Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine.

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.

Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

Thermostability of seven hepatitis C virus genotypes in vitro and in vivo.

Hepatitis C virus (HCV) is transmitted primarily through percutaneous exposure to contaminated blood especially in healthcare settings and among people who inject drugs. The environmental stability of HCV has been extrapolated from studies with the bovine viral diarrhoea virus or was so far only addressed with HCV genotype 2a viruses. The aim of this study was to compare the environmental and thermostability of all so far known seven HCV genotypes in vitro and in vivo. Incubation experiments at room temperature revealed that all HCV genotypes showed similar environmental stabilities in suspension with viral infectivity detectable for up to 28 days. The risk of HCV infection may not accurately be reflected by determination of HCV RNA levels. However, viral stability and transmission risks assessed from in vitro experiments correlated with viral infectivity in transgenic mice containing human liver xenografts. A reduced viral stability for up to 2 days was observed at 37 °C with comparable decays for all HCV genotypes confirmed by thermodynamic analysis. These results demonstrate that different HCV genotypes possess comparable stability in the environment and that noninfectious particles after incubation in vitro do not cause infection in an HCV in vivo model. These findings are important for estimation of HCV cross-transmission in the environment and indicate that different HCV genotypes do not display an altered stability or resistance at certain temperatures.

Metabolic studies with promagnon, methylclostebol and methasterone in the uPA+/+-SCID chimeric mice.

The chimeric uPA(+/+)-SCID mouse model, transplanted with human hepatocytes, was previously validated as an alternative tool to study in vivo the human steroid metabolism. This humanized mouse model was now applied, in the framework of anti-doping research, to test different nutritional supplements containing steroids. These steroids, intentionally or accidentally added to a nutritional supplement, usually are derivatives of testosterone. Information about the metabolism of these derivatives, which is important to assure their detection, is quite limited. However, due to ethical constraints, human volunteers cannot be used to perform experimental excretion studies. Therefore the chimeric mice were selected to perform three separated excretion studies with superdrol (methasterone), promagnon and also methylclostebol. The urine of the humanized mice was collected 24h after a single dose administration and analyzed by gas chromatography-mass spectrometry (GC-MS). The results indicated the presence of several metabolites including a 3-keto reduced metabolite and numerous hydroxylated metabolites. Also phase 2 metabolism was investigated to update the complete picture of their metabolism.

Development and validation of a quantitative gas chromatography-mass spectrometry method for the detection of endogenous androgens in mouse urine.

A quantitative method based on gas chromatography-mass spectrometry (GC-MS) has been developed for the detection of 16 endogenous androgens in the urine of mice. The substances are extracted from 100 microL urine with freshly distilled diethyl ether after alkalinisation. The substances are derivatised with a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide/NH(4)I/ethanethiol (383/1/2, v/w/v) and detected by GC-MS in the selected ion monitoring mode. The results of the method validation indicate good linearity, accuracy and precision, making the method suitable for the quantification of endogenous androgens in mouse urine. The selectivity of the method showed that no interfering peaks were observed at the retention times of the analytes. The method allows for the direct quantification and identification of testosterone and 15 other endogenous androgens at low concentrations (ng/mL) in mouse urine. The applicability of the method is shown by the analysis of a mouse urine. Several endogenous steroids could be detected.

The interferon gamma secretion assay: a reliable tool to study interferon gamma production at the single cell level.

Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracellular cytokine staining, cytokine secretion assay, etc.) have been analyzed. In this paper we present the data of a thorough validation of the IFNgamma Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNgamma is bound to the cell surface and is then stained as an artificial surface molecule and analyzed by flow-cytometry. The introduction of five quality criteria markedly improved the reproducibility of this assay and made it very reliable (intra-assay variability<5%; inter-assay variability<20%). Recovery experiments further demonstrated that almost 100% of IFNgamma(+) labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNgamma-production, we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNgamma-secretion patterns could be discerned. In Pattern 1 there is a predominant and almost equal contribution of T cells and NK cells with a minor contribution of CD3(+)CD56(+) and B cells. Pattern 2, which is most abundant, is characterized by a predominance of NK cells (60-70%). Pattern 3 differs from the previous one in its minor contribution of NK cells. Here T cells predominate the IFNgamma secretion. These results clearly demonstrate that the IFNgamma(+) subset distribution after VZAg-stimulation is not uniform and differs individually. Furthermore, the ISA-technology proves to be very useful in vaccine research. This was demonstrated by testing the IFNgamma(+) secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals.

A simple and rapid method to determine the zygosity of uPA-transgenic SCID mice.

Successful transplantation of xenogeneic hepatocytes into uPA-transgenic SCID mice depends on the zygosity of the recipient mice. Normally, the difference between homozygous and heterozygous animals is determined via a quantitative Southern blot. We sequenced a part of the mouse genome that is eliminated upon integration of the transgene in the genome. Based on that sequence we developed a multiplex PCR that allows the unambiguous discrimination of negative, heterozygous, and homozygous uPA-transgenic SCID mice in a single day procedure. The speed of the procedure is an essential quality because transplantation of xenogeneic hepatocytes into uPA-SCID mice should be done as soon as possible after birth.

In vivo inhibition of anti-hepatitis B virus core antigen (HBcAg) immunoglobulin G production by HBcAg-specific CD4(+) Th1-type T-cell clones in a hu-PBL-NOD/SCID mouse model.

Hepatitis B virus (HBV) core antigen (HBcAg)-specific CD4(+) T-cell responses are believed to play an important role in the control of human HBV infection. In the present study, HBcAg-specific, HLA-DR13*-restricted CD4(+) Th1-type T-cell clones were generated which secreted both gamma interferon and tumor necrosis factor alpha after in vitro antigen stimulation. These HBcAg-specific CD4(+) Th1-type T cells were able to lyse HBc peptide-loaded Epstein-Barr virus-transformed lymphoblastoid target cells in vitro. To examine whether these HLA-DR13*-restricted human CD4(+) Th1 T cells also display the same cytotoxic effects in vivo, we transferred peripheral blood leukocytes (PBL) derived from HBV-infected donors or an HBV-naïve donor sharing the DR13*, together with the HBcAg-specific CD4(+) Th1-type T cells and HBcAg, directly into the spleen of optimally conditioned Nod/LtSz-Prkdc(scid)/Prkdc(scid) (NOD/SCID) mice. The production of both secondary anti-HBc-immunoglobulin G (anti-HBc-IgG) and primary HBcAg-binding IgM in hu-PBL-NOD/SCID mice was drastically inhibited by HBcAg-specific CD4(+) Th1-type T cells. No inhibition was observed when CD4(+) Th1 cells and donor PBL did not share an HLA-DR13. These results suggest that HBcAg-specific CD4(+) Th1 T cells may be able to lyse HBcAg-binding, or -specific, B cells that have taken up and presented HBcAg in a class II-restricted manner. Thus, HBcAg-specific CD4(+) Th1-type T cells can modulate the function and exert a regulatory role in deleting HBcAg-binding, or -specific, human B cells in vivo, which may be of importance in controlling the infection.

Prevention of hepatitis B infections: vaccination and its limitations.

Infection with hepatitis B virus has become a vaccine-preventable disease. The recombinant hepatitis B vaccines available today are safe and immunogenic. In order for these vaccines to eradicate HBV a universal vaccination of neonates and/or children needs to be implemented. Major obstacles on the road to global hepatitis B vaccination are poverty and scarcity of human resources in those parts of the world who are most badly in need of these vaccines. Despite their proven immunogenicity hepatitis B vaccines are unable to induce an adequate immune response in 5-10% of healthy adults. The non-responsiveness of these subjects is a selective phenomenon and not the expression of a general immune deficiency. Studies that correlated the HLA haplotype of vaccine recipients with their anti-HBs response patterns has led to the identification of markers of good and non/poor response. Universal vaccination of neonates and children has elicited questions about the durability of antibody persistence and the need for booster doses later in life. The European Consensus Group on Hepatitis B Immunity has proposed a series of recommendations that are summarized in this review.

Murine IL-2 receptor beta chain blockade improves human leukocyte engraftment in SCID mice.

Severe combined immunodeficient (SCID) mice accept human xenografts and can act as a model for human immune functions. Murine natural killer cells (NK), however, represent an important barrier for the reconstitution of SCID mice with human peripheral blood leukocytes (Hu-PBL). We investigated the effect on Hu-PBL survival of pretreatment with TM-beta1, a rat monoclonal antibody for the mouse IL-2 receptor beta chain. TM-beta1 greatly improved the survival of Hu-PBL. Human lymphocytes, predominantly T cells, survived in the peritoneum and infiltrated spleen and lungs already 1 week after engraftment and liver and thymus from 2 weeks on. Secondary humoral responses were evaluated with Hu-PBL from a donor immune to hepatitis-B surface Ag (HBsAg) and tetanus toxoid (TT). TM-beta1 pretreatment enhanced the recall Ig response to HBsAg and did not affect the baseline anti-TT Ig production. In conclusion, TM-beta1 pretreatment of SCID mice significantly improves the survival and functionality of the Hu-PBL graft.

Evaluation of Glucocard Memory 2 and Accutrend sensor blood glucose meters.

The performance and practicability of 2 blood glucose meters (Glucocard Memory 2 and Accutrend sensor) were evaluated. Both glucose meters produced acceptably precise results in the hyper- and normoglycaemic concentration ranges. In the hypoglycaemic concentration range, the imprecision of Accutrend sensor was much higher than recommended by the American Diabetes Association. Within-run coefficients of variation for Glucocard Memory 2 were 6.3%, 3.9% and 2.4% at glucose concentrations of 1.7 mmol/l, 5.8 mmol/l and 11.7 mmol/l, respectively: for Accutrend sensor these were 15.2%, 5.0% and 1.2% at respective concentrations of 0.9 mmol/l, 4.2 mmol/l and 19.6 mmol/l. Between-day coefficients of variation for Glucocard Memory 2 were 4.8% and 3.5% at glucose concentrations of 3.9 mmol/l and 17.2 mmol/l, respectively and for Accutrend sensor they were 3.8% and 2.9% at glucose concentrations of 3.8 mmol/l and 18.7 mmol/l, respectively. Results were linear over a range of 1.6 mmol/l -29.7 mmol/l for Glucocard Memory 2 and 1.6 mmol/l -33.3 mmol/l for Accutrend sensor. Results of both blood glucose meters correlated closely with the hexokinase/glucose-6-phosphate dehydrogenase laboratory method. Ninety-eight percent of both Glucocard Memory 2 and Accutrend sensor results were within 20% of the comparison method values. Ninety-three percent of the Glucocard Memory 2 and 96% of the Accutrend sensor results were within 15% of the comparison method results. An inverse relation between the glucose readings and haematocrit values was observed for both blood glucose meters in the hyperglycaemic range and this effect was more pronounced for Accutrend sensor. In the normo- and hypoglycaemic ranges the effect was insignificant and absent, respectively. Minimum sample volume for Glucocard Memory 2 was 3 microliters and for Accutrend sensor it was 9 microliters. Lower sample volumes gave erroneous results. Presenting more than the required volume had no effect on results.

Bioprosthetic valve endocarditis caused by Neisseria elongata subspecies nitroreducens.

A new case of Neisseria elongata ssp. nitroreducens bacteremia and endocarditis in a 74-year-old woman who had undergone aortic valve replacement in 1992 is reported in detail. N. elongata ssp. nitroreducens differs from the other subspecies of N. elongata in the additional reduction of nitrate without gas formation. Like most Neisseria spp. except Neisseria meningitidis and Neisseria gonorrhoeae, this N. elongata ssp. nitroreducens is usually classified in the group of "non-pathogenic" Neisseria spp. This case report indicates that the presence of subspecies of this group is significant when isolated from normally sterile sites and can cause severe disease in susceptible individuals.