Therapeutic vaccination with lentiviral vector in HBV-persistent mice and two inactive HBsAg carriers.

BACKGROUND & AIMS: Immunotherapy for chronic hepatitis B virus (HBV) infection has not yet demonstrated sufficient efficacy. We developed a non-integrative lentiviral-vectored therapeutic vaccine for chronic hepatitis B and tested its antiviral effects in HBV-persistent mice and two inactive HBsAg carriers. METHODS: Lentiviral vectors (LVs) encoding the core, preS1, or large HBsAg (LHBs) proteins of HBV were evaluated for immunogenicity in HBV-naïve mice and therapeutic efficacy in a murine model of chronic HBV infection. In addition, two inactive HBsAg carriers each received two doses of 5×107 transduction units (TU) or 1×108 TU of lentiviral-vectored LHBs (LV-LHBs), respectively. The endpoints were safety, LHBs-specific T-cell responses, and serum HBsAg levels during a 24-week follow-up. RESULTS: In the mouse models, LV-LHBs was the most promising in eliciting robust antigen-specific T cells and in reducing the levels of serum HBsAg and viral load. By the end of the 34-week observation period, six out of ten (60%) HBV-persistent mice vaccinated with LV-LHBs achieved serum HBsAg loss and significant depletion of HBV-positive hepatocytes in the liver. In the two inactive HBsAg carriers, vaccination with LV-LHBs induced a considerable increase in the number of peripheral LHBs-specific T cells in one patient, and a weak but detectable response in the other, accompanied by a sustained reduction of HBsAg (-0.31 log10 IU/ml and -0.46 log10 IU/ml, respectively) from baseline to nadir. CONCLUSIONS: A lentiviral-vectored therapeutic vaccine for chronic HBV infection demonstrated the potential to improve HBV-specific T-cell responses and deplete HBV-positive hepatocytes, leading to a sustained loss or reduction of serum HBsAg. IMPACT AND IMPLICATIONS: Chronic HBV infection is characterized by an extremely low number and profound hypo-responsiveness of HBV-specific T cells. Therapeutic vaccines are designed to improve HBV-specific T-cell responses. We show that immunization with a lentiviral-vectored therapeutic HBV vaccine was able to expand HBV-specific T cells in vivo, leading to reductions of HBV-positive hepatocytes and serum HBsAg.

A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection.

SARS-CoV-2 infection results in impaired interferon response in patients with severe COVID-19. However, how SARS-CoV-2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS-CoV-2-infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus-derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3'UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 can potentially employ a virus-derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon-mediated immune response.

An intranasal lentiviral booster reinforces the waning mRNA vaccine-induced SARS-CoV-2 immunity that it targets to lung mucosa.

As the coronavirus disease 2019 (COVID-19) pandemic continues and new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines starts waning and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal, humoral, and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization owing to its non-cytopathic, non-replicative, and scarcely inflammatory properties. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months after vaccination, were boosted intranasally with LV::SBeta-2P. A strong boost effect was detected on cross-sero-neutralizing activity and systemic T cell immunity. In addition, mucosal anti-spike IgG and IgA, lung-resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19. LV::SBeta-2P vaccination was also fully protective against Omicron infection of the lungs and central nervous system, in the highly susceptible B6.K18-hACE2IP-THV transgenic mice.

A Single Dose of NILV-Based Vaccine Provides Rapid and Durable Protection against Zika Virus.

Zika virus, a member of the Flaviviridae family, is primarily transmitted by infected Aedes species mosquitoes. In 2016, Zika infection emerged as a global health emergency for its explosive spread and the remarkable neurological defects in the developing fetus. Development of a safe and effective Zika vaccine remains a high priority owing to the risk of re-emergence and limited understanding of Zika virus epidemiology. We engineered a non-integrating lentiviralvector(NILV)-based Zika vaccine encoding the consensus pre-membrane and envelope glycoprotein of circulating Zika virus strains. We further evaluated the immunogenicity and protective efficacy of this vaccine in both immunocompromised and immunocompetent mouse models. A single immunization in both mouse models elicited a robust neutralizing antibody titer and afforded full protection against Zika challenge as early as 7 days post-immunization. This NILV-based vaccine also induced a long-lasting immunity when immunized mice were challenged 6 months after immunization. Altogether, our NILV Zika vaccine provides a rapid yet durable protection through a single dose of immunization without extra adjuvant formulation. Our data suggest a promising Zika vaccine candidate for an emergency situation, and demonstrate the capacity of lentiviral vector as an efficient vaccine delivery platform.

Human hematopoietic reconstitution and HLA-restricted responses in nonpermissive alymphoid mice.

We generated a new humanized mouse model to study HLA-restricted immune responses. For this purpose, we created unique murine hosts by enforcing the expression of human SIRPα by murine phagocytes in murine MHC-deficient HLA-transgenic alymphoid hosts, an approach that allowed the immune reconstitution of nonpermissive mice following injection of human hematopoietic stem cells. We showed that these mouse/human chimeras were able to generate HLA-restricted responses to immunization. These new humanized mice may offer attractive models to study immune responses to human diseases, such as HIV and EBV infections, as well as to assay new vaccine strategies.

TG1050, an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBV-persistent mice.

OBJECTIVE: To assess a new adenovirus-based immunotherapy as a novel treatment approach to chronic hepatitis B (CHB). METHODS: TG1050 is a non-replicative adenovirus serotype 5 encoding a unique large fusion protein composed of a truncated HBV Core, a modified HBV Polymerase and two HBV Envelope domains. We used a recently described HBV-persistent mouse model based on a recombinant adenovirus-associated virus encoding an over length genome of HBV that induces the chronic production of HBsAg, HBeAg and infectious HBV particles to assess the ability of TG1050 to induce functional T cells in face of a chronic status. RESULTS: In in vitro studies, TG1050 was shown to express the expected large polyprotein together with a dominant, smaller by-product. Following a single administration in mice, TG1050 induced robust, multispecific and long-lasting HBV-specific T cells detectable up to 1 year post-injection. These cells target all three encoded immunogens and display bifunctionality (i.e., capacity to produce both interferon γ and tumour necrosis factor α as well as cytolytic functions). In addition, control of circulating levels of HBV DNA and HBsAg was observed while alanine aminotransferase levels remain in the normal range. CONCLUSIONS: Injection of TG1050 induced both splenic and intrahepatic functional T cells producing cytokines and displaying cytolytic activity in HBV-naïve and HBV-persistent mouse models together with significant reduction of circulating viral parameters. These results warrant clinical evaluation of TG1050 in the treatment of CHB.

Adenoviral delivery of recombinant hepatitis B virus expressing foreign antigenic epitopes for immunotherapy of persistent viral infection.

UNLABELLED: We previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign-antigen recombinant HBV (rHBV) as a gene therapy vector. Targeted elimination of wild-type HBV (wtHBV)-infected cells could be achieved by functionally activating an in situ T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also feared that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious, as demonstrated in primary tupaia hepatocytes. These results greatly expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign-antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying the wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign-antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection. IMPORTANCE: Adenovirus-delivered rHBV activated a foreign-antigen-specific T-cell response that abrogated HBV persistence in a mouse model. Our study provides further evidence of the potential of foreign-antigen-based immunotherapy for the treatment of chronic HBV infection.

Therapeutic vaccines in treating chronic hepatitis B: the end of the beginning or the beginning of the end?

The antiviral treatment of chronic hepatitis B virus (HBV) infection has greatly improved over the last 20 years since it has allowed a disappearance of cirrhosis decompensation and a significant reduction of the incidence of hepatocellular carcinoma. However, a complete HBV cure has not been achieved, and alternative treatments are still needed to optimize the current treatments. Therapeutic vaccination is a promising new strategy for controlling persistent infections and tumors. However, this approach has not been as successful as initially anticipated for chronic hepatitis B. General impairment of the immune responses generated during persistent HBV infection, with exhausted T cells not responding correctly to therapeutic vaccination, is most likely responsible for the poor clinical responses observed to date. We describe here the past approaches of therapeutic vaccination, in the hope that useful lessons will emerge from these previous clinical trials. Intensive research efforts are now focusing on a better understanding of immune responses in liver, on mechanisms by which HBV escapes innate immunity and on an accurate selection of the patients susceptible to benefit of immune therapy, which could increase the efficacy of therapeutic vaccination.

Adeno-associated virus-mediated gene transfer leads to persistent hepatitis B virus replication in mice expressing HLA-A2 and HLA-DR1 molecules.

Hepatitis B virus (HBV) persistence may be due to impaired HBV-specific immune responses being unable to eliminate efficiently or cure infected hepatocytes. The immune mechanisms that lead to HBV persistence have not been completely identified, and no appropriate animal model is available for such studies. Therefore, we established a chronic HBV infection model in a mouse strain with human leukocyte antigen A2/DR1 (HLA-A2/DR1) transgenes and an H-2 class I/class II knockout. The liver of these mice was transduced with adeno-associated virus serotype 2/8 (AAV2/8) carrying a replication-competent HBV DNA genome. In all AAV2/8-transduced mice, hepatitis B virus surface antigen, hepatitis B virus e antigen, and HBV DNA persisted in serum for at least 1 year. Viral replication intermediates and transcripts were detected in the livers of the AAV-injected mice. The hepatitis B core antigen was expressed in 60% of hepatocytes. No significant inflammation was observed in the liver. This was linked to a higher number of regulatory T cells in liver than in controls and a defect in HBV-specific functional T-cell responses. Despite the substantial tolerance resulting from expression of HBV antigens in hepatocytes, we succeeded in priming functional HBV-specific T-cell responses in peripheral tissues, which subsequently reached the liver. This AAV2/8-HBV-transduced HLA-A2/DR1 murine model recapitulates virological and immunological characteristics of chronic HBV infection, and it could be useful for the development of new treatments and immune-based therapies or therapeutic vaccines for chronic HBV infections.

Lentiviral Vectors as a Vaccine Platform against Infectious Diseases.

Lentiviral vectors are among the most effective viral vectors for vaccination. In clear contrast to the reference adenoviral vectors, lentiviral vectors have a high potential for transducing dendritic cells in vivo. Within these cells, which are the most efficient at activating naive T cells, lentiviral vectors induce endogenous expression of transgenic antigens that directly access antigen presentation pathways without the need for external antigen capture or cross-presentation. Lentiviral vectors induce strong, robust, and long-lasting humoral, CD8+ T-cell immunity and effective protection against several infectious diseases. There is no pre-existing immunity to lentiviral vectors in the human population and the very low pro-inflammatory properties of these vectors pave the way for their use in mucosal vaccination. In this review, we have mainly summarized the immunological aspects of lentiviral vectors, their recent optimization to induce CD4+ T cells, and our recent data on lentiviral vector-based vaccination in preclinical models, including prophylaxis against flaviviruses, SARS-CoV-2, and Mycobacterium tuberculosis.

Preclinical proof of concept of a tetravalent lentiviral T-cell vaccine against dengue viruses.

Dengue virus (DENV) is responsible for approximately 100 million cases of dengue fever annually, including severe forms such as hemorrhagic dengue and dengue shock syndrome. Despite intensive vaccine research and development spanning several decades, a universally accepted and approved vaccine against dengue fever has not yet been developed. The major challenge associated with the development of such a vaccine is that it should induce simultaneous and equal protection against the four DENV serotypes, because past infection with one serotype may greatly increase the severity of secondary infection with a distinct serotype, a phenomenon known as antibody-dependent enhancement (ADE). Using a lentiviral vector platform that is particularly suitable for the induction of cellular immune responses, we designed a tetravalent T-cell vaccine candidate against DENV ("LV-DEN"). This vaccine candidate has a strong CD8+ T-cell immunogenicity against the targeted non-structural DENV proteins, without inducing antibody response against surface antigens. Evaluation of its protective potential in the preclinical flavivirus infection model, i.e., mice knockout for the receptor to the type I IFN, demonstrated its significant protective effect against four distinct DENV serotypes, based on reduced weight loss, viremia, and viral loads in peripheral organs of the challenged mice. These results provide proof of concept for the use of lentiviral vectors for the development of efficient polyvalent T-cell vaccine candidates against all DENV serotypes.

Full eradication of pre-clinical human papilloma virus-induced tumors by a lentiviral vaccine.

Human papillomavirus (HPV) infections are the cause of all cervical and numerous oropharyngeal and anogenital cancers. The currently available HPV vaccines, which induce neutralizing antibodies, have no therapeutic effect on established tumors. Here, we developed an immuno-oncotherapy against HPV-induced tumors based on a non-integrative lentiviral vector encoding detoxified forms of the Early E6 and E7 oncoproteins of HPV16 and 18 genotypes, namely, "Lenti-HPV-07". A single intramuscular injection of Lenti-HPV-07 into mice bearing established HPV-induced tumors resulted in complete tumor eradication in 100% of the animals and was also effective against lung metastases. This effect correlated with CD8+ T-cell induction and profound remodeling of the tumor microenvironment. In the intra-tumoral infiltrates of vaccinated mice, the presence of large amounts of activated effector, resident memory, and transcription factor T cell factor-1 (TCF-1)+ "stem-like" CD8+ T cells was associated with full tumor eradication. The Lenti-HPV-07-induced immunity was long-lasting and prevented tumor growth after a late re-challenge, mimicking tumor relapse. Lenti-HPV-07 therapy synergizes with an anti-checkpoint inhibitory treatment and therefore shows promise as an immuno-oncotherapy against established HPV-mediated malignancies.

Mice humanized for MHC and hACE2 with high permissiveness to SARS-CoV-2 omicron replication.

Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike. The murine ACE2 does not interact efficiently with SARS-CoV-2 Spike and therefore the laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in "HHD-DR1" background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. These new hACE2 transgenic strains display high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants, while the previously available B6.K18-ACE22Prlmn/JAX mice have been reported to be poorly susceptible to infection with Omicron. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral-based COVID-19 vaccine.

Plasmid vector-linked maturation of natural killer (NK) cells is coupled to antigen-dependent NK cell activation during DNA-based immunization in mice.

Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1(+) CD27(-) NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1(+) CD27(+) NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity.

Full-Lung Prophylaxis against SARS-CoV-2 by One-Shot or Booster Intranasal Lentiviral Vaccination in Syrian Golden Hamsters.

Following the breakthrough of numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in recent months and the incomplete efficiency of the currently available vaccines, development of more effective vaccines is desirable. Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models and are particularly suitable for mucosal vaccination, which is acknowledged as the most effective in reducing viral transmission. Here, we demonstrate that a single intranasal administration of a vaccinal lentiviral vector encoding a stabilized form of the original SARS-CoV-2 Spike glycoprotein induces full-lung protection of respiratory tracts and strongly reduces pulmonary inflammation in the susceptible Syrian golden hamster model against the prototype SARS-CoV-2. In addition, we show that a lentiviral vector encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P) prevents pathology and reduces infectious viral loads in lungs and nasal turbinates following inoculation with the SARS-CoV-2 Omicron variant. Importantly, an intranasal boost with LV::SBeta-2P improves cross-seroneutralization much better in LV::SBeta-2P-primed hamsters than in their counterparts primed with an LV-encoding Spike from the ancestral SARS-CoV-2. These results strongly suggest that an immune imprint with the original Spike sequence has a negative impact on cross-protection against new variants. Our results tackle the issue of vaccine effectiveness in people who have already been vaccinated and have vanished immunity and indicate the efficiency of LV-based intranasal vaccination, either as a single dose or as booster.

Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: perspectives and challenges.

The treatment of chronic hepatitis B virus (HBV) infection has greatly improved over the last 10 years, but alternative treatments are still needed. Therapeutic vaccination is a promising new strategy for controlling chronic infection. However, this approach has not been as successful as initially anticipated for chronic hepatitis B. General impairment of the immune responses generated during persistent HBV infection, with exhausted T cells not responding correctly to therapeutic vaccination, is probably responsible for the poor clinical responses observed to date. Intensive research efforts are now focusing on increasing the efficacy of therapeutic vaccination without causing liver disease. Here we describe new approaches to use with therapeutic vaccination, in order to overcome the inhibitory mechanisms impairing immune responses. We also describe innovative strategies for generating functional immune responses and inducing sustained control of this persistent infection.

Lentiviral vector induces high-quality memory T cells via dendritic cells transduction.

We report a lentiviral vector harboring the human ß2-microglobulin promoter, with predominant expression in immune cells and minimal proximal enhancers to improve vector safety. This lentiviral vector efficiently transduces major dendritic cell subsets in vivo. With a mycobacterial immunogen, we observed distinct functional signatures and memory phenotype in lentiviral vector- or Adenovirus type 5 (Ad5)-immunized mice, despite comparable antigen-specific CD8+ T cell magnitudes. Compared to Ad5, lentiviral vector immunization resulted in higher multifunctional and IL-2-producing CD8+ T cells. Furthermore, lentiviral vector immunization primed CD8+ T cells towards central memory phenotype, while Ad5 immunization favored effector memory phenotype. Studies using HIV antigens in outbred rats demonstrated additional clear-cut evidence for an immunogenic advantage of lentiviral vector over Ad5. Additionally, lentiviral vector provided enhance therapeutic anti-tumor protection than Ad5. In conclusion, coupling lentiviral vector with ß2-microglobulin promoter represents a promising approach to produce long-lasting, high-quality cellular immunity for vaccinal purposes.

Brain cross-protection against SARS-CoV-2 variants by a lentiviral vaccine in new transgenic mice.

COVID-19 vaccines already in use or in clinical development may have reduced efficacy against emerging SARS-CoV-2 variants. In addition, although the neurotropism of SARS-CoV-2 is well established, the vaccine strategies currently developed have not taken into account protection of the central nervous system. Here, we generated a transgenic mouse strain expressing the human angiotensin-converting enzyme 2, and displaying unprecedented brain permissiveness to SARS-CoV-2 replication, in addition to high permissiveness levels in the lung. Using this stringent transgenic model, we demonstrated that a non-integrative lentiviral vector, encoding for the spike glycoprotein of the ancestral SARS-CoV-2, used in intramuscular prime and intranasal boost elicits sterilizing protection of lung and brain against both the ancestral virus, and the Gamma (P.1) variant of concern, which carries multiple vaccine escape mutations. Beyond induction of strong neutralizing antibodies, the mechanism underlying this broad protection spectrum involves a robust protective T-cell immunity, unaffected by the recent mutations accumulated in the emerging SARS-CoV-2 variants.

Intranasal vaccination with a lentiviral vector protects against SARS-CoV-2 in preclinical animal models.

To develop a vaccine candidate against coronavirus disease 2019 (COVID-19), we generated a lentiviral vector (LV) eliciting neutralizing antibodies against the Spike glycoprotein of SARS-CoV-2. Systemic vaccination by this vector in mice, in which the expression of the SARS-CoV-2 receptor hACE2 has been induced by transduction of respiratory tract cells by an adenoviral vector, confers only partial protection despite high levels of serum neutralizing activity. However, eliciting an immune response in the respiratory tract through an intranasal boost results in a >3 log10 decrease in the lung viral loads and reduces local inflammation. Moreover, both integrative and non-integrative LV platforms display strong vaccine efficacy and inhibit lung deleterious injury in golden hamsters, which are naturally permissive to SARS-CoV-2 replication and closely mirror human COVID-19 physiopathology. Our results provide evidence of marked prophylactic effects of LV-based vaccination against SARS-CoV-2 and designate intranasal immunization as a powerful approach against COVID-19.

Hepatitis B virus as a gene delivery vector activating foreign antigenic T cell response that abrogates viral expression in mouse models.

UNLABELLED: Chronic hepatitis B virus (HBV) infection is characterized by functionally impaired T cell responses. To ensure active immunotherapy, the immune response must be switched from exhausted T cells to functional effectors that can attain the liver and cure the viral infection. We thus designed a recombinant HBV (rHBV) containing a modified viral core gene that specifically delivers a foreign antigenic polyepitope to the liver. This recombinant virus could only be self-maintained in hepatocytes already infected by HBV through capsid complementation. A strong foreign epitope-specific T cell response was first primed in the periphery by way of DNA immunization in human leukocyte antigen (HLA)-A2/DR1 transgenic mice. After the hydrodynamic (hyd.) injection of rHBV, expression of the foreign antigenic polyepitope in hepatocytes attracted/reactivated a vigorous T cell response in situ. Most liver-infiltrating CD8(+) T cells proved to be functional effectors. Following DNA priming and hyd. injection, the rHBV-based expression of hepatitis B surface antigen (HBsAg) in mouse liver was almost completely inhibited without causing major liver injury. Studies in HBsAg/HLA-A2/DR1 transgenic mice further validated our approach. CONCLUSION: For the first time, HBV was used as a gene delivery vector, which strongly triggered functional T cell response and subsequently controlled the viral expression in the liver of surrogate mouse models for HBV infection. It might represent an innovative and promising strategy of active immunotherapy during HBV persistent infection. This concept could even be more generally extended to other chronic viral diseases.