Pharmacokinetics of a Novel Anagrelide Extended-Release Formulation in Healthy Subjects: Food Intake and Comparison With a Reference Product.

Anagrelide is an established therapy for essential thrombocythemia. Common adverse effects have been linked to peak plasma concentrations of anagrelide and its 3OH metabolite. Our study was performed to investigate the pharmacokinetics (PK) of a novel anagrelide extended-release (AER) formulation and its active metabolites. Thirty healthy volunteers were randomized to receive either 2 mg AER (under fasting and fed conditions) or 2 mg commercially available reference product (CARP) in an open-label, 3-way crossover trial with washout periods of 6 days. Plasma concentrations of anagrelide and its active metabolites were assessed by tandem mass spectrometry. The PK differed significantly between all treatment periods. Bioavailability of AER was 55% of the CARP under fasting conditions and 60% under fed conditions. Cmax , AUCt, and AUC∞ were significantly higher and Tmax and T1/2 were significantly shorter after the CARP compared with AER. Food had a significant impact on the PK of AER, increasing the Cmax and AUCt while reducing the T1/2 , plateau, and mean residence time. Both formulations were well tolerated, with a trend toward more frequently occurring adverse events after the CARP. The PK of AER and the CARP differed significantly in all parameters. Food enhanced the bioavailability of AER.

Frequency, Private Specificity, and Cross-Reactivity of Preexisting Hepatitis C Virus (HCV)-Specific CD8+ T Cells in HCV-Seronegative Individuals: Implications for Vaccine Responses.

UNLABELLED: T cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response toward vaccines is conceivable. However, little is known about their frequency, repertoire, and impact on vaccination. We performed a detailed characterization of CD8(+) T cells specific to a hepatitis C virus (HCV) epitope (NS3-1073) in 121 HCV-seronegative individuals. We show that in vitro HCV NS3-1073-specific CD8(+) T cell responses were rather abundantly detectable in one-third of HCV-seronegative individuals irrespective of risk factors for HCV exposure. Ex vivo, these NS3-1073-specific CD8(+) T cells were found to be both naive and memory cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073-specific CD8(+) T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that preexisting NS3-1073-specific CD8(+) T cells can impact the T cell response toward peptide vaccination. Healthy, vaccinated individuals who showed an in vitro response toward NS3-1073 already before vaccination displayed a more vigorous and earlier response toward the vaccine. IMPORTANCE: Preventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Preexisting T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8(+) T cells are abundant in HCV-seronegative individuals but that their repertoire is highly diverse due to the involvement of both naive precursors and cross-reactive memory cells of different specificities, which can influence the response to vaccines. The data may emphasize the need to personalize immune-based therapies based on the individual's T cell repertoire that is present before the immune intervention.

Sustained viral load reduction in treatment-naive HCV genotype 1 infected patients after therapeutic peptide vaccination.

BACKGROUND: Novel antivirals augment treatment efficacy in chronic HCV infection, to overcome limitations on safety profile alternative approaches are warranted. The effect of a therapeutic peptide vaccine on HCV viral load was investigated in treatment-naïve genotype 1 HCV patients. METHODS: Fifty patients received 8 intradermal IC41 vaccinations biweekly with topical application of the TLR7 agonist imiquimod (Group A). In Group B, 21 patients received a condensed schedule of 16 subcutaneous vaccinations weekly without imiquimod. RESULTS: At Week 16 Group A (n=44) showed a statistically significant (p=0.0013) HCV viral load decline of 0.21 log. 24 weeks after the last vaccination the viral load decreased by 0.47 log (p<0.0001) in 34 subjects. This effect was more pronounced in 17 patients with high baseline HCV (>2×10(6)IU/ml) with a 0.61 log decline, which was statistically significant (p<0.02) starting two weeks after the third vaccination. No apparent effect on HCV viral load was observed in Group B (n=21). In Group A eight patients (24%) showed a viral load response defined as a decline of >0.8 log. Overall, about 30-55% of patients showed T cell responses during the vaccination series and up to six months in both groups. No significant correlations between the HCV viral load decrease and T cell immune response were detected. CONCLUSIONS: This is the first report on a significant antiviral effect of a peptide vaccine in HCV infected patients. Response kinetics with increased HCV RNA decline 24 weeks after the last IC41 vaccination is encouraging.

Correlation of protection against Japanese encephalitis virus and JE vaccine (IXIARO(®)) induced neutralizing antibody titers.

Immune sera from volunteers vaccinated in a blinded Phase 3 clinical trial with JE-VAX(®) and a new Japanese encephalitis virus (JEV) vaccine (IC51 or IXIARO), were tested for the ability to protect mice against lethal JEV challenge. Sera from IXIARO vaccinated subjects were pooled into four batches based on neutralizing antibody measured by plaque reduction neutralization test (PRNT(50) titer): high (∼200), medium (∼40-50), low (∼20) and negative (<10). Pooled sera from JE-VAX(®) vaccinated subjects (PRNT(50) titer∼55) and pooled JEV antibody negative pre-vaccination sera were used as controls. Groups of ten 6- to 7-week-old female ICR mice were injected intraperitoneally with 0.5 ml of each serum pool diluted 1:2 or 1:10, challenged approximately 18 h later with a lethal dose of either JEV strain SA14 (genotype III) or strain KE-093 (genotype I) and observed for 21 days. All mice in the non-immune serum groups developed clinical signs consistent with JEV infection or died, whereas high titer sera from both IXIARO and JE-VAX(®) sera protected 90-100% of the animals. Statistical tests showed similar protection against both JEV strains SA14 and KE-093 and protection correlated with the anti-JEV antibody titer of IXIARO sera as measured by PRNT(50). Ex vivo neutralizing antibody titers showed that almost all mice with a titer of 10 or greater were fully protected. In a separate study, analysis of geometric mean titers (GMTs) of the groups of mice vaccinated with different doses of IXIARO and challenged with JEV SA14 provided additional evidence that titers≥10 were protective.

Long term immunity following a booster dose of the inactivated Japanese Encephalitis vaccine IXIARO®, IC51.

INTRODUCTION: IXIARO (IC51), a recently approved inactivated Japanese Encephalitis vaccine, is immunogenic and safe in a 0/28 days primary immunization schedule. Neutralizing antibody titers decline with time and booster doses are likely needed to enhance persistence of immunity. OBJECTIVES: To assess the effect of a booster dose on neutralizing JE antibody titers for up to 12 months after boostering. METHODS: In this phase III trial, 198 subjects, who had received primary immunization in a preceding randomized trial, were boosted with IXIARO 15 months after the primary immunization. Neutralizing antibody titers were assessed by plaque-reduction neutralisation test, PRNT. RESULTS: Prior to the booster dose, 69.2% (137/198) of subjects had PRNT50 titers ≥ 1:10. One month after the booster, the rate of subjects with PRNT50 ≥ 1:10 (recognized as a protective titer) was 100%. This rate remained high at 98.5% at 6 and 12 months; GMTs were 22.5 before the booster and 900, 487 and 361 at 1, 6 and 12 months after the booster, respectively. CONCLUSION: A booster dose of IXIARO at 15 months after primary immunization was highly immunogenic with GMTs >5-fold higher than those seen immediately after primary immunization, and remained at high levels for at least 12 months after the booster.

Evaluation of serological diagnostic test systems assessing the immune response to Japanese encephalitis vaccination.

A new commercial anti-Japanese encephalitis virus IgM and IgG indirect immunofluorescence test (IIFT) was evaluated for the detection of the humoral immune response after Japanese encephalitis vaccination. The IgM IIFT was compared to two IgM capture ELISAs and the IgG IIFT was analysed in comparison to a plaque reduction neutralization test (PRNT50) and an IgG ELISA. Moreover, the course of the immune reaction after vaccination with an inactivated JEV vaccine was examined. For the present study 300 serum samples from different blood withdrawals from 100 persons vaccinated against Japanese encephalitis were used. For the IgM evaluation, altogether 78 PRNT50 positive samples taken 7 to 56 days after vaccination and 78 PRNT50 negative sera were analyzed with the Euroimmun anti-JEV IgM IIFT, the Panbio Japanese Encephalitis - Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA. For the IgG evaluation, 100 sera taken 56 days after vaccination and 100 corresponding sera taken before vaccination were tested in the PRNT50, the Euroimmun anti-JEV IgG IIFT, and the InBios JE Detect IgG ELISA. The Euroimmun IgM IIFT showed in comparison to the Panbio ELISA a specificity of 95% and a sensitivity of 86%. With respect to the InBios ELISA, the values were 100% and 83.9%, respectively. The analysis of the Euroimmun IgG IIFT performance and the PRNT50 results demonstrated a specificity of 100% and a sensitivity of 93.8%, whereas it was not possible to detect more than 6.6% of the PRNT50 positive sera as positive with the InBios JE Detect IgG ELISA. Thus, the IIFT is a valuable alternative to the established methods in detecting anti-JEV antibodies after vaccination in travellers and it might prove useful for the diagnosis of acutely infected persons.

Immunogenicity and safety of different injection routes and schedules of IC41, a Hepatitis C virus (HCV) peptide vaccine.

BACKGROUND: An effective vaccine would be a significant progress in the management of chronic HCV infections. This study was designed to examine whether different application schedules and injection routes may enhance the immunogenicity of the HCV peptide vaccine IC41. METHODS: In this randomized trial 54 healthy subjects received either subcutaneous (s.c.) or intradermal (i.d.) vaccinations weekly (16 injections) or every other week (8 injections). One group additionally received imiquimod, an activator of the toll-like receptor (TLR) 7. The T cell epitope-specific immune response to IC41 was assessed using [(3)H]-thymidine CD4+ T cell proliferation, interferon-gamma (IFN-gamma) CD8+ and CD4+ ELIspot and HLA-A*0201 fluorescence-activated cell sorting (FACS) tetramer-binding assays. RESULTS: More than 60% of vaccinees responded in the CD4+ T cell proliferation assay in all groups. An HLA-A*0201 FACS tetramer-binding assay and IFN-gamma CD8+ ELIspot class I response of more than 70% was induced in four and three groups, respectively. IC41 induced significant immunological responses in all groups with responder rates of up to 100%. Interestingly, topical imiquimod was not able to enhance immunogenicity but was associated with a lower immune response. Local injection site reactions were mostly transient. Intradermal injections caused more pronounced reactions compared to s.c., especially erythema and edema. CONCLUSION: Compared to a previous study intensified dosing and/or i.d. injections enhanced the response rates to the vaccine IC41 in three assays measuring T cell function. Immunization with IC41 was generally safe in this study. These results justify testing IC41 in further clinical trials with HCV-infected individuals.

Therapeutic vaccine IC41 as late add-on to standard treatment in patients with chronic hepatitis C.

We examined the effect of the hepatitis C virus (HCV) peptide vaccine IC41 on HCV-specific T-cell responses and virological relapse rates in patients with chronic HCV genotype 1 infection when added to pegylated interferon plus ribavirin standard therapy. 35 patients received 6 vaccinations with IC41 from weeks 28 to 48 of standard antiviral treatment and were followed-up for another 6 months. IC41 vaccination did not prevent HCV-RNA relapse in patients with ongoing interferon standard treatment but HCV-specific T-cell responses were inducible and were associated with lower relapse rates. An increase of HCV-specific T-cell responses occurred in 73% of patients, responses were more frequent and stronger in patients with sustained virologic response than in patients who relapsed. Optimized vaccine responses may enhance sustained virologic response rates obtained with standard treatment of chronic hepatitis C.

Comparison of a single, high-dose vaccination regimen to the standard regimen for the investigational Japanese encephalitis vaccine, IC51: a randomized, observer-blind, controlled Phase 3 study.

The standard administration of the investigational Japanese encephalitis vaccine IC51 is 2 doses of 6 microg with a 28-day interval. This study investigated the immunogenicity of a single-immunization, high-dose regimen (1 x 12 microg) compared to the 2-injection, standard regimen to determine the immune response that one, high-dose injection can confer. The single, high-dose regimen resulted in about 60% seroconversion rate (SCR) at 10 days after administration, but it did not reach the almost 100% SCR achieved by the 2-dose standard administration at Day 35. The standard regimen conferred essentially 100% seroconversion already 7 days after the second immunization.

Analysis of the human cytomegalovirus pp65-directed T-cell response in healthy HLA-A2-positive individuals.

Human cytomegalovirus (HCMV) is contained by T-lymphocyte responses focused towards the major tegument protein pp65. To systematically identify T-cell epitopes, we applied the following strategy: 441 overlapping 15mer peptides spanning the entire HCMV pp65 antigen in 1-aa steps were screened in enzyme-linked immunospot (ELispot) assays for interferon gamma (IFN-gamma) secretion by peripheral blood mononuclear cells (PBMCs) from nine healthy HCMV-seropositive subjects expressing human leukocyte antigen (HLA)-A2. This analysis confirmed a number of previously known epitopes and revealed several new ones. A total of 26 epitopes were identified, including 14 HLA-A2, four HLA-B7, -B35, -812 and -B44 restricted class I epitopes, six class II epitopes, and two epitopes of unknown restriction. Three novel HLA-A2 epitopes were confirmed using T2-cells, and one peptide for which only binding data had been published so far was verified. Two novel class II epitopes were confirmed by intracellular cytokine staining. Responses were usually oligoclonal against up to seven HLA-A2 epitopes, albeit with a few dominating epitopes. Clusters of overlapping epitopes (hot-spots) were identified. These and the newly identified T-cell epitopes may be of great value for epitope-based immunotherapeutic approaches, including peptide vaccines.

Effect of pre-existing anti-tick-borne encephalitis virus immunity on neutralising antibody response to the Vero cell-derived, inactivated Japanese encephalitis virus vaccine candidate IC51.

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia with a case fatality rate up to 35% and long-term sequelae up to 75%. This active-controlled, randomized, multi-centre, observer-blind, phase III trial investigated the neutralising antibody response to the new Japanese encephalitis (JE) vaccine IC51 in subjects with (N=81) and without (N=339) pre-existing tick-borne encephalitis (TBE) vaccine induced antibodies as determined by TBE enzyme-linked immunosorbent assay IgG (ELISA). Neutralising antibody response was statistically superior in TBE ELISA-positive subjects compared to TBE ELISA-negative subjects after the first (p<0.0001) but not after the second vaccination with IC51. Thus, pre-existing vaccine-induced TBE immunity enhances the neutralising JEV-specific antibody response after a single IC51 vaccination.

Therapeutic vaccination of chronic hepatitis C nonresponder patients with the peptide vaccine IC41.

BACKGROUND & AIMS: IC41 is a synthetic peptide vaccine containing 7 relevant hepatitis C virus (HCV) T-cell epitopes and the T helper cell (Th)1/Tc1 adjuvant poly-L-arginine. IC41 has been shown to be safe and to induce HCV-specific interferon (IFN)-gamma-secreting CD4+ and CD8+ T cells in healthy volunteers. We aimed to investigate whether IC41 is able to induce HCV-specific T-cell responses also in chronic hepatitis C patients. METHODS: Sixty HLA-A2-positive chronic HCV patients not responding to or relapsing from standard therapy were randomized in a double-blind phase II study into 5 groups to receive 6 vaccinations of IC41 (3 different dose groups), HCV peptides alone, or poly-L-arginine alone. RESULTS: IC41 was well tolerated, and no drug-related serious adverse events or induction of hepatitis were observed. T-cell proliferation was recorded in up to 67% of patients in the 3 IC41 vaccine groups but only in 17% of patients treated with peptides alone. IFN-gamma enzyme-linked immunospot assay responses were observed exclusively in the IC41 groups with response rates up to 42%. There were 3 RNA responders with transient >1-log declines of HCV serum RNA associated with the strongest IFN-gamma enzyme-linked immunospot assay values within all 60 patients. CONCLUSIONS: This study showed that the HCV peptide vaccine IC41 can induce HCV-specific Th1/Tc1 responses in a subset of difficult to treat HCV nonresponder patients despite persisting viremia. However, changes in HCV RNA occurred only in single patients. Because strongest T-cell responses were associated with HCV RNA decline, further studies with optimized vaccine regimens and combination therapies have been initiated.

Functional and phenotypic characterization of peptide-vaccine-induced HCV-specific CD8+ T cells in healthy individuals and chronic hepatitis C patients.

Only very limited information on phenotype and function of vaccine-induced CD8+ T cells is available for humans. We investigated hepatitis C virus-specific CD8+ T cells after vaccination with the HCV peptide-vaccine IC41 which includes 5 MHC-class I and 3 MHC class-II-restricted epitopes. In healthy subjects, IC41 induced both HCV-specific central memory as well as effector CD8+ T cells which rapidly expanded upon antigen exposure in vitro. IFNgamma production was dependent on formulation of the synthetic peptides with the adjuvant poly-l-arginine. In chronic HCV patients, the frequency of HCV-specific CD8+ T cells increased after vaccination with a decline of CD45RA-positive effector memory cells in some but not all patients. Thus, this study suggests that HCV-specific memory cells can be induced by peptide vaccination and that a reversion of functional impaired phenotypes by therapeutic vaccination is possible in chronic HCV infection.

Experimental discovery of T-cell epitopes: combining the best of classical and contemporary approaches.

T cells specifically recognize antigens as peptide epitope-MHC complexes on the surface of target cells. The inherent complexities of antigen processing and presentation, the polygenic and polymorphic nature of MHC and the technical hurdles in working with T cells have made epitope discovery challenging. Here, we review significant experimental advances in recent years. These include new and sensitive assays and the availability of human cells and high numbers of synthetic peptides for screening, which have allowed for the first time comprehensive analysis of antigens and whole virus genomes. Such studies have provided important insights into the immunobiology of a number of diseases. The newly gathered detailed information on T-cell epitopes will aid vaccine design and immunological monitoring in clinical trials.

Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.

As interferon/ribavirin-based standard therapy is curative in only about half of HCV patients, there remains an important need for alternatives including vaccines. The novel peptide vaccine IC41 consists of five synthetic peptides harboring HCV T cell epitopes and poly-L-arginine as synthetic adjuvant. In this randomized, placebo-controlled trial, 128 HLA-A2 positive healthy volunteers received four s.c. vaccinations of seven different doses IC41, HCV peptides alone, poly-l-arginine alone or saline solution, every 4 weeks. IC41 was safe and well tolerated. Mild to moderate local reactions were transient. Immunogenicity was assessed using T cell epitope specific [3H]-thymidine proliferation, IFN-gamma ELIspot and HLA-tetramer assays. IC41 induced responses in all dose groups. Higher responder rates were recorded in higher dose groups and increasing number of vaccinations were associated with higher responder rates and more robust responses. Poly-L-arginine was required for the aimed-for Th1/Tc1-type immunity (IFN-gamma secreting T cells).

Proteomics approaches towards antigen discovery and vaccine development.

Vaccines have proved to be powerful medical interventions, and recent advances in immunology and in microbial pathogen genomics now allow the rational design of molecularly defined vaccines. Classical proteomics and immunoproteomics approaches such as serological proteome analysis (SERPA) have already provided first vaccine candidate antigens. The same is true for approaches based on recombinant DNA technology, and in the future protein arrays may further accelerate this kind of research. Taken together, these technologies will allow identification of the full set of antigens (ie, the immunome) targeted by the immune system in a certain pathological situation. The comparison of multiple immunomes may allow the discovery of immunogenic structural features shared and conserved between different pathogens, which could form the basis of broadly protective vaccines. In this review, recent proteomics and other post-genomics approaches for the identification of antigens in infectious disease and cancer, as well as strategies for their characterization, are discussed.

Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis.

In this study we applied serological proteome analysis (Klade, C. S. et al. Proteomics 2001, 1, 890-898) for identification of bacterial vaccine candidate antigens. First, approximately one hundred sera from healthy individuals and patients suffering from Staphylococcus aureus infections were screened for antibodies against staphylococcal lysates and recombinant proteins representing surface antigens. Two pools (healthy donors, patients) each consisting of five sera with the highest antiproteinaceous IgG reactivity were selected. Second, S. aureus COL was grown under different conditions and the number of antigens expressed was monitored by Western blot analysis. Third, surface proteins were enriched by digesting the bacterial cell wall under isotonic conditions and subsequent removal of protoplasts. These protein preparations were resolved by two-dimensional electrophoresis (2-DE) (pI 4-7). 2-DE immunoblotting using the preselected serum pools at 1:10 000-1:100 000 dilutions revealed a number of highly immunogenic staphylococcal proteins. Twenty-one spots were isolated by preparative 2-DE, and analysed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. This led to the identification of 15 proteins including known and novel vaccine candidates. Seroreactivity of several antigens including serine-aspartate repeat containing protein D, immuno-dominant staphylococcal antigen and a novel 309 amino acid lipoprotein was independently confirmed by enzyme-linked immunosorbent assay and Western blot analysis of purified recombinant proteins. In conclusion, serological proteome analysis proved to be a powerful tool for the identification of novel staphylococcal antigens, which provide a basis for rational vaccine design.

Identification and characterization of 9D7, a novel human protein overexpressed in renal cell carcinoma.

With the objective of discovering novel tumor-associated antigens of the cancer/testis type, we compared the transcriptional profiles of renal cell carcinoma (RCC) and non-tumorous kidney and further screened for genes expressed in RCC and testis, but not other normal tissues. In a first step, a representational difference analysis library consisting of approximately 1,900 RCC cDNA clones was generated. Clones were then spotted onto filters and hybridized with cDNA probes derived from a testis-specific cDNA library, a pool of RCCs and a pool of 10 healthy normal tissues, respectively. Based on strong hybridization signals with both RCC and testis, but not normal tissue probes, 185 clones were sequenced and annotated. After EST-database comparison, 35 clones were selected for experimental analysis, including conventional and quantitative RT-PCR as well as Northern blotting. Clone 9D7 showed strong mRNA expression in RCC as well as in several other major tumor types. In normal tissues there was little or no mRNA expression with the exception of heart. 9D7 was cloned to full-size and found to represent a novel human gene containing 5 exons residing on chromosome 14. Alternative splicing within exon 1 generates 2 open-reading-frames consisting of 717 or 435 bp corresponding to predicted proteins of 239 or 145 amino acids. 9D7 shows high homology (227/239 amino acids or 95% identity) to a growth factor-inducible gene of Rattus norvegicus involved in apoptosis. In situ hybridization as well as immunohistochemical analysis using 9D7-specific antisera confirmed overexpression of 9D7 in RCCs as compared to normal kidney tissue.