose lenalidomide. Given its immunomodulatory properties, it is hypothesized that co-administration of lenalidomide will enhance the T cell-mediated immune response induced by PVX-410. DisclosuresThomas:Novartis, Celgene, Millenium, Idera Pharmaceuticals: Consultancy, Research Funding. Weber:OncPep: Research Funding. Kaufman:Millennium; Celgene; Novartis; Onyx; Janssen; Spectrum: Consultancy; Novartis; Onyx; Merck; Celgene: Research Funding; Millennium; Celgene; Novartis; Onyx; Janssen; Spectrum: Honoraria. Lonial:Millennium, Celgene, Novartis, BMS, Onyx: Consultancy, Research Funding. Raje:Eli Lilly, Acetylon: Research Funding; novartis, Amgen, Celgene, Millenium, Onyx: Consultancy.

The human CD8+ T cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes.

The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8(+) T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8(+) T cell responses after live attenuated dengue vaccination and show that CD8(+) T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV. The development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that seroconversion is the only reliable correlate of protection. Here, we show that CD8(+) T cell responses in vaccinees were readily detectable and comparable to natural dengue virus infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV.

HIV control is mediated in part by CD8+ T-cell targeting of specific epitopes.

We investigated the hypothesis that the correlation between the class I HLA types of an individual and whether that individual spontaneously controls HIV-1 is mediated by the targeting of specific epitopes by CD8(+) T cells. By measuring gamma interferon enzyme-linked immunosorbent spot (ELISPOT) assay responses to a panel of 257 optimally defined epitopes in 341 untreated HIV-infected persons, including persons who spontaneously control viremia, we found that the correlation between HLA types and control is mediated by the targeting of specific epitopes. Moreover, we performed a graphical model-based analysis that suggested that the targeting of specific epitopes is a cause of such control--that is, some epitopes are protective rather than merely associated with control--and identified eight epitopes that are significantly protective. In addition, we use an in silico analysis to identify protein regions where mutations are likely to affect the stability of a protein, and we found that the protective epitopes identified by the ELISPOT analysis correspond almost perfectly to such regions. This in silico analysis thus suggests a possible mechanism for control and could be used to identify protective epitopes that are not often targeted in natural infection but that may be potentially useful in a vaccine. Our analyses thus argue for the inclusion (and exclusion) of specific epitopes in an HIV vaccine. Some individuals naturally control HIV replication in the absence of antiretroviral therapy, and this ability to control is strongly correlated with the HLA class I alleles that they express. Here, in a large-scale experimental study, we provide evidence that this correlation is mediated largely by the targeting of specific CD8(+) T-cell epitopes, and we identify eight epitopes that are likely to cause control. In addition, we provide an in silico analysis indicating that control occurs because mutations within these epitopes change the stability of the protein structures. This in silico analysis also identified additional epitopes that are not typically targeted in natural infection but may lead to control when included in a vaccine, provided that other epitopes that would otherwise distract the immune system from targeting them are excluded from the vaccine.

Characterization of T-cell responses to cryptic epitopes in recipients of a noncodon-optimized HIV-1 vaccine.

Cryptic epitopes (CEs) can be encoded by any of the 5 alternative reading frames (ARFs, 2 sense and 3 antisense) of a known gene. Although CE responses are commonly detected during HIV-1 infection, it is not known whether these responses are induced after vaccination. Using a bioinformatic approach, we determined that vaccines with codon-optimized HIV inserts significantly skewed CE sequences and are not likely to induce crossreactive responses to natural HIV CE. We then evaluated the CE- and protein-specific T-cell responses using Gag, Pol, and ARF peptide pools among participants immunized with a non-codon optimized vaccine regimen of 2 pGA2/JS7 DNA primes followed by 2 MVA/HIV62 Gag-Pol-Env vector boosts or 4 saline injections. Vaccinees had significantly more interferon gamma enzyme-linked immunosorbent spot (IFNγ ELISpot) responses toward Gag (P = 0.003) but not toward Pol protein than did placebo recipients. However, CE-specific T-cell responses were low in magnitude, and their frequencies did not differ significantly between vaccine and placebo recipients. Additionally, most positive CE responses could not be mapped to individual peptides. After expanding responses in a cultured assay, however, the frequency and the median magnitude of responses to ARF peptides were significantly greater in vaccinees (P < 0.0001), indicating that CE-specific T-cell responses are present but below an ex vivo assay's limit of detection. Our data demonstrate that HIV-1 vaccines currently in clinical trials are poorly immunogenic with regard to CE-specific T-cell responses. Therefore, the context of HIV-1 immunogens may need to be modified as a comprehensive strategy to broaden vaccine-induced T-cell responses.

Occupational Exposure to Hepatitis C Virus: Early T-Cell Responses in the Absence of Seroconversion in a Longitudinal Cohort Study

T-cell responses have been described in seronegative patients who test negative for hepatitis C virus (HCV) RNA despite frequent HCV exposure. However, the cross-sectional design of those studies did not clarify whether T cells were indeed induced by low-level HCV exposure without seroconversion or whether they resulted from regular acute infection with subsequent antibody loss. Over a 10-year period, our longitudinal study recruited 72 healthcare workers with documented HCV exposure. We studied viremia and antibody and T-cell responses longitudinally for 6 months. All healthcare workers remained negative for HCV RNA and antibodies. However, 48% developed proliferative T-cell response and 42% developed responses in interferon-gamma enzyme-linked immunosorbent spot assays, with 29 healthy HCV-unexposed controls used to define assay cutoffs. The response prevalence was associated with the transmission risk score. T-cell responses peaked at week 4 and returned to baseline by week 12 after exposure. They predominantly targeted nonstructural HCV proteins, which are not part of the HCV particle and thus must have been synthesized in infected cells. Subclinical transmission of HCV occurs frequently, resulting in infection and synthesis of nonstructural proteins despite undetectable systemic viremia. T-cell responses are more sensitive indicators of this low-level HCV exposure than antibodies.

Evolution of CD8+ T cell responses after acute PARV4 infection.

PARV4 is a small DNA human virus that is strongly associated with hepatitis C virus (HCV) and HIV infections. The immunologic control of acute PARV4 infection has not been previously described. We define the acute onset of PARV4 infection and the characteristics of the acute-phase and memory immune responses to PARV4 in a group of HCV- and HIV-negative, active intravenous drug users. Ninety-eight individuals at risk of blood-borne infections were tested for PARV4 IgG. Gamma interferon enzyme-linked immunosorbent spot assays, intracellular cytokine staining, and a tetrameric HLA-A2-peptide complex were used to define the T cell populations responding to PARV4 peptides in those individuals who acquired infection during the study. Thirty-five individuals were found to be PARV4 seropositive at the end of the study, eight of whose baseline samples were found to be seronegative. Persistent and functional T cell responses were detected in the acute infection phase. These responses had an active, mature, and cytotoxic phenotype and were maintained several years after infection. Thus, PARV4 infection is common in individuals exposed to blood-borne infections, independent of their HCV or HIV status. Since PARV4 elicits strong, broad, and persistent T cell responses, understanding of the processes responsible may prove useful for future vaccine design.

T-Cell Responses in Children to Internal Influenza Antigens, 1 Year After Immunization With Pandemic H1N1 Influenza Vaccine, and Response to Revaccination With Seasonal Trivalent–inactivated Influenza Vaccine

During seasonal influenza epidemics, 5-15% of the population are affected with an illness having a nontrivial mortality, morbidity and economic burden. Inactivated influenza vaccines are routinely used to prevent influenza infection, primarily by inducing humoral immunity. In addition, trivalent-inactivated influenza vaccines have previously been shown to boost influenza-specific T-cell responses in a small percentage of adults. We investigate here the influenza-specific T-cell response, in children, 1 year after pandemic H1N1 vaccination and the ability to boost the T-cell response with trivalent-inactivated influenza immunization. Peripheral blood mononuclear cells (PBMCs) were isolated from children previously vaccinated with pandemic H1N1 vaccine, pre- and postseasonal 2010-2011 trivalent influenza vaccine (TIV) vaccination. Samples were analyzed by interferon-gamma enzyme-linked immunosorbent spot for reactogenicity toward internal influenza antigens (nucleoprotein, matrix protein 1 and nonstructural protein 1). Basal ex vivo T-cell responses to nucleoprotein, matrix protein 1 and nonstructural protein 1 measured by interferon-gamma enzyme-linked immunosorbent spot assay were significantly higher in those children who had previously received an AS03B-adjuvanted split virion pandemic vaccine 12 months earlier rather than a nonadjuvanted whole virion vaccine. Boosting of these responses, 21 days after 2010/2011 seasonal TIV vaccination was observed regardless of age or prior pandemic vaccination regime, although boosting was greater in those groups with the lowest initial response. We show here that children previously vaccinated with the 2009 pandemic H1N1 vaccine have measurable T-cell responses 1 year after vaccination. The magnitudes of these responses are dependent on both age of vaccine and type of pandemic H1N1 vaccine used. After 2010/2011 seasonal TIV vaccination, these T-cell responses undergo a small but significant boost.

Specific CD8+ T cell responses correlate with control of simian immunodeficiency virus replication in Mauritian cynomolgus macaques.

Specific major histocompatibility complex (MHC) class I alleles are associated with an increased frequency of spontaneous control of human and simian immunodeficiency viruses (HIV and SIV). The mechanism of control is thought to involve MHC class I-restricted CD8(+) T cells, but it is not clear whether particular CD8(+) T cell responses or a broad repertoire of epitope-specific CD8(+) T cell populations (termed T cell breadth) are principally responsible for mediating immunologic control. To test the hypothesis that heterozygous macaques control SIV replication as a function of superior T cell breadth, we infected MHC-homozygous and MHC-heterozygous cynomolgus macaques with the pathogenic virus SIVmac239. As measured by a gamma interferon enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT) using blood, T cell breadth did not differ significantly between homozygotes and heterozygotes. Surprisingly, macaques that controlled SIV replication, regardless of their MHC zygosity, shared durable T cell responses against similar regions of Nef. While the limited genetic variability in these animals prevents us from making generalizations about the importance of Nef-specific T cell responses in controlling HIV, these results suggest that the T cell-mediated control of virus replication that we observed is more likely the consequence of targeting specificity rather than T cell breadth.