Marie-Louise Michel

Direct gene transfer in skeletal muscle : plasmid DNA-based immunization against the hepatitis B virus surface antigen

Direct gene transfer by intramuscular injection of plasmid DNA encoding an antigenic protein may be used for the purpose of immunization. DNA-based immunization may be of value for basic immunological research and vaccine development. Several factors influence the uptake and expression of plasmid DNA in skeletal muscle, which in turn influence the immune response to the expressed protein. Physical barriers and other factors may impede diffusion of the DNA within the muscle tissue or its entry into the muscle fibres. Although the efficiency of gene transfer in normal mouse muscle is low (< 100 fibres per injection site), a humoral response to the hepatitis B surface antigen (HBsAg) is obtained after expression of a transferred gene. Direct gene transfer is ten times more efficient in regenerating than in normal mouse muscle. DNA-based immunization in such regenerating muscles results in an earlier and stronger humoral response to HBsAg than is seen in normal mature muscle. A needleless jet injection system (Biojector) is able to deliver DNA into normal muscle in rats and rabbits such that a substantial immune response is obtained.

DNA-mediated immunization to hepatitis B surface antigen : longevity of primary response and effect of boost

Intramuscular (i.m.) injection of mice with plasmid DNA expression vectors containing all or part of the hepatitis B virus (HBV) gene encoding the envelope proteins induces a strong humoral response to the HBV surface antigen (HBsAg) which is sustained for up to 74 weeks without boost. After a single i.m. injection of 100 micrograms DNA, antibodies to HBsAg (anti-HBs) reach ELISA titers of 4 x 10(4) in C57BL/6 mice and 10(4) in BALB/c mice, or somewhat less in older mice. Although antibody levels induced by a single injection of DNA do not diminish significantly over time, they can be further increased 10-200-fold by boosting with a second injection of DNA or an injection of recombinant HBsAg protein. Prior injection of DNA does not affect the strength or timing of the boosting effect, suggesting that there is no immune response against the vector itself. Boosting with a second injection of DNA is possible even in BALB/c mice, which are known to have a strong cytotoxic T-lymphocyte response against an epitope on the major HBV envelope protein, indicating that possible destruction of newly transfected muscle fibers is not so quick and efficient as to abort the boosting effect. A single injection of DNA results in a stronger and longer lasting humoral response than does a single injection of recombinant protein.

Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers

Despite the availability of effective hepatitis B vaccines for many years, over 370 million people remain persistently infected with hepatitis B virus (HBV). Viral persistence is thought to be related to poor HBV‐specific T‐cell responses. A phase I clinical trial was performed in chronic HBV carriers to investigate whether HBV DNA vaccination could restore T‐cell responsiveness. Ten patients with chronic active hepatitis B nonresponder to approved treatments for HBV infection were given 4 intramuscular injections of 1 mg of a DNA vaccine encoding HBV envelope proteins. HBV‐specific T‐cell responses were assessed by proliferation, ELISpot assays, and tetramer staining. Secondary end points included safety and the monitoring of HBV viraemia and serological markers. Proliferative responses to hepatitis B surface antigen were detected in two patients after DNA injections. Few HBV‐specific interferon γ–secreting T cells were detectable before immunization, but the frequency of such responses was significantly increased by 3 DNA injections. Immunization was well tolerated. Serum HBV DNA levels decreased in 5 patients after 3 vaccine injections, and complete clearance was observed in 1 patient. In conclusion, this study provides evidence that HBV DNA vaccination is safe and immunologically effective. We demonstrate that DNA vaccination can specifically but transiently activate T‐cell responses in some chronic HBV carriers who do not respond to current antiviral therapies. Supplementary material for this article can be found on the HEPATOLOGYwebsite (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:874–882.)

Multiepitopic HLA-A*0201-Restricted Immune Response Against Hepatitis B Surface Antigen After DNA-Based Immunization

CTL together with anti-envelope Abs represent major effectors for viral clearance during hepatitis B virus (HBV) infection. The induction of strong cytotoxic and Ab responses against the envelope proteins after DNA-based immunization has been proposed as a promising therapeutic approach to mediate viral clearance in chronically infected patients. Here, we studied the CTL responses against previously described hepatitis B surface Ag (HBsAg)-HLA-A*0201-restricted epitopes after DNA-based immunization in HLA-A*0201 transgenic mice. The animal model used was Human Human Db (HHD) mice, which are deficient for mouse MHC class I molecules (β2-microglobulin−/− Db−/−) and transgenic for a chimeric HLA-A*0201/Db molecule covalently bound to the human β2-microglobulin (HHD+/+). Immunization of these mice with a DNA vector encoding the small and the middle HBV envelope proteins carrying HBsAg induced CTL responses against several epitopes in each animal. This study performed on a large number of animals described dominant epitopes with specific CTL induced in all animals and others with a weaker frequency of recognition. These results confirmed the relevance of the HHD transgenic mouse model in the assessment of vaccine constructs for human use. Moreover, genetic immunization of HLA-A2 transgenic mice generates IFN-γ-secreting CD8+ T lymphocytes specific for endogenously processed peptides and with recognition specificities similar to those described during self-limited infection in humans. This suggests that responses induced by DNA immunization could have the same immune potential as those developing during natural HBV infection in human patients.

DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice

Hepatitis B virus (HBV) remains a serious worldwide health problem and the possibility to control it will depend on the availability of safe, effective and affordable vaccines. Recombinant protein or plasma-derived vaccines containing HBV surface antigen (HBsAg) are safe and generally efficacious, however, they are too expensive for widespread use in areas of HBV endemicity and are only partially effective for treatment of HBV chronic carriers. Immunization of mice by injection of HBsAg-expressing plasmid DNA results in rapid induction of strong and long-lasting humoral and cell-mediated immune responses. Here we report optimization of the humoral response with the use of necrotizing agents, co-expression of cytokines or co-stimulatory molecules and formulation of the DNA with cationic liposomes. DNA-based immunization of HBsAg-transgenic mice can also overcome non-response to HBsAg. Thus, DNA vaccines against HBV may be useful for both prophylactic and therapeutic purposes.

Use of Plasmid DNA for Direct Gene Transfer and Immunization

Direct gene transfer by intramuscular injection of plasmid DNA encoding an antigenic protein may be used for the purpose of immunization. Several factors influence the uptake and expression of plasmid DNA in skeletal muscle, which in turn influence the immune response to the expressed protein. Physical barriers and other factors may impede the diffusion of the DNA within the muscle tissue or its entry into the muscle fibers. Although the efficiency of gene transfer in normal mouse muscle is low (< 100 fibers per injection site), both humoral and cell-mediated immune responses to the hepatitis B surface antigen (HBsAg) are obtained after the expression of a transferred gene, and these are dose dependent. The efficacy of the immune response can be improved by injection of the DNA in or following pretreatment with a hypertonic solution or with the local anesthetic bupivacaine, and even more so by injecting the DNA into regenerating muscle.

Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site

ABSTRACT A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.

Changes to the natural killer cell repertoire after therapeutic hepatitis B DNA vaccination.

Background Improvements to the outcome of adaptive immune responses could be achieved by inducing specific natural killer (NK) cell subsets which can cooperate with dendritic cells to select efficient T cell responses. We previously reported the induction or reactivation of T cell responses in chronic hepatitis B patients vaccinated with a DNA encoding hepatitis B envelope proteins during a phase I clinical trial. Methodology/Principal Findings In this study, we examined changes in the peripheral NK cell populations occurring during this vaccine trial using flow cytometry analysis. Despite a constant number of NK cells in the periphery, a significant increase in the CD56bright population was observed after each vaccination and during the follow up. Among the 13 different NK cell markers studied by flow cytometry analysis, the expression of CD244 and NKG2D increased significantly in the CD56bright NK population. The ex vivo CD107a expression by CD56bright NK cells progressively increased in the vaccinated patients to reach levels that were significantly higher compared to chronically HBV-infected controls. Furthermore, modifications to the percentage of the CD56bright NK cell population were correlated with HBV-specific T cell responses detected by the ELISPOT assay. Conclusions/Significance These changes in the CD56bright population may suggest a NK helper effect on T cell adaptive responses. Activation of the innate and adaptive arms of the immune system by DNA immunization may be of particular importance to the efficacy of therapeutic interventions in a context of chronic infections. Trial Registration ClinicalTrials.gov NCT00988767

Hepatitis B Virus Splice-Generated Protein Induces T-Cell Responses in HLA-Transgenic Mice and Hepatitis B Virus-Infected Patients

ABSTRACT Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8+ and CD4+ human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression.

Immunization with a plasmid encoding a modified hepatitis B surface antigen carrying the receptor binding site for hepatocytes

Intramuscular injection of a plasmid encoding a modified hepatitis B surface antigen (HBsAg) induced humoral and cytotoxic responses in C57BL/6 mice. This modified HBsAg contains a preS1-derived peptide (amino acids 21 to 47), that carries the HBV receptor binding site for hepatocytes fused to the C-terminus of the small protein (at the position of amino acid 223). After a single DNA injection, the immunized mice elicited high titers of anti-HBs and anti-preS1 antibodies, and produced strong HBV specific cytotoxic T-lymphocyte (CTL) responses. The immune response induced after a single injection of this modified HBsAg gene in HBsAg-transgenic mice resulted in the clearance of circulating HBsAg and the appearance of anti-HBs and anti-preS1 antibodies. The high titers of preS1 antibody in transgenic mice were comparable to those found in non-transgenic controls and may be efficient to clear Dane particles existing in sera from chronic carriers. These data indicated that a genetic vaccine consisting of this modified HBsAg gene may have a potential use for both prophylactic and therapeutic purposes.