icon-folder.gif   Conference Reports for NATAP  
  American Association for the Study of Liver Diseases 2003 Conference
Boston, MA
Oct 24-28, 2003
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  HCV Vaccines: early research
  Reported by Jules Levin
Geert Leroux-Roels and colleagues from Innogenetics NV, Ghent, Belgium reported on research examining the tolerability and immunogenicity of a candidate HCV vaccine, based on the envelope 1 (E1) protein of the hepatitis C virus (HCV) when administered as three sub-epidermal injections.
In a phase I/II, prospective, open label, single center, exploratory study, 10 healthy male volunteers (group A), 3 men with a history of resolved HCV infection (group B), and 8 patients (3 men and 5 women) suffering from therapy-resistant chronic HCV (group C) were given sub-epidermal doses of non-adjuvanted E1 (4 µg E1 in 0.1 ml PBS per dose) at weeks 0,4, and 8. Safety evaluations were performed after each dose.
The humoral immune response to E1 was assessed at weeks -3, 0, 4, 8, 10, and 12 by measuring the serum levels of anti-E1 Ab. The E1-specific cellular immune responses were monitored at weeks -3 and 10 by measuring the in vitro proliferative responses of PBMC stimulated with 0.03, 0.3, and 3 µg of E1. These lympoproliferative responses were measured by H-thymidine incorporation and expressed as stimulation indices (SI) with a SI ≥ 3 considered significant.
Local reactions at the injection site were seen in 14 of the 21 participants. Four out of 8 group C patients and 1 group B volunteer reported transient flu-like symptoms after injection. No other noteworthy side effects were observed. In group A, all 10 volunteers were negative for anti-E1 antibodies prior to vaccination and only three mounted a detectable anti-E1 antibody response by week 10. The subject with the highest anti-E1 titer (1203 mU/ml at week 10) was the only one with a SI≥3 at week 10.
In group B one subject had low-titer anti-E1 prior to vaccination. This subject mounted the highest anti-E1 response at week 10 (251 mU/ml) and also had a SI ≥3 at week 10. The other two subjects mounted an early anti-E1 antibody response after two vaccine doses but displayed no cellular response.
In group C 7 out of 8 patients had anti-E1 antibodies at baseline. At week 10 a rise in titer was observed in only 2 subjects. An E1-specific lymphoproliferative response was measured at week 10 in the patient with the highest antibody response (1058 mU/ml).
Sub-epidermal injection of three doses of 4 µg of non-adjuvanted E1 vaccine induces much weaker humoral and cellular immune responses in healthy subjects and chronic HCV patients than the intramuscular administration of 20 µg E1 formulated in alum (Leroux-Roels et al. Hepatology 2001;34(4Pt.2), 449A, abstract 1110, and Nevens et al. J. Hepatol 2003;38(Suppl.2), 17, abstract 47).
However, in three subjects who cleared HCV infection, subepidermal administration of this low dose of E1 induced rapid and clear anamnestic responses. These data demonstrate that E1-specific immune responses may be induced during resolving HCV infections and that memory (B and T) cells can be restimulated with suboptimal doses of E1 antigen. It is tempting to speculate that these E1-specific immune responses contribute to the clearance of the infection.
Noriyoshi Kuzushita and researchers at the Liver Research Center, RI Hospital and Brown Medical School, Providence, RI reported on findings into a potential HCV vaccine technique using dendtritic cells.
It is estimated that 3% of the world’s population is infected with hepatitis C virus (HCV). HCV infection contributes to the development of acute and chronic hepatitis, end-stage cirrhosis, and hepatocellular carcinoma. Combination therapy with interferon and ribavirin is the treatment method of choice for chronic HCV but is only modestly effective.
No vaccine is currently available. Individuals with self-limiting HCV infection exhibit strong, broad-based CD4+ and CD8+ T cell responses against nonstructural (NS) viral proteins. Dendritic cells (DCs) are professional antigen-presenting cells capable of eliciting a vigorous anti-viral response of naïve T cells. The administration of antigen-loaded DCs offers a potential approach inducing high level anti-HCV immunity.
The DC population in BALB/c mice was expanded by hydrodynamic delivery of naked DNA that encoded the secreted form of human fms-like tyrosine kinase 3 ligand (Flt3L). The CD11c-enriched DC population obtained from the spleen by positive selection with magnetic beads was transduced in vitro with recombinant NS5 protein (a.a.2054-2995) using a macromolecular-based protein delivery system. Transduction efficiency and cell-surface markers were analyzed by flow cytometry.
Mice were inoculated subcutaneously three times at 2-week intervals with DCs transduced or not transduced with NS5. Two and 10 weeks following the final inoculation, the cytolytic activity expressed by splenocytes was assessed in vitro using NS5-expressing myeloma cells as target cells in a conventional 51Cr-release assay. Additionally, the splenocytes obtained from vaccinated mice were co-cultured with NS5, and the quantities of IFN- and IL-4 present in the culture supernatants collected 48 hours later were determined by ELISA. The percent of cytokine producing cells were quantified by flow cytometric analyses.
An approximate 80% transduction efficiency of NS5 protein was consistently found in DCs using our method of transfer. The transduced DCs constituted a relatively mature population based upon the large percent of cells that expressed MHC class II and co-stimulatory molecules; a substantial portion of these cells expressed both CD11b and CD8.
Relative to mice inoculated with non-transduced DCs, splenocytes derived from mice immunized with NS5-transduced DCs exhibited 3- to 5-fold more NS5 specific CTL activity. These same cells exhibited elevated production of IFN-, but not IL-4, typical of a TH1 type response when cultured in the presence of NS5; IFN- production was completely suppressed by anti-CD4 mAb. Elevations in CTL activity and IFN- production were maintained for at least at 10 weeks post-vaccination.
These findings suggest that DCs transduced with NS5 protein represent a promising approach for vaccination and generation of high level CTL activity and TH1 type immune response during persistent HCV infection.
Stephan Gehring and colleagues from Liver Research Center, RI Hospital and Brown Medical School, Providence, RI reported on additional HCV vaccine research they are conducting.
Type 1 interferons (IFNs) are antiviral agents used in the treatment of chronic liver diseases associated with persistent Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) infection. IFNs are mainly known for their direct anti-viral and anti-cellular properties, but they may also promote a) NK cell activity, b) dendritic cell maturation, c) CD8+ CTLs activation and generation, and d) MHC class I and class II molecule expression.
Eradication of chronic HCV infection depends upon a broad based host cellular immune response to epitopes that reside on viral structural and non-structural proteins. Genetic immunization is known to stimulate such immune responses but activity is generally weak.
The aim of this study was to assess the capacity of type 1 IFNs to augment the cellular immune response to DNA vaccination against HCV core protein.
Six- to 8-week-old Balb/c mice were vaccinated with an HCV core-expressing plasmid. The effects of three types of IFNs were explored i) non-species-specific hybrid IFN A/D, ii) human PegIFN characterized by a long half-life, and iii) pORFmIFN, a plasmid that encodes mouse IFN.
We used a conventional chromium release assay to measure the cellular immune response of splenocytes to syngeneic HCV core expressing SP2O target cells and a tumor challenge model to estimate the generation of CTL activity in vivo.
We determined that the optimal concentration of hybrid IFN A/D was achieved in vivo by administering 1 x 104 U every other day during the period of vaccination. This resulted in 29.6% specific CTL activity compared to 7.4% activity when the HCV core expressing plasmid was administered without IFN A/D augmentation.
In a tumor challenge model, CD8 T cell-dependent suppression of tumor cell growth correlated with in vitro CTL activity implicating that these CTLs were highly effective in vivo. Further increasing the hybrid IFN A/D dose to 1 x 105 U led to a significant reduction in CTL activity. Similar dose-dependent findings were observed when human PegIFN was used.
Co-immunization with pORFmIFN (100 ug/injection) yielding very high level of murine IFN also resulted in a marked reduction in the CTL response to vaccination with the HCV core-expressing plasmid indicating that there is an optimal dose to facilitate genetic immunization.
Suprisingly, low doses of IFN A/D and human PegIFN augment the cellular immune response to DNA-based vaccination against HCV core protein. High doses, however, suppress the CTL response in a manner similar to that found in humans treated with IFN. These findings have important clinical implications with regards to antiviral responses generated by DNA-based vaccines.