Induction of systemic immune responses in sheep by topical application of cholera toxin to skin

Cholera (and related) toxins (CT) when applied topically on unbroken skin induce systemic immune responses in mice, a procedure called transcutaneous immunization (TCI). The current study examined the capacity for TCI to induce systemic immune responses in sheep. Three groups (n=5 per group) were immunized at day 0 (priming) and day 28 (boosting) with 250 microg of CT in water by TCI, with 25 microg of CT in alum by intramuscular injection, or not immunized. Serum samples were taken at days 0, 28, 42, 56 and 70 after immunization for measurement of CT-specific IgG as well as CT-specific IgG1, IgG2, IgA and IgM antibodies by ELISA. After immunization, IgG, IgG1 and IgG2 antibody in immunized groups were significantly higher than in the control group, and boosting further increased these titres. IgG, IgG1 and IgG2 in the injection group were significantly higher than in the TCI group. There was a preponderance of IgG1 antibody, relative to IgG2, in both immunized groups. CT-specific IgA and IgM were detected in both immunized groups. Lymphocyte proliferation to CT was measured at day 90. A CT-specific lymphocyte proliferative response (stimulation index>2) was detected in all sheep from the injection group, in two sheep from the TCI group and in none of the controls. Results demonstrated that TCI induces primary and secondary antibody responses and specific proliferative responses to CT in sheep.     

Antibody isotype profiles in serum and circulating antibody-secreting cells following mucosal and peripheral immunisations of sheep

The isotype-specific antibody responses of sheep immunised with keyhole limpet hemocyanin by a peripheral route (intramuscular (i.m.) injection) were compared to those induced by immunisation via different mucosal routes: (1) intra-nasal spray; (2) rectal deposition with cholera toxin; (3) injection into the mucosa of the small intestine or rectum. Antigen-specific IgG1 antibodies were induced in the i.m., intra-intestinal and intra-rectal injection groups and in a proportion of the cholera toxin immunised sheep, but not in the intra-nasal immunisation group. IgA was the only antibody isotype detected in serum collected from the intra-nasal immunisation group. No significant differences in serum IgA levels were detected in any of the mucosal immunisation groups as compared to the i.m. injection group. In contrast, analysis of the in vitro antibody profiles secreted by circulating antibody-secreting cells (ASC) revealed significantly higher IgA responses in the supernatants from all mucosal immunisation groups. This suggests that the measurement of antibodies secreted by circulating ASCs may be a better correlate of local mucosal responses in ruminants, as has been previously demonstrated in human studies. In addition to IgG1 and IgA responses, immunisation by direct injection of antigen formulations into the intestinal and rectal mucosa were the only groups to induce consistently high IgG2 antibodies in serum and ASC cultures.     

Synthesis of immunoglobulin by normal and antigenically stimulated fetal sheep.

The rate of appearance, quantity and immunochemical character of serum immunoglobulins which appear normally during the development of fetal sheep and following the injection of antigens at different stages of gestation have been studied. After 70 days' gestation a percentage of normal fetal sheep synthesise IgM. Although the concentration of IgM in the circulation of these animals was very low, the precentage with IgM increased with fetal age. A few late term fetuses were detected which also had IgG1 in their circulation, although none were detected with IgG2 or IgA. Fetuses injected with antigens before 71 days' gestation only synthesised IgM, while fetuses injected after 79 days' gestation synthesised both IgM and IgG1. Neither IgG2 nor IgA were detected by single-radial immunodiffusion analyses during the first 14 days of primary immune responses to a variety of antigens, although trace amounts of IgG2 were detected late in responses occurring in older fetuses. The immunoglobulins synthesised by antigenically stimulated fetal sheep appeared identical to adult sheep 19S IgM, 7S IgG1 and 7S IgG2 respectively, when analysed by immunoelectrophoresis, isoelectric focusing and G200 Sephadex column chromatography.     

The intestinal and serum humoral immune response of mice to orally administered antigens in liposomes: II. The response to liposome-entrapped bacterial proteins.

Effective oral adjuvants are needed to improve the intestinal immune responses to oral vaccines that are based on relatively low molecular weight antigens refined from veterinary pathogens. Liposomes prepared by different methods and composed of phospholipids of varying transition temperature were used to entrap cholera toxin (CT) and fed to mice. No significant increase in the intestinal antibody nor the serum IgA antibody response was detected but levels of serum IgG anti-CT antibody were significantly elevated in the group fed CT in phosphatidylcholine-based liposomes. Levels of antibody were significantly reduced in the groups fed CT in dipalmitoylphosphatidylcholine liposomes. Escherichia coli wall extract (ECWE) entrapped in certain liposome types and fed to mice elicited significantly increased serum anti-ECWE antibody responses but intestinal antibody responses were insignificantly different from the controls. These results suggest that orally administered liposomes fail to act as potent intestinal adjuvants for the entrapped antigens of bacterial origin used in this study.     

Serum and mucosal antibody responses and protection in pigs vaccinated against Mycoplasma hyopneumoniae with vaccines containing a denatured membrane antigen pool and adjuvant

Objective To investigate the protective efficacy of a pool of denatured membrane protein antigens of Mycoplasma hyopneumoniae (J strain) in the molecular size range 70 to 85 kDa (F3 antigen) in combination with adjuvants for pigs challenged with M hyopneumoniae .
Design A vaccine efficacy experiment with assessment of serum and respiratory tract antibody responses.
Procedure F3 antigens were emulsified with five different adjuvants. To groups of three pigs per vaccine, four vaccines were given by intramuscular injection, and two vaccines, including one of those given intramuscularly, were given by intraperitoneal injection.
Results Compared to six unvaccinated pigs, animals vaccinated with F3 antigen displayed significantly reduced pneumonia (54% reduction in mean lung score) following experimental challenge. Analysis of post-vaccination, pre-challenge IgG and IgA ELISA antibody absorbances in serum and respiratory tract washings revealed no correlation with lung score. Six weeks after challenge, pigs previously vaccinated intramuscularly mostly demonstrated greater IgG and IgA responses in respiratory tract washings, and greater IgG serum antibody responses, than those vaccinated by intraperitoneal injection.
Conclusion Pigs vaccinated with M hyopneumoniae antigens in the molecular size range of 70 to 85 kDa showed a significant reduction in lung lesions compared with unvaccinated control animals after experimental challenge. IgG and IgA antibody concentrations in serum and respiratory tract washings after vaccination do not provide a useful prognostic indicator of protection from enzootic pneumonia.     

The effect of parenteral immunisation on antibody production in the pig colon

Local and systemic antibody production was studied in pigs to compare responses to live and killed bacterial antigen and purified protein antigen, with and without prior mucosal stimulation. Recovery from challenge with live bacteria and intramuscular injection with killed bacteria gave rise to similar high levels of serum IgG antibody, but the ratio of specific IgA to IgG in the colon was significantly higher after infection than following vaccination with killed bacteria. Vaccination with a protein antigen gave rise to serum and local antibody production. Prior feeding of the antigen had a tolerising effect on the serum antibody response, but production of IgG and IgA antibody by the colon was not suppressed.     

Production of antibodies in murine mucosal immunization with Toxoplasma gondii excreted/secreted antigens

Toxoplasmagondii RH strain excreted/secreted antigens (ESA) were administrated weekly by the oral route, to two groups of 40 OF1 mice for 4 weeks. One group received ESA associated with cholera toxin (CT+) and the other, ESA only (CT-). Five animals from each group were sacrificed from day 4 (D4) to D49 following the first immunization and their feces and sera were collected and tested by ELISA for IgA, IgG and IgM antibody detection. In feces, IgA antibodies were detected on D4 and on D12 in the CT+ and CT- groups, respectively, and they persisted up to D49. IgG antibodies were detected from D12 to D41 in the CT+ group and on D12 only in the CT- group. No IgM antibodies were detected. In sera, IgA antibodies were detected on D27, D41 and D49 only in the CT+ group. IgG and IgM antibodies were found on D12 and D4, respectively, in the CT+ group and starting from D27 in the CT- group. To our knowledge, this is the first demonstration that ESA, with or without CT, are immunogenic when administrated by the oral route.     

Local and systemic immune responses to Echinococcus granulosus in experimentally infected dogs

Local and systemic immune responses were studied in six dogs experimentally infected with the dog/sheep tapeworm Echinococcus granulosus. All dogs developed similar IgG antibody response to parasite antigens. In contrast, IgE and IgA responses differed widely. No relationship between IgA responses and parasite burden at the end of the infection were observed. Further, clear differences in the anti-parasite IgA response in serum as compared with specific IgA forming cells in mesenteric lymph nodes were observed within the same dog. An inverse association of anti-parasite IgE and parasite load seemed to be present, with the strongest IgE response in the one dog that had no worms in the intestine at the end of the experiment. No differences were observed in the numbers of intestinal mast cells and goblet cells among all infected dogs. However, the dog with no detectable parasite load had a marked reduction of detected mast cells in the submuscular and muscular layer of the mucosa. Our data give new insight into the immune response of dogs during E. granulosus infection and provide information that may be useful for the rational design of vaccines for the control of hydatid disease.     

The immunogenicity and efficacy of replication-defective and replication-competent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle

Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.     

Local antibody responses in the bile and faeces of sheep infected with Fasciola hepatica

The effect of infection with the liver fluke Fasciola hepatica on serum, bile and faecal immunoglobulin and antibody levels was studied in Scottish Blackface sheep. In the serum the immunoglobulins showing the most marked increase were IgG1 and IgG2 and their maximal values were reached at 16 weeks after infection. In the bile IgG2 rose to peak values at two weeks and IgG1, IgA and IgM were maximal at four weeks after infection. The levels of faecal IgG and IgA were low after primary infection but after reinfection a rapid increase in IgA concentration was observed within one to two weeks. Haemagglutinating antibody levels against egg antigens, juvenile and adult excretory-secretory antigens and adult fluke somatic antigens were evaluated. In the sera high titres were observed starting from two to four weeks after infection and persisting until 14 to 16 weeks. Bile haemagglutinating antibodies against excretory-secretory antigens showed the highest level at two and four weeks after infection while antibodies against adult somatic antigens reached maximal titres between four and eight weeks. Faecal antibody levels after primary infection were low but increased rapidly within two weeks after reinfection, coinciding with the elevation in faecal IgA concentration. However, there was no reduction in the number of flukes established in reinfected animals.     

Class-specific circulating antibodies in infections with Eimeria tenella

Using a three-layer immunofluorescent test, class-specific, parasite-specific circulating antibody responses to Eimeria tenella were investigated following oocyst infection, drug-truncated oocyst infection and the injection of non-living antigens. Following all three means of antigenic stimulation, IgG, IgM and IgA antibodies were detected. The response to drug-truncated infections was dose-dependent. The sequence of appearance of antibody was IgM, IgA, IgG, whilst the relative quantities were IgG greater than or equal to IgM greater than IgA.     

Immunity to bovine reproductive infections.

Protective immune responses in the genital tract are robust, as shown by convalescent and vaccine-induced immunity. Systemic immunity is crucial for systemic infections that result in reproductive failure (such as brucellosis, leptospirosis, and the systemic forms of C. fetus and H. somnus infection). Although IgA responses can protect against sexually transmitted or venereal infections, systemically induced IgG antibody responses also protect. IgA responses can be induced by immunization of the genital tract, where inductive sites develop after antigenic stimulation. The common mucosal immune system can also be used to induce a genital IgA response, as shown by intranasal vaccination. Lastly, it is necessary to determine which antigens of each infectious agent are protective and which types of immune responses protect best.     

猪流感病毒多表位抗原的小鼠黏膜免疫试验

为探讨经黏膜途径免疫接种猪流感病毒(SIV)多肽疫苗的可行性,选择STV主要结构蛋白血凝素(HA)的T、B抗原表位,将多个表位片段串联,定向克隆到原核表达载体pET-30a(+)中,对串联表达的重组猪流感病毒蛋白SIV-ep1、SW-ep2、SIV-el2进行了小鼠免疫试验。重组蛋白以包涵体形式表达,经洗涤定量后,采用灌胃途径免疫6周龄KM小鼠,检测免疫小鼠抗体及细胞免疫水平。结果发现三免后各免疫组血清IgG抗体与PBS对照组差异极显著(P<0.01);黏膜分泌型IgA抗体,所有免疫蛋白组与PBS组差异均极显著(P<0.01);细胞免疫检测,所有免疫组小鼠外周血T淋巴细胞增殖明显;猪流感病毒血凝抗体检测,SIV-ep1免疫组、SIV-ep2免疫组、SIV-ep1与LT蛋白免疫组抗SIVH1N1亚型HI效价达1:1024,SIV-el2蛋白免疫组HI效价达1:512。结果表明,猪流感病毒重组多表位抗原灌胃免疫小鼠,能产生理想的黏膜免疫抗体和血清抗体。     

Serum anti-trichostrongyle antibody responses of first and second season grazing calves

Serum anti-Ostertagia ostertagi and anti-Cooperia oncophora antibody responses were assessed in first season and second season calves grazing permanent paddocks. Calves without previous exposure to trichostrongyles were found to mount significant parasite-specific IgG1 antibody responses within two months of introduction to the pastures. A significant serum IgA response to O ostertagi and IgG2 responses to both O ostertagi and C oncophora antigens were also observed, but these responses were weaker. No consistent serum anti-trichostrongyle IgM responses were discernible in either age group. Second season grazing calves had significantly elevated IgG1, IgG2 and IgA antibody levels at turnout when compared to first season calves, but only IgA antibody levels against O ostertagi increased during the second grazing season. Comparison of serum antibody levels in first and second season calves grazed separately or together suggests that mixed grazing had no discernible effect on antigen priming.     

Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

Antigen dose modulates the immunoglobulin isotype responses of pigs against intramuscularly administered F4-fimbriae

Parenteral immunisation normally induces a systemic antibody response characterised by high IgG and low IgA responses. In the present study, the effect of different doses of F4-fimbriae on the isotype-specific antibody response after intramuscular immunisation was studied in pigs. Pigs were injected twice with a 9 weeks interval with either 1, 0.1 or 0.01 mg of F4-ETEC fimbriae. The dose of 1mg F4 induced significantly lower primary F4-specific IgG and IgM responses than the doses of 0.1 and 0.01 mg F4, but primed for an enhanced F4-specific IgM serum antibody response after the booster immunisation. Furthermore, the dose of 0.1mg induced the highest F4-specific IgA serum response which was significantly higher than after injection with 0.01 and 1mg F4. Moreover, both lower doses (0.1 and 0.01 mg) showed a higher number of F4-specific IgA and IgG antibody secreting cells (ASC) in the local draining lymph nodes of the pigs. This study demonstrated that low doses of purified F4-ETEC fimbriae, especially the 0.1mg dose, are optimal for inducing F4-specific IgA responses after IM immunisation.     

Kinetics and type of immune response following intranasal and subcutaneous immunisation of calves

Protection of animals against respiratory infections has long been known to depend on respiratory mucosal immunity. However, few studies have been reported on the immune response following intranasal (i.n.) immunisation with non-living, soluble antigens. This study determined the kinetics of the humoral and cellular immune responses in calves after i.n. immunisation with Limulus haemocyanin (LH) with cholera toxin adjuvant, or subcutaneous (s.c.) immunisation with LH in incomplete Freund's adjuvant. A proliferative response of peripheral blood mononuclear cells cultured in vitro with LH was observed in animals immunised 7-10 days after i.n. and s.c. immunisations with no significant differences between the two immunised groups. LH -specific antibody was present in the serum of animals immunised s.c. (IgM, IgG1 and IgG2) and i.n. (IgA). Although significant IgA responses were observed, i.n. immunisations in cattle with soluble protein antigens and cholera toxin as an adjuvant did not induce a strong systemic immune response.     

表面表达猪水肿病毒素保护性抗原重组大肠杆菌激发仔猪黏膜免疫反应的研究

以菌体表面表达猪水肿病毒素保护性抗原(SLT-ⅡeB)的重组大肠杆菌(PZBSPAX),对1日龄、15日龄仔猪口服免疫,用ELISA法对口服免疫后10 d、15 d、30 d仔猪肠黏液中特异性SIgA和血清中特异性IgA、IgG进行检测,结果显示口服免疫猪能产生较高水平的肠黏液特异性SIgA及血清特异性IgA和IgG,证明该重组菌能有效激发肠道黏膜免疫反应,同时也能激发全身性体液免疫反应.     

Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar)

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens – dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) – administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.     

Systemic and mucosal immune responses to H1N1 influenza virus infection in pigs

Influenza is a common respiratory disease in pigs, and since swine influenza viruses are zoonotic pathogens, they also pose human health risks. Pigs infected with influenza virus mount an effective immune response and are protected from subsequent challenge, whereas the currently available, inactivated-virus vaccine does not consistently confer complete protection to challenge. To develop and evaluate new vaccination strategies, it is imperative to fully understand the immune responses that are associated with protection following natural infection. Therefore, we have evaluated the phenotype and kinetics of immune responses to primary and re-challenge infection with H1N1 swine influenza virus in the pig. Through the use of isotype-specific antibody secreting cell ELISPOT assays, interferon-gamma ELISPOT assays and isotype-specific ELISAs on serum, nasal wash and bronchoalveolar lavage samples, we defined the humoral and cellular immune responses, both locally in the respiratory tract and systemically, to this viral infection. Virus-specific serum IgG, IgA, and HI titers all peaked 2-3 weeks after primary infection and did not substantially increase after re-challenge. The predominant virus-specific isotype in serum was IgG. Pigs responded with virus-specific IgG and IgA in both the upper (nasal washes) and lower (bronchoalveolar lavages) airways; IgA was the predominant isotype in both sites. Despite the fact that the pigs were completely protected from re-challenge, the antibody titers in the nasal washes increased. Results of the antibody-secreting cell ELISPOT assays demonstrated that the numbers of both IgG and IgA secreting cells in the nasal mucosa were dramatically higher than in any other tissue examined. In contrast, IFN-gamma secreting cells were predominantly localized to the spleen and tracheobronchial lymph nodes. These data will be helpful in the future development and evaluation of novel vaccines.