Previous studies have demonstrated the feasibility of implantation of human blood cells or tissues in lethally irradiated mice or rats, radioprotected with SCID mouse bone marrow cells: The Trimera system. In the present study, we describe the development of a mouse Trimera model for human hepatitis B virus (HBV) infection. In this model, viremia is induced by transplantation of ex vivo HBV-infected human liver fragments. Engraftment of the human liver fragments, evaluated by hematoxylin-eosin staining and human serum albumin mRNA expression, was observed in 85% of the transplanted animals 1 month postimplantation. Viremia levels were determined in these mice by measuring serum HBV DNA using polymerase chain reaction (PCR), followed by dot-blot hybridization. HBV DNA is first detected 8 days after liver transplantation. Viremia attains a peak between days 18 and 25 when HBV infection is observed in 85% of the transplanted animals. Hepatitis B virus (HBV) infection is a major public health problem affecting millions of people worldwide. 1 Following acute HBV infection, 5% to 10% of the adult patients will develop persistent infection that may lead to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 2-4 Whereas considerable progress has been achieved regarding the identification and characterization of the virus, the development of new, effective therapies has been impeded because of the lack of a practical small HBV animal model. Attempts to establish animal models to study HBV infection in rats, 5 nude mice, 6 and transgenic mice 7-9 have been described. Other animal models, based on HBV-related hepadnaviruses that infect nonprimates, were developed and successfully used for assessment of antiviral drugs. These models, however, involve relatively large animals that are difficult to handle in most laboratories. In addition, testing of antiviral agents such as nucleoside analogs could produce aberrant results as a consequence of virus-specific differential susceptibility of the viral polymerase. 10 Chimpanzees provide a good HBV animal model in which effects of vaccines and therapeutic agents can be evaluated. 11 Nonetheless, the limited availability and the high cost of these primates severely restrict their use for such purposes.Recently, we have developed a human-mouse radiation chimera in which normal mice, preconditioned by lethal total body irradiation and radioprotected with SCID mouse bone marrow cells, are permissive for engraftment of human hematopoietic cells and tissues. [12][13][14][15][16] This resulting humanmouse model that comprises three genetically disparate sources of tissue is therefore termed ''Trimera.'' The Trimera mouse, engrafted with human peripheral blood lymphocytes, has been adapted successfully to generate human monoclonal antibodies. 17 Likewise, transplantation of Trimera mice with hepatitis C virus-infected human liver tissue was used for the development of an hepatitis C virus infection model. 14 In the present study, we describe in detail the development of an HBV ani...

The lack of small-animal models that are suitable for evaluation of agents used to treat infection with hepatitis C virus (HCV) severely hinders the assessment of potential new therapies for the disease. This study created such a model, termed the "HCV-Trimera" model. The HCV-Trimera model was developed by using lethally irradiated mice, reconstituted with SCID mouse bone marrow cells, in which human liver fragments infected ex vivo with HCV had been transplanted. Viremia (positive-strand HCV RNA levels) in HCV-Trimera mice peaked at approximately day 18 after liver transplantation, and an infection rate of 85% was reached. Viral replication in liver grafts was evidenced by the presence of specific negative-strand HCV RNA. The usefulness of this model for evaluation of anti-HCV agents was demonstrated by the ability of a small molecule (an HCV internal ribosomal entry site inhibitor) and an anti-HCV human monoclonal antibody (HCV AB(XTL)68) to reduce virus loads in HCV-Trimera mice in a dose-dependent manner.

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Hepatitis B virus (HBV) infection presents a major public health problem worldwide. Persistent HBV infection occurs in 5% to 10% of patients after acute hepatitis and may progress to chronic liver disease including chronic active hepatitis, cirrhosis, and hepatocellular carcinoma. 1 Newborns to HBV carrier mothers and immunocompromised individuals such as liver transplant recipients are at high risk for contracting HBV infection. 2,3 Recombinant HBV vaccines provide a safe and effective means for prevention of HBV conferring long-term immunity through active immunization. 4 In contrast to the gradual onset of protection following active immunization against HBV, passive immunization with antibodies to hepatitis B immunoglobulin (HBIG) provides immediate but short-term protection against viral transmission and infection. Administration of HBIG is commonly used in recipients of liver transplants who were HBV carriers to prevent HBV reinfection of the graft. 5-9 HBIG is a plasma derived, polyclonal antibody preparation obtained from blood donors who were anti-hepatitis B surface antigen (HBsAg) antibody positive. The limited availability of anti-HBsAg high-titer donors as well as safety concerns as to the use of a blood derived product, especially in immunosuppressed patients, urge for an alternative source for such immunotherapy. Human monoclonal antibodies (mAbs) to HBV may offer such an alternative. For clinical use, mAbs would be advantageous by presenting a stable and reproducible source for prolonged immunotherapy. Human mAbs to HBV generated by fusion of B cells derived from peripheral blood lymphocytes of vaccine immunized patients were described. [10][11][12][13][14][15] It was shown that mAbs might protect chimpanzees against challenge with HBV. 14,16,17 Recently we have described the development of high affinity, fully human mAbs against HBsAg generated in the Trimera system. These mAbs were previously characterized in terms of their affinity, specificity, and ability to bind HBV-infected human liver tissue. 18 In the present report, we have characterized the anti-HBV effect of 2 human mAbs, 17.1.41 and 19.79.5 by evaluating their specificity and reactivity to HBsAg derived from different viral subtypes and genotypes. The anti-HBV effect was evaluated in vivo in 2 different animal model systems, in HBVTrimera mice, [19][20][21] and in HBV chronic-carrier chimpanzees. The antibodies were able to reduce viral load in both animal models and to inhibit HBV infection in the HBV-Trimera model. The 2 mAbs were more potent than Hepatect (Biotest Pharma, Dreieich, Germany) in reducing viral load and inhibiting HBV infection in this model, suggesting a potential clinical use.Abbreviations: HBV, hepatitis B virus; HBIG, hepatitis B immunoglobulin; HBsAg, hepatitis B surface antigen; mAb, monoclonal antibody; ␤-L-5FddC, ␤-L-5-fluoro-2Ј, 3Ј-dideoxycytidine; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin.From

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno-incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 10(2)-10(3)-fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific-antibody titres and cells were fused to a human-mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high-affinity, IgG, anti-hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested.