Ieva Kotlarski

Entecavir Therapy Combined with DNA Vaccination for Persistent Duck Hepatitis B Virus Infection

ABSTRACT This study was designed to test the efficacy of antiviral treatment with entecavir (ETV) in combination with DNA vaccines expressing duck hepatitis B virus (DHBV) antigens as a therapy for persistent DHBV infection in ducks. Ducks were inoculated with 109 DHBV genomes at 7 days of age, leading to widespread infection of the liver and viremia within 7 days, and were then treated orally with either ETV (0.1 mg/kg of body weight/day) or distilled water from 21 days posthatch for 244 days. Treatment with ETV caused a 4-log drop in serum DHBV DNA levels within 80 days and a slower 2- to 3-log drop in serum DHBV surface antigen (DHBsAg) levels within 120 days. Following withdrawal of ETV, levels of serum DHBV DNA and DHBsAg rebounded to match those in the water-treated animals within 40 days. Sequential liver biopsy samples collected throughout the study showed that ETV treatment reduced DHBV DNA replicative intermediates 70-fold in the liver, while the level of the stable, template form, covalently closed circular DNA decreased only 4-fold. ETV treatment reduced both the intensity of antigen staining and the percentage of antigen-positive hepatocytes in the liver, but the intensity of antigen staining in bile duct cells appeared not to be effected. Intramuscular administration of five doses of a DNA vaccine expressing the DHBV presurface, surface, precore, and core antigens, both alone and concurrently with ETV treatment, on days 50, 64, 78, 127, and 141 did not result in any significant effect on viral markers.

Amino-terminal domain of the El Tor haemolysin of Vibrio cholerae O1 is expressed in classical strains and is cytotoxic

Previous studies have shown that the classical isolates of Vibrio cholerae possess an 11 bp deletion in the structural gene for the El Tor haemolysin leading to the production of a 27 kDa non-haemolytic truncated product HlyA* compared to the 82 kDa haemolysin, HlyA. These studies were designed to assess whether this truncated product had any biological activity. A KmR cartridge was introduced into the hlyA gene effectively eliminating the haemolysin. This was recombined into the chromosome of a variety of strains and isogenic pairs were examined in a number of systems. These studies suggest that the haemolytic (cytolytic) domain of HlyA resides at the C-terminus and that the N-terminus, which is conserved as HlyA* in classical strains, possesses enterotoxic (cytotoxic) activity. Experiments with the cholera-toxinless vaccine candidate JBK70 and its hlyA::KmR mutant suggest that HlyA* may be responsible for the residual diarrhoea observed in cholera-toxinless vaccine strains.

Identification of duck T lymphocytes using an anti-human T cell (CD3) antiserum

Duck lymphocytes have not been classified into cells resembling B or T cells of mammals. Reagents used in the past to identify lymphocyte populations in other species have not been useful for this purpose and antibodies raised to duck immunoglobulin bind in high proportions to blood and organ lymphocytes of ducks as well as to their red blood cells. Here we report that a polyclonal rabbit antiserum reacting to the CD3 marker on human T cells has been used to identify duck T lymphocytes. These antibodies react with the intracytoplasmic portion of the human CD3 epsilon chain (amino acids 156-168), an epitope highly conserved between mammals. Immunohistochemical staining with this antiserum of sections of duck lymphoid organs and FACScan analysis of duck lymphoid cell suspensions identified a population of duck lymphocytes with a staining pattern similar to that seen for mammalian T cells. This anti-human CD3 immunoprecipitated a 23 kDa protein from a duck lymphoblast lysate: a size similar to the human CD3 epsilon chain. This is the first direct identification of duck T lymphocytes.

Effect of antiviral treatment with entecavir on age- and dose-related outcomes of duck hepatitis B virus infection.

ABSTRACT Entecavir (ETV), a potent inhibitor of the hepadnaviral polymerases, prevented the development of persistent infection when administered in the early stages of duck hepatitis B virus (DHBV) infection. In a preliminary experiment, ETV treatment commenced 24 h before infection showed no significant advantage over simultaneous ETV treatment and infection. In two further experiments 14-day-old ducks were inoculated with DHBV-positive serum containing 104, 106, 108, or 5 × 108 viral genomes (vge) and were treated orally with 1.0 mg/kg of body weight/day of ETV for 14 or 49 days. A relationship between virus dose and infection outcome was seen: non-ETV-treated ducks inoculated with 104 vge had transient infection, while ducks inoculated with higher doses developed persistent infection. ETV treatment for 49 days did not prevent initial infection of the liver but restricted the spread of infection more than ∼1,000-fold, a difference which persisted throughout treatment and for up to 49 days after withdrawal. Ultimately, three of seven ETV-treated ducks resolved their DHBV infection, while the remaining ducks developed viremia and persistent infection after a lag period of at least 63 days. ETV treatment for 14 days also restricted the spread of infection, leading to marked and sustained reductions in the number of DHBV-positive hepatocytes in 7 out of 10 ducks. In conclusion, short-term suppression with ETV provides opportunity for the immune response to successfully control DHBV infection. Since DHBV infection of ducks provides a good model system for HBV infection in humans, it seems likely that ETV may be useful in postexposure therapy for HBV infection aimed at preventing the development of persistent infection.

Isolation of koala lymphoid cells and their in vitro responses to mitogens

Baseline parameters have been established for the successful in vitro culture of mononuclear cells from the peripheral blood (PMC) of koalas. To minimise stress-related influences and allow repeated testing of cells from the same animals, most studies were performed using blood samples from captive koalas which had become accustomed to regular handling. Ficoll-Paque density gradient fractionation of whole blood was required to prepare cell suspensions which responded well to the T-lymphocyte mitogens phytohaemagglutinin, concanavalin A and pokeweed mitogen. In contrast, very low or negligible proliferative responses were induced by the B-lymphocyte mitogens lipopolysaccharide, jacalin and protein A, even when purified PMC were cultured with a wide range of concentrations of these molecules. Using the standard approaches established with T-lymphocytes of eutherian animals, it was shown that concanavalin A-stimulated PMC produced an interleukin 2-like growth factor. The significance of these findings is discussed in the context of current knowledge and understanding of similar studies carried out using the lymphoid cells of eutherian and other metatherian animals.

Further characterisation of the immune response of the koala

Sensitive enzyme immunoassays (EIA) were developed to monitor antibody (Ab) production in the koala, in response to both soluble and particulate antigens (Ag). When compared with a eutherian mammal, the rabbit, both the dynamics and kinetics of Ab production in the koala were found to be severely retarded. In vitro, Ag specific lymphocyte proliferative responses were demonstrated for the first time in this animal by sensitising koalas in vivo with Bacillus Calmet-Guerin (BCG), with the level and timing of this cell mediated immune (CMI) response comparable with those seen in non-metatherian mammals. Levels of circulating B lymphocytes were examined in an attempt to clarify the retarded humoral responses to foreign Ags. In addition, peripheral mononuclear cells (PMC) from koalas, were examined for their reactivity to a range of monoclonal Abs and lectins in an attempt to characterise these cells further. The lectins examined, demonstrated an all or none reactivity with koala lymphocytes and were therefore considered unsuitable as markers for identifying lymphoid subsets in this animal. A monoclonal Ab directed at class II MHC Ags in the mouse, demonstrated cross reactivity with a high percentage of all koala monocytes tested. Using this Ab to probe CMI responses in vitro, it is concluded that immune interactions required for such responses in the koala parallel those seen in other mammals.

Structural and functional homology between duck and chicken interferon-gamma.

The Duck interferon gamma (DuIFN-gamma) cDNA was cloned from a phytohaemaglutinin-stimulated duck spleen cDNA library screened using a chicken IFN-gamma (ChIFN-gamma) cDNA probe. The DuIFN-gamma cDNA is 1392 nt long and shows 99% and 80% sequence identity with another cloned DuIFN-gamma cDNA, and with ChIFN-gamma cDNA, respectively. The cDNA contains a 495 bp ORF that encodes a putative 164 amino acid (AA) protein that shares 67% identity with ChIFN-gamma, but only 30-35% identity with mammalian IFN-gamma. The predicted three-dimensional (3D) structures of DuIFN-gamma and ChIFN-gamma are similar when analysed by comparative protein modelling. Culture supernatant collected from COS cells transfected with DuIFN-gamma cDNA was able to activate nitrite secretion from a chicken macrophage cell line (HD11) in a dose-dependent fashion. This activity could not be neutralised by an anti-ChIFN-gamma monoclonal antibody (Mab 85) that was able to neutralise the activity of ChIFN-gamma. Recombinant DuIFN-gamma (rDuIFN-gamma) protein was expressed in E. coli as an N-terminally His-tagged protein and was purified on a nickel affinity column. The eluted protein, which was detected as a approximately 18 kDa band with a purity of >90%, was also detected by Western blot using the anti-ChIFN-gamma monoclonal antibody (Mab 9.1). The rDuIFN-gamma was shown to activate nitrite secretion by HD11 cells in a dose-dependent fashion with a specific activity that was approximately 16-fold lower than a rChIFN-gamma control. Two rabbit antisera raised against rDuIFN-gamma were able to neutralise COS cell-expressed DuIFN-gamma activity; one of these also neutralised ChIFN-gamma activity. These findings indicate that DuIFN-gamma shares structural and functional identity with ChIFN-gamma, which is consistent with our previous results which demonstrated cross reactivity with other lymphokines from the two species.

Optimization of an in vitro assay which measures the proliferation of duck T lymphocytes from peripheral blood in response to stimulation with PHA and ConA.

The in vitro proliferative responses of duck PBMCs purified from Ficoll-Paque density gradients to the mitogens PHA and ConA show a great deal of duck-to-duck variation. Better responses were consistently obtained by using nylon wool fractionation to increase the proportion of duck T lymphocytes in PBMC preparations and then culturing these preparations with homologous monocytes, purified from PBMC preparations by their adherence properties. We have also established that the addition of homologous red blood cells enhances the in vitro proliferative responses of duck T lymphocytes, especially when limiting doses of PHA and ConA are used. Duck T lymphocytes showed greater and more consistent proliferation when cultures were incubated at 37 degrees C as compared to incubation at 41.6 degrees C. The improved consistency of higher proliferative responses with this assay should make it more suitable for detecting in vitro proliferative responses of antigen-specific T lymphocytes, as a measure of in vivo induced cell mediated immune responses.

Identification of koala T lymphocytes using an anti-human CD3 antibody.

Previous attempts to identify T lymphocytes in the koala using cross reacting monoclonal antibodies (mAbs) against other species T cell antigens (Ags) and classical T cell lectins have proved unsuccessful. Recently a polyclonal rabbit Ab preparation directed at the epsilon chain of the human CD3 complex (anti-CD3) has become commercially available and has been shown to have broad cross-species reactivity. We have demonstrated that this anti-CD3 is suitable for labelling T cells from peripheral blood of koalas if the purified peripheral mononuclear cells (PMC) are first made permeable with mild fixation in buffered formal acetone. We have also been able to identify koala T cells in both formalin-fixed and frozen lymphoid tissues. Immunoprecipitation and Western blotting studies demonstrated that this anti-CD3 bound to a single 23 kDa polypeptide, probably representing the koala homologue of the human epsilon chain. This is the first report of successful identification of koala T cells and the first reported use of this anti-CD3 for the identification of peripheral circulating T lymphocytes in any species.

Characterisation of duck thromhocytes

Abstract Duck thrombocytes were initially identified in peripheral blood mononuclear cells (PBMCS) purified from whole blood on Ficoll-Paque density gradients by examining stained smears of these cells. These thrombocytes could be readily purified from lymphocytes on the FACStar cell sorter by their increased side-scatter. They were similar to chicken thrombocytes in both appearance and function; they had a diameter of 4.5–6 μm and contained large vacuoles and were able to phagocytose carbon and Staphylococcus aureus. A monoclonal antibody (mAb) BA3 was generated which binds specifically to duck thrombocytes and has facilitated the characterisation of these cells which comprise up to 50–60 per cent of cells in Ficoll-Paque purified duck PBMCS.