Nili Daudi

Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.

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 102–103‐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.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization

A femtosecond laser beam gene transduction (SG‐LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 μg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg‐specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNγ, and IL‐4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG‐LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV‐infected replicating cells. Tumor growth retardation was induced in vacci‐nated mice challenged with an HBsAg‐expressing syn‐geneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.—Zeira, E., Manevitch, A., Manevitch, Z., Kedar, E., Gropp, M., Daudi, N., Barsuk, R., Harati, M., Yotvat, H., Troilo, P. J., Griffiths, T. G., II, Pacchione, S. J., Roden, D. F., Niu, Z., Nussbaum, O., Zamir, G., Papo, O., Hemo, I., Lewis, A., Galun, E. Femtosecond laser: a new intradermal DNA delivery method for efficient, long‐term gene expression and genetic immunization. FASEB J. 21, 3522–3533 (2007)

Genotypic and phenotypic resistance: Longitudinal and Sequential analysis of hepatitis B virus polymerase mutations in patients with lamivudine resistance after liver transplantation

OBJECTIVE:Lamivudine-resistant strains appear in 27–62.5% of liver transplant recipients treated with lamivudine for hepatitis B virus (HBV) recurrence, and may lead to failure of antiviral therapy. In an extension of our previous study, we investigated the molecular events associated with the emergence of lamivudine-resistant mutants in this population.METHODS:Sequential serum samples from 10 consecutive patients with lamivudine resistance after liver transplantation were analyzed for viral genotype, precore mutants, and viral polymerase gene mutants (L528M, M552V, M552I) using restriction fragment length polymorphism. Quantitative analysis of HBV DNA was performed using hybridization assay and polymerase chain reaction.RESULTS:Eight patients (80%) were infected with genotype D and two (20%) with genotype C. Polymerase mutants (genotypic resistance) were identified in all the patients. Phenotypic resistance (rise in serum HBV DNA titers above the detection limit of the hybridization assay) developed in five patients (50%); of the remainder, three (30%) did not have phenotypic resistance, and two were primary nonresponders. Genotypic resistance was detected earlier than phenotypic resistance (median 285 days [range 42–510] vs median 387 days [range 320–420], p = 0.055). In five patients (50%), the emergence of the YMDD mutants took over the wild type; in three (30%), the YMDD mutant took over the wild type, but the wild type re-emerged during lamivudine therapy; and in two (20%), the YMDD mutants were detected in a mixture with the wild type (in different percentages). The mean pretreatment serum ALT level was significantly lower in the patients who did not develop phenotypic resistance (p = 0.0002). The M552I pure viral population was found mainly in these patients, and all retained stable graft function (median follow-up 33 months). A high pretreatment HBV DNA level (>50 × 106 copies/ml) was highly statistically significantly correlated with the rapid occurrence of phenotypic resistance (r = −0.90, p = 0.04).CONCLUSIONS:We reached the following conclusions: 1) In our area, liver transplant recipients who develop resistance to lamivudine given for recurrent HBV infection seem to be mainly infected with genotype D. 2) Re-emergence of the wild type can occur during lamivudine therapy. 3) Genotypic resistance precedes phenotypic resistance, although phenotypic resistance does not always follow genotypic resistance. 4) Quantitative determination of viremia and analysis of polymerase gene mutants are recommended for monitoring antiviral therapy of liver transplant patients with HBV reinfection in the graft.

Genotype prevalence, viral load and outcome of hepatitis B virus precore mutant infection in stable patients and in patients after liver transplantation

Abstract:  Objective:  The precore mutant is detectable in most Israeli patients with persistent hepatitis B virus (HBV) infection. The aim of this study was to determine the prevalence of HBV genotypes, viral load and outcome of precore mutant infection in stable patients and in patients after liver transplantation.

Breastmilk hepatitis A virus RNA in nursing mothers with acute hepatitis A virus infection.

Breastmilk specimens from three women with acute hepatitis A virus (HAV) infection were studied. Anti-HAV immunoglobulin M and immunoglobulin G antibodies were detected in serum and breastmilk specimens of the three women. The three women also had serum HAV RNA. However, HAV RNA was detected only in two of the three breastmilk specimens. It is interesting that none of the three infants contracted clinical HAV infection. Furthermore, mothers with HAV infection should not be encouraged to discontinue breastfeeding.

735. Femtosecond Laser- A New Intradermal DNA Delivery Method for Gene Expression and Vaccination

The skin is an ideal target for naked DNA delivery for gene expression and vaccination, due to ease of administration and presence of a large number of antigen-presenting cells within the tissue. There are several different methods to transfer naked DNA to the skin. However, some of these delivery methods encounter low level of transfection efficiency and lack of sustained expression. We propose a new and highly efficient method of dermal gene transduction - the Laser Beam Gene Transduction method (LBGT). Methods. Using a femtosecond infrared titanium sapphire laser beam as a laser source illumination, the shaved back skin of Balb/c mice was exposed for assessment of LBGT. The pCAG/Luc construct was used for luc expression. For in vivo real time continuous luc expression, the bioluminescence CCCD system was used (Zeira et al. Mol. Ther. 4:239, 2001). For assessment of this delivery method for vaccination, we used the pCMV/HBsAg vector. Results. A single intra-dermal injection of 10mg of pCAG/Luc followed by laser beam illumination for 2 minutes, resulted in an increase of transgene expression for up to an average of 1000-fold on day 43, relative to naked DNA injection. Characterization studies revealed the optimal conditions for LBGT into the mice dermis. Gene expression following LBGT continued for > 50 days. In mice immunized with the HBsAg plasmid, (10|[mu]|g) on day 0 and 21, HBsAg antibody titers were detected shortly after the first injection and continued to increase over one month, and were significantly higher than titers obtained by naked DNA administration without LBGT. Conclusion. Delivery of DNA plasmid into the dermis by LBGT is highly efficient for transgene expression and for elicitation of immune response. These results provide a proof of principle that femtosecond laser may appear as a novel method for the administration of DNA-based vaccines.