Standardized monitoring of cytomegalovirus-specific immunity can improve risk stratification of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation.

Recurrence of cytomegalovirus reactivation remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Monitoring cytomegalovirus-specific cellular immunity using a standardized assay might improve the risk stratification of patients. A prospective multicenter study was conducted in 175 intermediate- and high-risk allogeneic hematopoietic stem cell transplant recipients under preemptive antiviral therapy. Cytomegalovirus-specific cellular immunity was measured using a standardized IFN-γ ELISpot assay (T-Track® CMV). Primary aim was to evaluate the suitability of measuring cytomegalovirus-specific immunity after end of treatment for a first cytomegalovirus reactivation to predict recurrent reactivation. 40/101 (39.6%) patients with a first cytomegalovirus reactivation experienced recurrent reactivations, mainly in the high-risk group (cytomegalovirus-seronegative donor/cytomegalovirus-seropositive recipient). The positive predictive value of T-Track® CMV (patients with a negative test after the first reactivation experienced at least one recurrent reactivation) was 84.2% in high-risk patients. Kaplan-Meier analysis revealed a higher probability of recurrent cytomegalovirus reactivation in high-risk patients with a negative test after the first reactivation (hazard ratio 2.73; p=0.007). Interestingly, a post-hoc analysis considering T-Track® CMV measurements at day 100 post-transplantation, a time point highly relevant for outpatient care, showed a positive predictive value of 90.0% in high-risk patients. Our results indicate that standardized cytomegalovirus-specific cellular immunity monitoring may allow improved risk stratification and management of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation. This study was registered at www.clinicaltrials.gov as #NCT02156479.

Perinatal Gram-Positive Bacteria Exposure Elicits Distinct Cytokine Responses In Vitro.

During pregnancy, infections caused by the gram-positive bacteria Enterococcus faecalis (E. faecalis), Streptococcus agalacticae (S. agalacticae), and Staphylococcus aureus (S. aureus) are major reasons for preterm labor, neonatal prematurity, meningitis, or sepsis. Here, we propose cytokine responses to bacterial infections by the immature perinatal immune system as central players in the pathogenesis of preterm birth and neonatal sepsis. We aimed to close the gap in knowledge about such cytokine responses by stimulating freshly isolated umbilical blood mononuclear cells (UBMC) with lysates of E. faecalis, S. agalacticae, and S. aureus collected from pregnant women in preterm labor. Bacterial lysates and, principally, S. aureus and S. agalacticae distinctly triggered most of the eleven inflammatory, anti-inflammatory, TH1/TH2 cytokines, and chemokines quantified in UBMC culture media. Chemokines depicted the most robust induction. Among them, MIP-1ß was further enhanced in UBMC from female compered to male newborn infants. Due to its stability and high levels, we investigated the diagnostic value of IL-8. IL-8 was critically upregulated in cord blood of preterm neonates suffering from infections compared to gestational age-matched controls. Our results provide novel clues about perinatal immunity, underscoring a potential value of IL-8 for the timely detection of infections and suggesting that MIP-1ß constitutes an early determinant of sex-specific immunity, which may contribute, e.g., to male's vulnerability to preterm birth.

Clinical validation of a novel enzyme-linked immunosorbent spot assay-based in vitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study.

Impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of CMV reactivation and associated complications in solid-organ transplantation. Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T-Track® CMV, a novel IFN-γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE-I CMV proteins, to monitor CMV-CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate-risk renal transplant recipients. CMV-CMI, CMV viral load, and clinical complications were monitored over 6 months post-transplantation. Ninety-five percent and 88-92% ELISpot assays were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65-specific response was ninefold higher in patients with self-clearing viral load compared to antivirally treated patients prior to first viral load detection (P < 0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T-Track® CMV is a highly sensitive IFN-γ ELISpot assay, suitable for the immunomonitoring of CMV-seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV-related clinical complications (ClinicalTrials.gov Identifier: NCT02083042).

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity.

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 × 104 and 2 × 105 PBMC per well upon stimulation with T-activated® IE-1 (R2 = 0.97) and pp65 (R2 = 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.

Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients.

BACKGROUND: Uncontrolled cytomegalovirus (CMV) replication in immunocompromised solid-organ transplant recipients is a clinically relevant issue and an indication of impaired CMV-specific cell-mediated immunity (CMI). Primary aim of this study was to assess the suitability of the immune monitoring tool T-Track® CMV to determine CMV-reactive CMI in a cohort of hemodialysis patients representative of patients eligible for renal transplantation. Positive and negative agreement of T-Track® CMV with CMV serology was examined in 124 hemodialysis patients, of whom 67 (54%) revealed a positive CMV serostatus. Secondary aim of the study was to evaluate T-Track® CMV performance against two unrelated CMV-specific CMI monitoring assays, QuantiFERON®-CMV and a cocktail of six class I iTAg™ MHC Tetramers. RESULTS: Positive T-Track® CMV results were obtained in 90% (60/67) of CMV-seropositive hemodialysis patients. In comparison, 73% (45/62) and 77% (40/52) positive agreement with CMV serology was achieved using QuantiFERON®-CMV and iTAg™ MHC Tetramer. Positive T-Track® CMV responses in CMV-seropositive patients were dominated by pp65-reactive cells (58/67 [87%]), while IE-1-responsive cells contributed to an improved (87% to 90%) positive agreement of T-Track® CMV with CMV serology. Interestingly, T-Track® CMV, QuantiFERON®-CMV and iTAg™ MHC Tetramers showed 79% (45/57), 87% (48/55) and 93% (42/45) negative agreement with serology, respectively, and a strong inter-assay variability. Notably, T-Track® CMV was able to detect IE-1-reactive cells in blood samples of patients with a negative CMV serology, suggesting either a previous exposure to CMV that yielded a cellular but no humoral immune response, or TCR cross-reactivity with foreign antigens, both suggesting a possible protective immunity against CMV in these patients. CONCLUSION: T-Track® CMV is a highly sensitive assay, enabling the functional assessment of CMV-responsive cells in hemodialysis patients prior to renal transplantation. T-Track® CMV thus represents a valuable immune monitoring tool to identify candidate transplant recipients potentially at increased risk for CMV-related clinical complications.

Functional impairment of CMV-reactive cellular immunity during pregnancy.

Cytomegalovirus (CMV) is the most common congenital viral infection. Mother-to-child transmission can cause severe child disability. Intact CMV-specific cell-mediated immunity (CMI) was shown to prevent uncontrolled replication in healthy individuals. This study aimed to determine whether CMV-specific CMI is impaired in pregnant women, thus potentially increasing the overall risk for active CMV replication and transmission. CMV-specific CMI in peripheral blood of 60 pregnant women was determined using T-Track® CMV for detection of IE-1 and pp65-reactive effector cells by IFN-γ ELISpot, and compared to the CMV-IgG and -IgM serostatus. CMV-specific CMI was detected in 65% of CMV-seropositive pregnant women. Five percent of CMV-IgG seronegative women showed IE-1- but not pp65-reactive cells. The overall number of CMV-reactive cells in pregnant women was significantly lower compared to a matched non-pregnant control group (P < 0.001). No significant difference in CMV-specific CMI was detected in the course of the three trimesters of pregnancy of CMV-IgG seropositive women. Postpartum (median days postnatal = 123), IE-1- and pp65-specific CMI remained significantly lower than in the non-pregnant control group (P < 0.001 and 0.0032, respectively). Functional analysis of CMV-reactive immune cells using T-Track® CMV therefore suggests a systemic down-regulation of CMV-specific CMI in pregnant women. Further studies are needed to investigate whether this may be indicative of a higher susceptibility to CMV reactivation or transmission. J. Med. Virol. 89:324-331, 2017. © 2016 Wiley Periodicals, Inc.

The effect of Indomethacin and Betamethasone on the cytokine response of human neonatal mononuclear cells to gram-positive bacteria.

Intrauterine infections with gram-positive bacteria pose a serious threat to neonates since they can result in neonatal sepsis, induce a fetal inflammatory response and also cause preterm birth. Despite intensive care, prematurity remains a leading cause of neonatal death, and is often accompanied by a number of morbidities. In order to prevent premature birth, tocolytic agents like Indomethacin are administered. Betamethasone is used to promote lung maturation and prevent respiratory distress syndrome. A combination of both drugs is assumed to prevent premature delivery while simultaneously facilitating lung maturation. This study investigates the effect of Betamethasone, Indomethacin and a combination of both on the cytokine production of neonatal cord blood mononuclear cells (CBMC) after stimulation with lysates of the gram-positive pathogens Streptococcus agalactiae and Enterococcus faecalis. The aim of the study is to determine the impact of these drugs on the function of the neonatal immune system which should aid clinicians in choosing the optimal therapy in case of preterm birth associated with intrauterine infection. Betamethasone reduced the production of the pro-inflammatory cytokines IL-6, IL-12p40, MIP-1α and TNF and increased the expression of the anti-inflammatory cytokine IL-10, depending on the pathogen used for stimulation. In contrast to Betamethasone, Indomethacin almost exclusively increased IL-10 production. The combination of both drugs decreased the expression of IL-6, IL-12p40, MIP-1α and TNF while increasing IL-10 production, depending on the concentration of Indomethacin and the pathogen used for stimulation. Based on our results, the combination therapy with Indomethacin and Betamethasone has a similar effect on cytokine production as Betamethasone alone, which is generally administered in case of impending preterm birth. However, the combination therapy has the advantage of promoting lung maturation while simultaneously blocking preterm labor effectively.

Performance of T-Track® SARS-CoV-2, an Innovative Dual Marker RT-qPCR-Based Whole-Blood Assay for the Detection of SARS-CoV-2-Reactive T Cells.

T-cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a central role in the control of the virus. In this study, we evaluated the performance of T-Track® SARS-CoV-2, a novel CE-marked quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels in response to the S1 and NP SARS-CoV-2 antigens, in 335 participants with or without a history of SARS-CoV-2 infection and vaccination, respectively. Of the 62 convalescent donors, 100% responded to S1 and 88.7% to NP antigens. In comparison, of the 68 naïve donors, 4.4% were reactive to S1 and 19.1% to NP. Convalescent donors <50 and ≥50 years of age demonstrated a 100% S1 reactivity and an 89.1% and 87.5% NP reactivity, respectively. T-cell responses by T-Track® SARS-CoV-2 and IgG serology by recomLine SARS-CoV-2 IgG according to the time from the last immunisation (by vaccination or viral infection) were comparable. Both assays showed a persistent cellular and humoral response for at least 36 weeks post immunisation in vaccinated and convalescent donors. Our results demonstrate the very good performance of the T-Track® SARS-CoV-2 molecular assay and suggest that it might be suitable to monitor the SARS-CoV-2-specific T-cell response in COVID-19 vaccinations trials and cross-reactivity studies.

Performance of T-Track® TB, a Novel Dual Marker RT-qPCR-Based Whole-Blood Test for Improved Detection of Active Tuberculosis.

Tuberculosis (TB) is one of the leading causes of death by an infectious disease. It remains a major health burden worldwide, in part due to misdiagnosis. Therefore, improved diagnostic tests allowing the faster and more reliable diagnosis of patients with active TB are urgently needed. This prospective study examined the performance of the new molecular whole-blood test T-Track® TB, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels, and compared it to that of the QuantiFERON®-TB Gold Plus (QFT-Plus) enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy and agreement analyses were conducted on the whole blood of 181 active TB patients and 163 non-TB controls. T-Track® TB presented sensitivity of 94.9% and specificity of 93.8% for the detection of active TB vs. non-TB controls. In comparison, the QFT-Plus ELISA showed sensitivity of 84.3%. The sensitivity of T-Track® TB was significantly higher (p < 0.001) than that of QFT-Plus. The overall agreement of T-Track® TB with QFT-Plus to diagnose active TB was 87.9%. Out of 21 samples with discordant results, 19 were correctly classified by T-Track® TB while misclassified by QFT-Plus (T-Track® TB-positive/QFT-Plus-negative), and two samples were misclassified by T-Track® TB while correctly classified by QFT-Plus (T-Track® TB-negative/QFT-Plus-positive). Our results demonstrate the excellent performance of the T-Track® TB molecular assay and its suitability to accurately detect TB infection and discriminate active TB patients from non-infected controls.

Stimulatory Effect of CMV Immunoglobulin on Innate Immunity and on the Immunogenicity of CMV Antigens.

BACKGROUND: Cytomegalovirus (CMV) immunoglobulin (CMVIG) is used for the prophylaxis of CMV infection after transplantation. Beyond providing passive CMV-specific immunity, CMVIG exerts enhancing and suppressive immunomodulatory functions. Although the anti-inflammatory activities of CMVIG have been extensively documented, its immunostimulatory activities remain poorly characterized. METHODS: This exploratory study analyzed the capacity of CMVIG to modulate cell-mediated innate and adaptive immunities in vitro on freshly isolated peripheral blood mononuclear cells (PBMCs) of CMV-seropositive and -seronegative healthy individuals, using interferon-γ (IFN-γ) enzyme-linked immunospot and intracellular cytokine staining assays. RESULTS: We showed that CMVIG treatment increases the number of IFN-γ-secreting PBMCs of both CMV-seronegative and -seropositive individuals, indicating a global stimulatory effect on innate immune cells. Indeed, CMVIG significantly increased the frequency of natural killer cells producing the T helper cell 1-type cytokines tumor necrosis factor and IFN-γ. This was associated with the induction of interleukin-12-expressing monocytes and the activation of cluster of differentiation (CD) 4+ and CD8+ T cells, as measured by the expression of tumor necrosis factor and IFN-γ. Interestingly, stimulation of PBMCs from CMV-seropositive subjects with CMVIG-opsonized CMV antigens (phosphoprotein 65, CMV lysate) enhanced CD4+ and CD8+ T-cell activation, suggesting that CMVIG promotes the immunogenicity of CMV antigens. CONCLUSIONS: Our data demonstrate that CMVIG can stimulate effector cells of both innate and adaptive immunities and promote the immunogenicity of CMV antigens. These immunostimulatory properties might contribute to the protective effect against CMV infection mediated by CMVIG.

Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation.

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8+ counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.

Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

Early detection of Varicella-Zoster virus (VZV)-specific T-cells before seroconversion in primary varicella infection: case report.

Here we report the case of a 54-year old, immunocompetent German patient with primary varicella whose Varicella-Zoster Virus (VZV)-specific T-cell responses could be detected early in infection and before the onset of seroconversion. This case demonstrates that the detection of VZV-specific T-cells may under certain circumstances support the diagnosis of a primary varicella infection, as for example in cases of atypical or subclinical varicella or in the absence of detectable VZV DNA in plasma.

Comparative purification and characterization of hepatitis B virus-like particles produced by recombinant vaccinia viruses in human hepatoma cells and human primary hepatocytes.

This study describes the comparative expression and purification of hepatitis B surface antigen (HBsAg) particles produced upon infection of human primary hepatocytes and human hepatoma cell lines (HuH-7 and HepG2) with recombinant vaccinia viruses. The highest levels of HBsAg expression were found in HuH-7 hepatoma cells following infection with recombinant vaccinia viruses, which contain the S gene under control of a 7.5 k-promoter. Four different methods for purification of the HBsAg particles were examined: isopycnic ultracentrifugation, sucrose cushion sedimentation, isocratic column gel filtration, and binding to anti-HBs-coated microparticles. The highest degree of purity of HBsAg particles was reached by the method based on anti-HBs-coated microparticles. The resulting product was >98% pure. Biochemical analysis and characterization of purified HBsAg particles were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy. The HBsAg, purified from human hepatoma cell lines and from human primary hepatocytes, consisted of both the non-glycosylated (p25) and the glycosylated (gp27) form and assembled into typical 22-nm particles, and thus may be of great interest and importance for research, diagnostics, and medical treatments.

Cytokine profiles of umbilical cord blood mononuclear cells upon in vitro stimulation with lipopolysaccharides of different vaginal gram-negative bacteria.

Inflammatory immune responses induced by lipopolysaccharides (LPS) of gram-negative bacteria play an important role in the pathogenesis of preterm labor and delivery, and in neonatal disorders. To better characterize LPS-induced inflammatory response, we determined the cytokine profile of umbilical cord blood mononuclear cells (UBMC) stimulated with LPS of seven vaginal gram-negative bacteria commonly found in pregnant women with preterm labor and preterm rupture of membrane. UBMC from ten newborns of healthy volunteer mothers were stimulated with purified LPS of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter calcoaceticus, Citrobacter freundii, and Pseudomonas aeruginosa. UBMC supernatants were tested for the presence of secreted pro-inflammatory cytokines (IL-6, IL-1ß, TNF), anti-inflammatory cytokine (IL-10), TH1-type cytokines (IL-12, IFN-γ), and chemokines (IL-8, MIP-1α, MIP-1ß, MCP-1) by Luminex technology. The ten cytokines were differentially induced by the LPS variants. LPS of E. coli and E. aerogenes showed the strongest stimulatory activity and P. aeruginosa the lowest. Interestingly, the ability of UBMC to respond to LPS varied greatly among donors, suggesting a strong individual heterogeneity in LPS-triggered inflammatory response.

Recombinant HIV-1 Pr55gag virus-like particles: potent stimulators of innate and acquired immune responses.

Several previous reports have clearly demonstrated the strong effectiveness of human immunodeficiency virus (HIV) Gag polyprotein-based virus-like particles (VLP) to stimulate humoral and cellular immune responses in complete absence of additional adjuvants. Yet, the mechanisms underlying the strong immunogenicity of these particulate antigens are still not very clear. However, current reports strongly indicate that these VLP act as "danger signals" to trigger the innate immune system and possess potent adjuvant activity to enhance the immunogenicity of per se only weakly immunogenic peptides and proteins. Here, we review the current understanding of how various particle-associated substances and other impurities may contribute to the observed immune-activating properties of these complex immunogens.

The past, present and future of HIV-vaccine development: a critical view.

Despite the extensive efforts that have been made to combat AIDS, the global number of HIV-1 infections is still increasing. There is major consent among scientists worldwide, that the development of successful HIV vaccine strategies requires a profound understanding of the epidemiological principles of a viral pandemic, as well as deep insights into the molecular and immunological mechanisms of HIV pathogenesis. This review provides an overview of past and present developments, as well as future aspects of HIV vaccines, and also provides a summary of current clinical trials in man.

Virus-like particles: a novel tool for the induction and monitoring of both T-helper and cytotoxic T-lymphocyte activity.

T-cells play a crucial role in the control of various viral infections such as HIV and herpes viruses. Thus, the development of advanced techniques for the stimulation and measurement of both antigen-specific T-helper and CTL responses is one of most meaningful objectives in vaccinology. Herein, we present HIV-1 Pr55gag lipoprotein particles (VLPs) to be a potent antigen for introducing epitopes into the MHC-class-I and -II processing and presentation pathway. These VLPs can easily be produced in insect cells by using the baculovirus expression system. Immunization studies in mice revealed the strong capacity of these VLPs to stimulate Gag-specific T-helper-1 cell-biased humoral and cellular immune responses. In addition, these VLPs can be used as a stimulator antigen for the detection of Gag-specific T-helper and CTL responses, as determined by conventional ELISA, ELISpot, FACS, and 51Cr-release assays. These results strongly underline the value of VLPs as a stimulator of MHC-class-I and -II mediated epitope presentation for preventive, therapeutic, and diagnostic purposes.

Codon-optimized genes that enable increased heterologous expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA.

Many of the problems related with mammalian gene expression, such as low translation efficiency and mRNA halflife, can be solved by means of a rational gene design, based on modern bioinformatics, followed by the de novo generation of a synthetic gene. Moreover, high expression rates and prolonged mRNA stability are not only crucial for heterologous mammalian expression, but, in particular, are important for the generation of effective DNA vaccines. In this chapter we show that an optimized synthetic gene encoding the HIV-1 Pr55gag outperforms wild-type gene driven expression by several orders of magnitude. RNA analysis revealed that this positive effect was mostly due to increased mRNA stability of the optimized transcripts. Moreover, mice vaccinated with the optimized gag gene elicited a much stronger immune response against Pr55gag than the control groups immunized with the respective wild-type gene.

Application of single-cell cultures of mouse splenocytes as an assay system to analyze the immunomodulatory properties of bacterial components.

Recently, various bacterial components have been suggested as initiating and modulating immune activation, thereby substantially affecting the complex and dynamic host/pathogen interactions. Herein, we present a valuable and simple methodology for determining the capacity of bacteria as well as defined bacterial structures to stimulate cellular effectors of the innate and cognate immune system. This assay format is based on the exposure of freshly prepared single-cell cultures of splenic cells derived from naive mice with the immunogen of interest. Herein, the determination of exclusive panels of cytokines by the ELISA, ELISpot, and FACS technology will serve as an indicator for the activation of defined arms of the immune system. An increased knowledge about microbial components with immunomodulatory properties will substantially contribute to a more detailed understanding of the dynamic interplay between the host and potential pathogens and, based on this knowledge, to the development of novel substances for the prevention and therapy of microbial infections.