Immune responses of mice to inactivated rabies vaccine administered orally: potentiation by Quillaja saponin

Administered orally, Quillaja saponin markedly potentiated the humoral immune responses of mice fed inactivated rabies vaccine, and significantly increased their resistance to subsequent intracerebral challenge with live rabies virus. Although mean serum neutralizing antibody titres were generally 8- to 16-fold higher when vaccine was given intraperitoneally, orally administered vaccine, with saponin, stimulated production of high protective antibody levels that were maintained for at least 6 months. The potentiating effect of saponin appeared to be mediated through several mechanisms, one of which was by increasing the permeability of the intestinal mucosa, allowing increased uptake of viral antigen. The potentiating effect was enhanced when saponin was administered in advance (up to at least 16 h) of the oral vaccine. Mice tolerated the effective saponin doses without any visible signs of distress or injury. In view of the many favourable physiological activities and low toxicity of orally delivered saponins, it is suggested that they may find more general applications in the immunopotentiation of oral vaccines.     

Immune response in cattle vaccinated against rabies

In order to determine the best type of rabies vaccine to use as a booster, 78 serological samples from singly vaccinated cattle were analyzed by counterimmunoelectrophoresis technique. The animals were divided into several groups, received the first vaccine dose with modified live virus vaccine (ERA strain) and were revaccinated with inactivated virus or modified live virus vaccines. Boosters were given at 2, 4, 8, 12 and 16 weeks following first vaccination. Results showed high titres in the cases of booster with inactivated vaccine. In all cases, however, detectable antibody titres declined quickly.     

Induction of antigen-specific antibody response in human peripheral blood lymphocytes in vitro by a dog kidney cell vaccine against rabies virus (DKCV)

In the present report an in vitro method for obtaining a secondary human antibody response to a dog kidney cell vaccine against rabies virus (DKCV) is described. Cultures of peripheral blood mononuclear cells from normal rabies-immune and nonimmune donors were stimulated in vitro by DKCV. The production of virus-specific antibody in supernatant fluids was monitored by ELISA. Antibody was produced by lymphocytes from rabies-immune individuals, whereas those of nonimmune subjects consistently failed to produce anti-rabies antibodies after in vitro stimulation with DKCV. The generation of the anti-rabies virus antibody response of lymphocytes stimulated with DKCV was shown to be an antigen-dependent, as well as an antigen-specific process. Optimal antigen-specific responses were observed at relatively low concentrations of antigen (10(-1) to 10(-2) micrograms/culture). At increasing concentrations of antigen in culture (greater than 1 microgram/culture), the anti-rabies virus response was suppressed. Antibody produced upon stimulation was capable of neutralizing rabies virus. The response to rabies virus requires T cell help because lymphocytes depleted of SE rosetting cells did not respond to an antigenic stimulus. Studies in which the same individuals were followed over time showed a sequential development of circulating B cell subsets. The system may provide a model for the study of human B cell differentiation in vivo and in vitro and may be valuable for testing the potency of rabies vaccines in vitro.     

Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Maternal antibody blocks humoral but not T cell responses to BVDV.

Bovine viral diarrhoea virus (BVDV) contributes significantly to health-related economic losses in the beef and dairy industry. Antibodies of maternal origin can be protective against BVDV infection, however, calves with low titres of maternal antibody or that do not receive colostrum may be at risk for acute BVDV infection. Interference by high titres of maternal antibodies prevents the development of an antibody response following vaccination with either a killed or attenuated BVDV vaccine. However, the T cell mediated immune response to BVDV may be generated in the absence of a detectable serum neutralizing antibody response. Two trials were conducted to evaluate the potential to elicit T cell mediated immune responses to BVDV in calves with circulating maternal antibody to BVDV. In the first trial, calves with high levels of circulating maternal antibody to BVDV 1 and BVDV 2 were experimentally infected with BVDV 2 (strain 1373) at two to five weeks of age. The T-cell mediated immune responses of the experimentally infected calves and non-infected calves were monitored monthly until circulating maternal antibody was no longer detectable in either treatment group. Calves experimentally infected with BVDV developed BVDV specific CD4(+), CD8(+), and delta T cell responses while high levels of maternal antibody were circulating. A second challenge with BVDV 2 (strain 1373) was performed in the experimentally infected and control calves once maternal antibody could no longer be detected. Previous exposure to BVDV in the presence of maternal antibody protected calves from clinical signs of acute BVDV infection compared to the control calves. In the second trial, three groups of calves with circulating maternal antibody to BVDV were given either a modified live vaccine (MLV) containing BVDV 1 and BVDV 2, a killed vaccine containing BVDV 1 and BVDV 2, or no vaccine, at seven weeks of age. Serum neutralizing antibody levels and antigen specific T cell responses were monitored for 14 weeks following vaccination. Calves vaccinated with MLV BVDV developed BVDV 1 and BVDV 2 specific CD4(+)T cell responses, and BVDV 2 specific gammadelta T cell responses, in the presence of maternal antibody. Vaccination with killed BVDV did not result in the generation of measurable antigen specific T cell immune responses. In this trial, a second vaccination was performed at 14 weeks to determine whether an anamnestic antibody response could be generated when calves were vaccinated in the presence of maternal antibody. Calves vaccinated with either a MLV or killed BVDV vaccine while they had maternal antibody developed an anamnestic antibody response to BVDV 2 upon subsequent vaccination. The results of these trials indicate that vaccinating young calves against BVD while maternal antibody is present may generate BVDV specific memory T and B cells. The data also demonstrated that seronegative calves with memory T and B cells specific for BVDV may be immune to challenge with virulent BVDV.     

Chemiluminescence of mononuclear cells is enhanced during antigen recognition

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononulcear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48–72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.     

Response of T lymphocytes from previously infected calves to recombinant Cryptosporidium parvum P23 vaccine antigen

We had previously demonstrated that a Type-1-like immune response involving interferon-gamma expression in lamina propria lymphocytes accompanied by IgG2 subclass fecal antibodies to Cryptosporidium parvum p23 emerged in gut mucosa of calves recovering from cryptosporidiosis. Because a recombinant p23 had been shown to protect calves from cryptosporidiosis when administered as a vaccine antigen to late gestation cattle, this study was undertaken to determine if the same vaccine antigen could induce a Type-1-like, in vitro response by T cells from calves that had recovered from C. parvum infection. We isolated peripheral blood mononuclear cells from calves that had been previously infected with C. parvum oocysts and incubated them in the presence or absence of the recombinant C. parvum p23 vaccine antigen. We used flow cytometry to simultaneously detect cells in cell cycle and identify the T cell subset containing cycling cells. We also used flow cytometry to identify interferon-gamma positive cells and 2-dimensional gel electrophoresis to profile proteins made by PBMC stimulated with the recombinant p23 vaccine antigen. The results demonstrated that CD4+ T lymphocytes proliferated and that interferon-gamma was synthesized by a subset of stimulated cells. Two-dimensional gel electrophoresis revealed the presence of several cytoplasmic proteins in a size range of approximately 25-80 kDa that were detected in p23-stimulated, but not in unstimulated, cytoplasmic samples. Together, the results show that the recombinant p23 vaccine antigen can stimulate a Type-1-like immune response by T cells from calves that have recovered from C. parvum infection.     

Potency assessment of foot-and-mouth disease vaccines in cattle by means of antibody assays.

A tendency has emerged for some years to replace the challenge infection of cattle for the assessment of foot-and-mouth disease (FMD) vaccine potency. This can be actually evaluated by means of antibody assays on cattle sera, at about 3/4 weeks after the vaccination. Serological results can be worked out as single titres (to be compared with a pre-determined threshold level) or as mean antibody titres induced by different vaccine dilutions. However, the assessment of FMDV-specific antibody titres would not fully depict the extent and the efficacy of the immune response of cattle; moreover, the antibody response would not be proportional if potent vaccines are used (greater than or equal to 10-12 PD50). Thus, a particular approach is suggested for the serological procedures, which enable credible estimates of potent FMD vaccines to be formulated.     

Comparative immunogenecity of foot and mouth disease virus antigens in FMD-haemorrhagic septicaemia combined vaccine and FMD vaccine alone in buffalo calves

Humoral immune response was evaluated by monitoring the serum antibody titres and virus specific IgM titres against Foot and Mouth Disease (FMD) virus antigens in serum samples obtained from different groups of calves inoculated with combined vaccine or FMD vaccine alone, on 0, 7, 14, 21, 28, 42 and 56 days post-vaccination (DPV). The cellular immune response was monitored by MTT based lymphoproliferation in peripheral blood mononuclear cell cultures. Higher liquid phase blocking (LPB) ELISA antibody titres were observed in calves receiving combined vaccine as compared to calves immunized with FMD vaccine alone with the peak titres in both the groups obtained on 21 days post-vaccination. However, the virus specific IgM titres were significantly higher in group of calves inoculated with combined vaccine than FMD vaccine alone. The lymphoproliferative responses against FMDV types O, A22 and Asia 1 in the groups receiving combined vaccine and FMD vaccine alone started increasing gradually after day 14 and reached peak levels on 28 DPV followed by a gradual decline subsequently. The group receiving combined vaccine showed higher proliferative responses on in vitro stimulation with FMD virus type O, whereas, with FMD virus type Asia 1, the responses were significantly higher on 14 and 21 DPV as compared to the group immunized with FMD vaccine alone. However, in the group receiving combined vaccine, the responses on in vitro stimulation with FMD virus type A22 were significantly higher than FMD vaccine alone group on all DPV except on 42 DPV.     

Human leptin signaling in human peripheral blood mononuclear cells: activation of the JAK-STAT pathway

Leptin is an adipocyte-secreted hormone that centrally regulates weight control. However, the leptin receptor is expressed not only in the central nervous system, but also in other systems, such as reproductive, hematopoietic, and immune tissues, suggesting various roles in addition to the regulation of food intake and energy expenditure. The leptin receptor bears homology to members of the class I cytokine receptor family. Leptin has previously been shown to enhance cytokine production by murine peritoneal macrophages and human circulating monocytes, where human leptin promotes activation and proliferation. We have recently found that the leptin receptor is expressed not only in monocytes but also in both CD4(+) and CD8(+) T lymphocytes. Besides, leptin enhances proliferation and activation of T lymphocytes when they are costimulated by PHA or Con A. In this paper, we have studied the signal transduction of the leptin receptor in peripheral blood mononuclear cells. We found that leptin stimulation activates the JAK-STAT signaling pathway. More specifically, we found that JAK-2/3 and STAT-3 are activated by tyrosine phosphorylation upon leptin stimulation. Moreover, leptin stimulated tyrosine phosphorylation of the RNA binding protein Sam68 and its association with STAT-3. These effects were dose-dependent (0.1-10 nM) and transient (5-30 min). We also observed the leptin stimulated translocation of activated STAT-3 from the cytoplasm to the nucleus. These results indicate that human leptin receptor in circulating mononuclear cells has the signaling capacity to activate JAK-STAT cascade. This pathway may mediate, at least in part, the action of human leptin in human peripheral blood mononuclear cells.     

Novel single nucleotide polymorphisms of the bovine methyltransferase 3b gene and their association with meat quality traits in beef cattle

DNA methylation is essential for adipose deposition in mammals. We screened SNPs of the bovine DNA methyltransferase 3b (DNMT3b) gene in Snow Dragon beef, a commercial beef cattle population in China. Nine SNPs were found in the population and three of six novel SNPs were chosen for genotyping and analyzing a possible association with 16 meat quality traits. The frequencies of the alleles and genotypes of the three SNPs in Snow Dragon beef were similar to those in their terminal-paternal breed, Wagyu. Association analysis disclosed that SNP1 was not associated with any of the traits; SNP2 was significantly associated with lean meat color score and chuck short rib score, and SNP3 had a significant effect on dressing percentage and back-fat thickness in the beef population. The individuals with genotype GG for SNP2 had a 25.7% increase in lean meat color score and a 146% increase in chuck short rib score, compared with genotype AA. The cattle with genotype AG for SNP3 had 35.7 and 24% increases in dressing percentage and 28.8 and 29.2% increases in back-fat thickness, compared with genotypes GG and AA, respectively. Genotypic combination analysis revealed significant interactions between SNP1 and SNP2 and between SNP2 and SNP3 for the traits rib-eye area and live weight. We conclude that there is considerable evidence that DNMT3b is a determiner of beef quality traits.     

Rabies virus infects mouse and human lymphocytes and induces apoptosis.

Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response.     

狂犬病毒糖蛋白DNA疫苗的研制及其免疫效果的观察

构建了含有狂犬病毒（RV）CVS株糖蛋白（GP）基因的重组质粒pCMVCVSRG,将其转染至鼠NIH3T3细胞中,用间接免疫荧光法和APAAP法均证实RVGP能在真核细胞中表达。分别将合RV不同毒株的GP基因的质粒（DNA疫苗）及空白载体质粒（对照组）免疫小鼠,仅DNA疫苗免疫的小鼠产生了中和抗体。以RV攻击后,DNA疫苗免疫组小鼠的存活率与对照组相比,差异有极显著性意义（P＜0．01）;不同的启动子（CMV或SV40）与不同GP基因（来源于CVS株或ERA株）对DNA疫苗的免疫效果无明显影响。在注射120d后．用PCR方法仍可检测出RVGP基因。结果表明：狂犬病DNA疫苗能够诱生低水平的中和抗体和记忆性B淋巴细胞,并能保护小鼠抵抗RV的攻击。该疫苗能在体内稳定存在。狂犬病DNA疫苗的研制为狂犬病免疫开辟了一条新途径,并可为防治其他疾病的DNA疫苗的研制奠定基础。     

Analyses of reaction norms reveal new chromosome regions associated with tick resistance in cattle

Despite single nucleotide polymorphism (SNP) availability and frequent cost reduction has allowed genome-wide association studies even in complex traits as tick resistance, the use of this information source in SNP by environment interaction context is unknown for many economically important traits in cattle. We aimed at identifying putative genomic regions explaining differences in tick resistance in Hereford and Braford cattle under SNP by environment point of view as well as to identify candidate genes derived from outliers/significant markers. The environment was defined as contemporary group means of tick counts, since they seemed to be the most appropriate entities to describe the environmental gradient in beef cattle. A total of 4363 animals having tick counts (n=10 673) originated from 197 sires and 3966 dams were used. Genotypes were acquired on 3591 of these cattle. From top 1% SNPs (410) having the greatest effects in each environment, 75 were consistently relevant in all environments, which indicated SNP by environment interaction. The outliers/significant SNPs were mapped on chromosomes 1, 2, 5, 6, 7, 9, 11, 13, 14, 15, 16, 18, 21, 23, 24, 26 and 28, and potential candidate genes were detected across environments. The presence of SNP by environment interaction for tick resistance indicates that genetic expression of resistance depends upon tick burden. Markers with major portion of genetic variance explained across environments appeared to be close to genes with different direct or indirect functions related to immune system, inflammatory process and mechanisms of tissue destruction/repair, such as energy metabolism and cell differentiation.     

Immunogenicity of a killed whole Neospora caninum tachyzoite preparation formulated with different adjuvants

A killed whole Neospora caninum tachyzoite preparation was formulated with various adjuvants and tested for its immunogenicity in cattle. The adjuvants used were: Havlogen, a polymer of acrylic acid cross-linked with polyallylsucrose; Polygen, a non-particulate copolymer; a mixture of Havlogen and Bay R-1005, which is a preparation of free base synthetic glycolipids; and Montanide ISA 773, a water-in-oil emulsion made with a mixture of metabolisable and mineral oils. Immune responses in immunised cattle were compared with those of cattle experimentally infected with culture-derived N. caninum tachyzoites. The overall mean serum IFAT titres were significantly higher (P < 0.05) in experimentally infected cattle compared with all immunised cattle. Nonetheless, the maximum antibody titres of the immunised cattle, which were obtained following the third immunisation, were within the range of titres previously described for naturally infected cattle. The overall mean serum IFAT titres were significantly higher (P < 0.05) in cattle immunised with the killed tachyzoite preparation formulated with Polygen and with the mixture of Havlogen and Bay R-1005, compared with cattle immunised with the Havlogen- and Montanide-based preparations. Two of the four adjuvant preparations were able to induce cell-mediated immune responses similar to those of the experimentally infected cattle. The Havlogen-adjuvanted tachyzoite preparation elicited N. caninum-specific proliferation of peripheral blood mononuclear cells statistically similar (P = 0.095) to that of the infected animals. Peripheral blood mononuclear cells from animals immunised with the Polygen-adjuvanted tachyzoite preparation produced interferon-gamma concentrations of similar magnitude (P = 0.17) to those from the infected animals. Polygen was one of two adjuvants that elicited the highest antibody responses, and was the only adjuvant that induced interferon-gamma levels similar to those of the infected heifers.     

Hepatitis B virus-reactive human T lymphocyte clones: antigen specificity and helper function for antibody synthesis

To study immunity to hepatitis B surface antigen (HBsAg) at the cellular level, lymphocytes were obtained from the peripheral blood of hepatitis B vaccine recipients and were examined for various immune responses to HBsAg in vitro. The peripheral blood mononuclear cells (PBM) from most of the vaccinees did not proliferate to a great extent to HBsAg in vitro. However, HBsAg-reactive lymphocyte lines and clones were obtained from some of these individuals if the PBM were stimulated in vitro with HBsAg and were maintained in the presence of T cell growth supplement. Most of the HBsAg-reactive T cell clones obtained were found to be antigen-specific and some of them provided help in the production of anti-HBsAg antibodies by a cell population enriched for HBsAg-binding cells. These results indicate that HBsAg-specific T and B cells exist in the circulation of hepatitis B vaccine recipients, although they are at limiting concentrations for the in vitro cell proliferation and antibody production assays.     

The impact of a leptin gene SNP on beef calf weaning weights

Prior research indicates that a SNP at position 305 of exon 2 in the leptin gene affects milk production in dairy cows. Dairy cows with at least one copy of the T allele have been shown to have higher milk production than CC cows. If that effect carries over to beef breeds, it is reasonable to expect that CT and TT beef cows will wean heavier calves than CC beef cows. We tested this hypothesis for a herd of mixed breed cows using anova. Results indicated that both crossbred CT and TT beef cows wean significantly heavier beef calves than CC crossbred beef cows. A lack of observations generally hinders detection of significance in other breeds. However, two other comparisons were found to be significant. The results suggest further investigation into the link between leptin genotype and calf weaning weights. Aside from interest to animal scientists, these results have the potential to alter mating and replacement selection decisions by cow-calf producers, given the importance of weaning weights on profitability.