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Background: The need for rapid and accurate comparison of panels of adjuvanted vaccine formulations and subsequent rational down selection, presents several challenges for modern vaccine development. Here we describe a method which may enable vaccine and adjuvant developers to compare antigen/adjuvant combinations in a harmonized fashion. Three reference antigens: Plasmodium falciparum apical membrane antigen 1 (AMA1), hepatitis B virus surface antigen (HBsAg), and Mycobacterium tuberculosis antigen 85A (Ag85A), were selected as model antigens and were each formulated with three adjuvants: aluminium oxyhydroxide, squalene-in-water emulsion, and a liposome formulation mixed with the purified saponin fraction QS21., Results: The nine antigen/adjuvant formulations were assessed for stability and immunogenicity in mice in order to provide benchmarks against which other formulations could be compared, in order to assist subsequent down selection of adjuvanted vaccines. Furthermore, mouse cellular immune responses were analyzed by measuring IFN-γ and IL-5 production in splenocytes by ELISPOT, and humoral responses were determined by antigen-specific ELISA, where levels of total IgG, IgG1, IgG2b and IgG2c in serum samples were determined., Conclusions: The reference antigens and adjuvants described in this study, which span a spectrum of immune responses, are of potential use as tools to act as points of reference in vaccine development studies. The harmonized methodology described herein may be used as a tool for adjuvant/antigen comparison studies.

Background: Induction of broad immune responses at mucosal site remains a primary goal for most vaccines against mucosal pathogens. Abundance of evidence indicates that the co-delivery of mucosal adjuvants, including cytokines, is necessary to induce effective mucosal immunity. In the present study, we set out to evaluate the role of a chemokine, CCL20, as an effective mucosal adjuvant for HIV vaccine., Methods: To evaluate the role of CCL20 as a potent adjuvant for HIV vaccine, we examined its effects on antigen-specific antibody responses, level of antibody-secreting cells, cytokine production and intestinal homing of plasma cells in vaccine immunized mice., Results: CCL20-incorporated VLP administered by mucosal route (intranasal (n = 10, p = 0.0085) or intravaginal (n = 10, p = 0.0091)) showed much higher potency in inducing Env-specific IgA antibody response than those administered by intramuscular route (n = 10). For intranasal administration, the HIV Env-specific IFN-γ(751 pg/ml), IL-4 (566 pg/ml), IL-5 (811 pg/ml) production and IgA-secreting plasma cells (62 IgA-secreting plasma cells/10 6 cells) in mucosal lamina propria were significantly augmented in CCL20-incorporated VLP immunized mice as compared to those immunized with Env only VLPs (p = 0.0332, 0.0398, 0.033, 0.0302 for IFN-γ, IL-4, IL-5, and IgA-secreting plasma cells, respectively). Further, anti-CCL20 mAb partially suppressed homing of Env-specific IgA ASCs into small intestine in mice immunized with CCL20-incorporated VLP by intranasal (62 decreased to 16 IgA- secreting plasma cells/10 6 cells, p = 0.0341) or intravaginal (52 decreased to 13 IgA- secreting plasma cells/10 6 cells, p = 0.0332) routes., Conclusion: Our data indicated that the VLP-incorporated CCL20 can enhance HIV Env-specific immune responses in mice, especially those occurring in the mucosal sites. We also found that i.m. prime followed by mucosal boost is critical and required for CCL20 to exert its full function as an effective mucosal adjuvant. Therefore, co-incorporation of CCL20 into Env VLPs when combined with mucosal administration could represent a novel and promising HIV vaccine candidate.

Background: In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions., Methods: Objective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay., Results: Optimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 10 4 and 2 × 10 5 PBMC per well upon stimulation with T-activated® IE-1 (R 2  = 0.97) and pp65 (R 2  = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3 + CD4 + (Th), CD3 + CD8 + (CTL), CD3 - CD56 + (NK) and CD3 + CD56 + (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive., Conclusion: The combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.

Background: The FluoroSpot assay, an advancement of the ELISpot assay, enables simultaneous measurement of different analytes secreted at a single-cell level. This allows parallel detection of several cytokines secreted by immune cells upon antigen recognition. Easier standardization, higher sensitivity and reduced labour intensity render FluoroSpot assays an interesting alternative to flow-cytometry based assays for analysis of clinical samples. While the use of immunoassays to study immunological primary and secondary endpoints becomes increasingly attractive, assays used require pre-trial validation. Here we describe the assay validation (precision, specificity and linearity) of a FluoroSpot immunological endpoint assay detecting Interferon γ (IFNγ) and Interleukin 2 (IL2) for use in clinical trial immune monitoring., Methods: We validated an IFNγ/IL2 FluoroSpot assay to determine Epstein-Barr virus (EBV)-specific cellular immune responses (IFNγ, IL2 and double positive IFNγ + IL2 responses), using overlapping peptide pools corresponding to EBV-proteins BZLF1 and EBNA3A. Assay validation was performed using cryopreserved PBMC of 16 EBV-seropositive and 6 EBV-seronegative donors. Precision was assessed by (i) testing 16 donors using three replicates per assay (intra-assay precision/repeatability) (ii) using two plates in parallel (intermediate precision/plate-to-plate variability) and (iii) by performing the assays on three different days (inter-assay precision/reproducibility). In addition, we determined specificity, linearity and quantification limits of the assay. Further we tested precision across the two assay systems, IFNγ/IL2 FluoroSpot and the corresponding enzymatic single cytokine ELISpot., Results: The validation revealed: (1) a high intra-assay precision (coefficient of variation (CV) 9.96, 8.85 and 13.05 %), intermediate precision (CV 6.48, 10.20 and 12.97 %) and reproducibility (CV 20.81 %, 12,75 % and 12.07 %) depending on the analyte and antigen used; (2) a specificity of 100 %; (3) a linearity with R (2) values from 0.93 to 0.99 depending on the analyte. The testing of the precision across the two assay systems, adduced a concordance correlation coefficient p c  = 0.99 for IFNγ responses and p c  = 0.93 for IL2 responses, indicating a large agreement between both assay methods., Conclusions: The validated primary endpoint assay, an EBV peptide pool specific IFNγ/IL2 FluoroSpot assay was found to be suitable for the detection of EBV-specific immune responses subject to the requirement of standardized assay procedure and data analysis.

Background: Investigating the neutralizing antibody (NAb) titer against HSV-1 is essential for monitoring the immune protection against HSV-1 in susceptible populations, which would facilitate the development of vaccines against herpes infection and improvement of HSV-1 based oncolytic virotherapy., Results: In this study, we have developed a neutralization test based on the enzyme-linked immunospot assay (ELISPOT-NT) to determine the neutralizing antibody titer against HSV-1 in human serum samples. This optimized assay employed a monoclonal antibody specifically recognizing glycoprotein D to detect the HSV-1 infected cells. With this test, the neutralizing antibody titer against HSV-1 could be determined within one day by automated interpretation of the counts of cell spots. We observed good correlation in the results obtained from ELISPOT-NT and plaque reduction neutralization test (PRNT) by testing 22 human serum samples representing different titers. Moreover, 269 human serum samples collected from a wide range of age groups were tested, the average neutralizing antibody titer (log2NT50) was 8.3 ± 2.8 and the prevalence of NAbs was 83.6 % in this cohort, it also revealed that the average neutralizing antibody titer in different groups increased with the age, and no significant difference in neutralizing antibody titers was observed between males and females., Conclusions: These results prove that this novel assay would serve as an accurate and simple assay for the assessment of the neutralizing antibody titers against HSV-1 in large cohorts.

Background: The neotropical, non-human primates (NHP) of the genus Saimiri and Aotus are recommended by the World Health Organization as experimental models for the study of human malaria because these animals can be infected with the same Plasmodium that cause malaria in humans. However, one limitation is the lack of immunological tools to assess the immune response in these models. The present study focuses on the development and comparative use of molecular and immunological methods to evaluate the cellular immune response in Saimiri sciureus., Methods: Blood samples were obtained from nineteen uninfected Saimiri. Peripheral blood mononuclear cells (PBMC) from these animals and splenocytes from one splenectomized animal were cultured for 6, 12, 18, 24, 48, 72 and 96 hrs in the presence of phorbol-12-myristate-13-acetate and ionomycin. The cytokine levels in the supernatant were detected using human and NHP cytometric bead array Th1/Th2 cytokine kits, the Bio-Plex Pro Human Cytokine Th1/Th2 Assay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assays and intracellular cytokine secretion assays. Cytokine gene expression was examined through TaqMan® Gene Expression Real-Time PCR using predesigned human gene-specific primers and probes or primers and probes designed based on published S. sciureus cytokine sequences., Results: The use of five assays based on monoclonal antibodies specific for human cytokines facilitated the detection of IL-2, IL-4 and/or IFN-γ. TaqMan array plates facilitated the detection of 12 of the 28 cytokines assayed. However, only seven cytokines (IL-1A, IL-2, IL-10, IL-12B, IL-17, IFN-β, and TNF) presented relative expression levels of at least 70% of the gene expression observed in human PBMC. The use of primers and probes specific for S. sciureus cytokines facilitated the detection of transcripts that showed relative expression below the threshold of 70%. The most efficient evaluation of cytokine gene expression, in PBMC and splenocytes, was observed after 6-12 hrs of culture, except for LTA in PBMC, whose expression was best analysed after 24 hrs of culture., Conclusions: Real-time PCR facilitates the analysis of a large number of cytokines altered during malaria infection, and this technique is considered the best tool for the evaluation of the cellular immune response in S. sciureus.

Background: There are controversies regarding the accuracy of the tuberculin skin test (TST) and methods based on the production of interferon gamma by sensitized T cells for the diagnosis of latent tuberculosis infection (LTBI) in pediatrics and immunosuppressed patients. Our objectives are to study TST and ELISPOT/T. SPOT.TB in the diagnosis of LTBI in children and adolescents with JIA undergoing methotrexate, the correlation between both and the sensitivity and specificity of T. SPOT.TB., Methods: This is an observational prospective longitudinal study in which children and adolescents with JIA undergoing methotrexate therapy were assessed for clinical and epidemiological data for LTBI, in addition to performing TST and T. SPOT.TB at baseline and after 3 and 12months., Results: There were 24 patients. The prevalence of LTBI at inclusion was 20.8%, the incidence after initiation of immunosuppressions 26.3% and the prevalence at the end of the study 41.6%. Epidemiological history positive for TB showed a relative risk of 2.0 for the development of LTBI. Only 2 patients had positive T. SPOT.TB but only in one it was useful for detecting early LTBI. T. SPOT.TB presented a sensitivity of 10%, specificity of 92.8%, and low correlation with TST. No patient developed TB disease at a mean follow-up of 47months., Conclusions: We found a high prevalence of ILTB that doubled with immunosuppression and that epidemiological history was an important relative risk. T. SPOT.TB showed low sensitivity and high specificity, and no superiority over TST. There was low agreement and little influence of immunosuppression on the results of both tests.

Background: The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpots cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood., Methods: Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot., Results: Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 uL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing., Conclusions: A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation are required before processing.