Antiviral therapy with entecavir combined with post-exposure "prime-boost" vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection.

Short-term antiviral therapy with the nucleoside analogue entecavir (ETV), given at an early stage of duck hepatitis B virus (DHBV) infection, restricts virus spread and leads to clearance of DHBV-infected hepatocytes in approximately 50% of ETV-treated ducks, whereas widespread and persistent DHBV infection develops in 100% of untreated ducks. To increase the treatment response rate, ETV treatment was combined in the current study with a post-exposure "prime-boost" vaccination protocol. Four groups of 14-day-old ducks were inoculated intravenously with a dose of DHBV previously shown to induce persistent DHBV infection. One hour post-infection (p.i.), ducks were primed with DNA vaccines that expressed DHBV core (DHBc) and surface (pre-S/S and S) antigens (Groups A, B) or the DNA vector alone (Groups C, D). ETV (Groups A, C) or water (Groups B, D) was simultaneously administered by gavage and continued for 14 days. Ducks were boosted 7 days p.i. with recombinant fowlpoxvirus (rFPV) strains also expressing DHBc and pre-S/S antigens (Groups A, B) or the FPV-M3 vector (Groups C, D). DHBV-infected hepatocytes were observed in the liver of all ducks at day 4 p.i. with reduced numbers in the ETV-treated ducks. Ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection during ETV treatment, but in 60% of cases, infection became widespread after ETV was stopped. In contrast, at 14 and 67 days p.i., 100% of ducks treated with ETV and "prime-boost" vaccination had no detectable DHBV-infected hepatocytes and had cleared the DHBV infection. These findings suggest that ETV treatment combined with post-exposure "prime-boost" vaccination induced immune responses that eliminated DHBV-infected hepatocytes and prevented the development of persistent DHBV infection.

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.

Immune responses to duck hepatitis B virus infection.

The duck hepatitis B virus (DHBV) was the first hepatitis B virus identified from an avian host. It is a member of the Hepadnaviridae family of viruses. All members of this family display similar genomic organization and replication strategies and cause species-specific infections that result in either transient (acute) or persistent infection. Hepadnavirus infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious 'empty' surface antigen particles into the bloodstream. Hepadnavirus replication is non-cytopathic and immune responses to viral antigens are thought to be responsible for the liver damage seen in both transient and persistent infection and for the clearance of virus from infected cells. This has provided the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human hepatitis B virus (HBV) infection. It follows that detailed understanding of the immune responses induced during transient and persistent infection may well facilitate the development of more effective approaches to immunotherapy in patients with persistent infection and may also provide a means of reducing the liver damage associated with this infection, without reducing the effectiveness of the immunity required to eliminate the virus. Immune responses to hepadnavirus infection have been studied primarily in humans, following natural infection with HBV, but studies have also been performed with the woodchuck hepatitis virus (WHV) and the DHBV models. This manuscript reviews the recent studies of immune responses to DHBV infection.

Characterisation of duck thrombocytes.

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 microm 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.

An homologous in vitro assay to detect lymphokines released by PHA-activated duck peripheral blood lymphocytes and spleen cells.

When stimulated with phytohaemagglutinin duck lymphocytes released lymphokines which were detected by their ability to maintain the proliferation of duck lymphoblasts using an in vitro assay similar to that previously developed with the mammalian system to measure IL-2. The inability of duck lymphokines to maintain the proliferation of mammalian lymphoblasts (mouse) indicated that there was no functional homology between duck and mammalian lymphokines. However, duck lymphokines did maintain the proliferation of chicken lymphoblasts indicating functional homology of these growth factors between these two species. The duck lymphokine maintenance assay is a simple and reliable test, and should be useful as an in vitro assay for the detection of factors released by antigen-specific lymphocytes when cultured in the presence of viral antigens.

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.

Role of SefA subunit protein of SEF14 fimbriae in the pathogenesis of Salmonella enterica serovar Enteritidis.

In this study, the role of the SefA subunit protein of SEF14 fimbriae in the pathogenesis of Salmonella enterica serovar Enteritidis was investigated. This was accomplished by mutating the sefA gene in the chromosome of two strains of S. enterica serovar Enteritidis by allelic exchange with a copy that has been inactivated by interruption with a nonpolar kanamycin resistance (aphA-3) cassette. The effect of this mutation on the ability of the S. enterica serovar Enteritidis strains to colonize the intestinal epithelium and to invade other tissues was assessed in BALB/c mice and in vitro by adherence and invasion of HeLa cells. Our results show that an avirulent S. enterica serovar Enteritidis vaccine strain, 11RX (no somatic antigen; flagellum antigen phase 1, g,m; flagellum antigen phase 2, -), colonized better and persisted longer in the Peyer's patches of these mice than did its SefA-deficient counterpart. However, no such difference was observed between a highly virulent S. enterica serovar Enteritidis strain, 7314 (somatic antigen, O1, O9, O12; flagellum antigen phase 1, g,m; flagellum antigen phase 2 [1,7]), and its SefA-deficient isogenic mutant. These findings were correlated with in vitro adherence and invasion of HeLa cells. Furthermore, we could not demonstrate a role for SefA in the virulence of S. enterica serovar Enteritidis as assessed by 50% lethal dose determinations. The implications of these findings are discussed.

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.

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.

A Salmonella enteritidis 11RX pilin induces strong T-lymphocyte responses.

Our previous work, using proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to define antigens of Salmonella enteritidis 11RX able to stimulate T cells from S. enteritidis 11RX-primed (BALB/c x C57BL/6)F1 mice, had indicated the presence of a major antigenic determinant of 14 to 18 kDa (H.-M. Vordermeier and I. Kotlarski, Immunol. Cell. Biol. 68:299-305, 1990). The 14-kDa size is similar to that of the monomeric units of one of the fimbrial structures, SEF14, produced by a human enteropathogen, S. enteritidis 27655 (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Here we present data which indicate that S. enteritidis 11RX also produces this protein and that it is able to elicit delayed-type hypersensitivity reactions in S. enteritidis 11RX-primed animals and to stimulate in vitro proliferation of, and cytokine release from, T cells obtained from these animals, implying that this fimbrial protein is likely to be an important immunogen of S. enteritidis. The protein was purified to homogeneity and is free from contamination with lipopolysaccharide. Standard immunoblot analysis with unabsorbed S. enteritidis 11RX antiserum and antiserum absorbed with Salmonella typhimurium C5 and various strains of Escherichia coli, as well as a panel of anti-14-kDa-protein monoclonal antibodies, suggests that this fimbrial protein is not the common antigen expressed by a number of organisms belonging to the family Enterobacteriaceae. Immunogold electron microscopy with one of these monoclonal antibodies confirms that the 14-kDa protein and SEF14 are identical.

Immunogenicity of the Escherichia coli fimbrial antigen K99 when expressed by Salmonella enteritidis 11RX.

Salmonella strains expressing the Escherichia coli fimbrial protein K99 were used to immunize adult mice, and the resulting anti-K99 T-cell responses were examined. Immunized animals displayed delayed-type hypersensitivity responses when challenged with K99 in the footpad. Lymphoid cells from immunized animals proliferated and released cytokines when cultured in vitro with K99 or peptides generated by cyanogen bromide treatment; the T cells which responded had the CD4+ phenotype.

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.

Induction of Lyt-2+ cytotoxic T lymphocytes following primary and secondary Salmonella infection.

Investigations of the cytotoxic activity of T cells induced following one or two intraperitoneal doses of live Salmonella revealed that cytotoxicity was restricted to the Lyt-2+ T-cell subset and was enhanced following secondary infection with Salmonella. Initial studies using the lectin-dependent cellular cytotoxicity (LDCC) assay detected Lyt-2+ cytotoxic T cells in peritoneal cell suspensions of S. enteritidis 11RX (11RX)-infected mice, with the peak of activity occurring 5 days after infection. This did not correlate with the proliferative activity of these cells, which peaked 10-12 days after infection. Secondary challenge with 11RX or S. typhimurium C5 (C5) induced a rapid increase in the cytotoxic activity of Lyt-2+ peritoneal T cells and was detected even 21 days later. The antigen specificity of some of these cells was confirmed in cytotoxicity assays using P815 tumour cells infected with 11RX organisms as targets. No cytotoxic activity was detected in the spleen cell suspensions of infected (and normal) mice unless the cells were first activated by in vitro culture with concanavalin A (Con A). Both types of activated spleen cells showed LDCC but Salmonella-specific cytotoxic Lyt-2+ T cells were detected only in spleen cell (SC) cultures prepared from mice challenged with a second dose of Salmonella.

Detection of Salmonella-specific L3T4+ and Lyt-2+ T cells which can proliferate in vitro and mediate delayed-type hypersensitivity reactivity.

This study was based on an initial observation that, although culture of T cells from Salmonella-infected mice with concanavalin A induced both L3T4+ T cells and Lyt-2+ T cells to proliferate, there was a relative increase in the responsiveness of the Lyt-2+ T cells in suspensions harvested from mice with secondary infection. Accordingly, primed T cells, obtained from the peritoneal cavities and spleens of mice that had received one or two intraperitoneal doses of Salmonella were examined for the presence of antigen-specific, class I major histocompatibility complex (MHC)-restricted Lyt-2+ T cells. After primary infection with avirulent Salmonella enteritidis 11RX (11RX) only L3T4+ T cells could be induced to proliferate in response to formalin-killed 11RX organisms, and a second dose of live 11RX did not change the phenotype of the responding T-cell population. In contrast, secondary challenge with S. typhimurium C5 (C5) generated cell populations where both L3T4+ and Lyt-2+ T cells proliferated when cultured with formalin-killed 11RX. Transfer of delayed-type hypersensitivity (DTH) using mixtures of primed T cells and either killed or live Salmonella organisms demonstrated that DTH was mediated by L3T4+ T cells, and secondary infection with either the 11RX or C5 strain did not change this result. However, antigen-specific Lyt-2+ T cells which mediated DTH reactivity were detected using a Salmonella-infected cell line which expressed MHC-coded class I but not class II products. These Lyt-2+ T cells were present in the spleen and peritoneal cavity after secondary infection and in the peritoneal cavity late after a primary infection with 11RX.

Antigen-presenting characteristics of peritoneal cells of Salmonella enteritidis 11RX-infected mice.

Investigation of the possibility that infection with intracellular bacterial parasites such as Salmonellae may modulate the function of antigen-presenting cells (APC) revealed no major change in APC function of peritoneal cells (PC) harvested from the peritoneal cavity of mice 1-3 days after intraperitoneal immunization with S. enteritidis 11RX. Analysis of the phenotype of the Salmonella-primed T cells which responded when cultured with PC from either normal or infected mice and Salmonella-antigen showed that only L3T4+ T cells proliferated. This was also true when PC from normal and infected mice were compared for their ability to induce allogeneic responses; both L3T4+ and Lyt-2.2+ T cells were induced to proliferate, with the majority belonging to the class I restricted, Lyt-2.2+ phenotype. Significant levels of alloantigen-specific Lyt-2.2+ cytotoxic T-cell activity were also induced in both types of cultures. However, a minor population of adherent cells which inhibited Salmonella antigen-specific T-cell proliferation in vitro was detected in peritoneal cell suspensions harvested 3 days after intraperitoneal immunization with S. enteritidis 11RX. Further characterization of these peritoneal cells revealed that they also inhibited the induction of in vitro T-cell responses to alloantigens. It is likely that the cells mediating these inhibitory effects belonged to a macrophage-like subset.

Koala lymphoid cells: analysis of antigen-specific responses.

Bovine serum albumin (BSA) and ovine immunoglobulin (OvIgG) were used to induce humoral immune responses in two koalas which were also painted with 2-4-dinitrofluorobenzene (DNFB) and subsequently tested for local delayed-type hypersensitivity (DTH) reactions. The responses observed support the suggestion that the koala is 'immunologically lazy'. Antibody responses to BSA and OvIgG were not detected until 12 weeks after the initial antigen injection and antigen-specific in vitro proliferative responses by the mononuclear cells (PMC) of immunised animals could not be induced, although these cells did respond to concanavalin A and phytohaemagglutinin. Similarly, DTH responses to DNFB could be elicited in vivo, but took a relatively long time to develop and the PMC of the sensitised koalas were unresponsive to the sensitising antigen in vitro. The absence of proliferation when mixed suspensions of PMC from different koalas were cultured in vitro is consistent with these results.

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.

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.

Partial purification and characterization of low molecular weight antigens of Salmonella enteritidis 11RX.

Sephadex G100 chromatography and preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 16% polyacrylamide gels were used for the partial purification of 16-18 kDa proteins able to stimulate Salmonella enteritidis 11RX-primed T cells of (BALB/c x C57BL/6J)F1 mice. A soluble antigen (Ag) extract of S. enteritidis 11RX (s11RX) was used as the starting material for purification for two reasons. First, s11RX had been previously shown to induce in vitro proliferation of Salmonella-primed T cells; second, initial analysis of SDS-PAGE fractionated s11RX Ag using the 'T cell western blot' technique indicated that T cell stimulatory activity was located only in the 16-18 kDa region. The partially purified antigens were able to elicit delayed-type hypersensitivity reactions in vivo, and stimulated in vitro proliferation and interleukin-2 release from 11RX-primed T cells and T cell lines and clones derived from these cells, indicating that they are major antigenic determinants of S. enteritidis 11RX. Testing of 16-18 kDa proteins of several other bacteria indicated that these antigens may be 'common' and expressed by a number of organisms belonging to the Enterobacteriaceae.

Identification of antigens which stimulate T lymphocytes of Salmonella enteritidis 11RX immunized mice.

The technique of using sodium dodecylsulfate-polyacrylamide gel electrophoresis fractionated antigens (Ag) transferred to nitrocellulose filters was adopted to analyse T cell responses to Salmonella enteritidis 11RX Ag. Employing in vitro proliferation assays with T cells from S. enteritidis 11RX-primed (BALB/c x C57BL/6J)F1 mice as the measure of T cell stimulation, we have identified Ag able to stimulate T cells in the regions containing 16, 24, 34 and 50-60 kDa proteins, with dominant Ag activity at about 16 kDa. These results were confirmed with long-term, Ag-specific L3T4+ T cell lines which responded to molecules in the same four Mr regions, suggesting that no selection by a single antigenic determinant had occurred during more than 3 months of in vitro culture, or that all the molecules which were stimulatory shared at least one antigenic determinant. Because the seven clones we examined responded only to 16 kDa molecules, the former alternative is the more likely. Standard immunoblot analysis indicated that these Ag also act as major B cell stimulating determinants. T cells of BALB/c mice, which are 5-10 times more resistant to S. enteritidis 11RX than C57BL/6J mice, showed the same pattern of reactivity as F1 mice whereas the major antigenic region for T cells of C57BL/6J mice was located between 50 and 60 kDa.