Serum Specific Antibody Responses Research Articles

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) enhances the immune responses against Helicobacter pylori in BALB/c mice

Objective To evaluate the adjuvant activities of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) in enhancing humoral and cellular responses against Helicobacter pylori (H.pylori). Methods BALB/c mice were immunized with the protein antigens including UreA, UreB and NapA of H. pylori in combination with cGAMP as the adjuvant on 0 d and 14 d by subcutaneous administration. Then, the serum-specific antibody responses were evaluated by ELISA. Flow cytometry (FCM) and enzyme-linked immunospot assay (ELISpot) were used to detect the cellular immune responses occurred in spleen and mesenteric lymph nodes (MLN). Results Subcutaneous administration of protein antigens of H. pylori together with cGAMP induced strong humoral and cellular immune responses in BALB/c mice. The levels of serum-specific IgG antibodies induced by adding cGAMP as the adjuvant were significantly higher than those by immunizing with antigens alone. The levels of splenic IFN-γ-producing lymphocytes in response to H. pylori antigens and cGAMP immunization were significantly higher than those in the corresponding groups without using cGAMP. Conclusion By using cGAMP as an adjuvant, H. pylori antigens could elicit significantly stronger humoral and cellular immune responses in mice than those induced by the antigens only. As a stable small molecular compound with strong adjuvant activity, cGAMP has the potential to be used for the development of H. pylori vaccine. Key words: cGAMP; Adjuvant; Helicobacter pylori; Vaccine

Vaccination at different anatomic sites induces different levels of the immune responses

This study was to evaluate the effects of anatomical sites for vaccination on the immune responses. In experiment A, rats were subcutaneously (s.c.) immunized with a quintuplet vaccine twice at houhai acupoint, underjaw, popliteal fossa or back with a two weeks interval. The serum specific antibody levels were determined 2, 4 and 6 weeks after second immunization. Splenocytes were separated for detection of lymphocyte proliferation and cytokine mRNA expression. In experiment B, 10 female Rottweiler puppies at their age of 34 ± 2 days were subcutaneously injected with a bivalent vaccine Nobivac® Puppy DP containing live attenuated canine distemper virus (CDV) and parvovirus (CPV) for primary vaccination, and a quadrivalent vaccine Nobivac® DHPPI containing live attenuated canine distemper virus (CDV), adenovirus type 2 (CAV-2), parvovirus (CPV) and parainfluenza virus (CPIV) for subsequent vaccination at houhai acupoint (4 dogs), the shoulder (3 dogs) or the nape (3 dogs) region. Blood samples were collected at 0, 2, 4 and 6 weeks after vaccination for determination of serum specific antibody responses by ELISA. The results showed that injection of a vaccine in houhai acupoint induced the highest antibody responses in both rats and dogs. When a vaccine was injected in houhai acupoint, significantly increased proliferative responses to Con A and LPS as well as mRNA expression of IL-4, IL-10, IL-12 and IFN-γ of splenocytes were detected in rats. Therefore, houhai acupoint is recommended for injection of a vaccine to improve the immune response in dogs.

Vaccination of lambs against Haemonchus contortus with the recombinant rHc23. Effect of adjuvant and antigen dose.

Haemonchus contortus is the most pathogenic gastrointestinal helminth of small ruminants. Natural or experimental repeated infections and several native antigens confer a partially protective immune response but vaccination with subunit antigens has been elusive. Promising results have been obtained with a recombinant form of a somatic antigen (rHc23). In this paper we present the results obtained in vaccination trials in lambs using two dosages of rHc23 and standard adjuvants. Six-months old Manchego females lambs were vaccinated with rHc23 (50 or 200 μg/dose) adjuvanted with 1mL aluminum hydroxide on days -42, -28 and -14 or with 200 μg/dose rHc23 and 5 mg Quil A on days -49, -28 and -7. Control lambs were kept receiving only the adjuvants the same days or no treatment. Moreover one group did not receive any treatment or infection. On day 0 vaccinated lambs, untreated animals and those receiving the adjuvant were infected per os with a monospecific single infection of 4000 L3 of H. contortus. Infection was kept for 58 days and follow-up included the determination of serum specific antibody response (ELISA, WB), hematological parameters (eosinophil counts, hematocrit) and fecal egg counts (epg). Absence of hematocrit alterations, reduction of helminth’s eggs output and abomasal parasite burden at the end of the experiment were the efficacy criteria of vaccination with the recombinant. Immunization with both adjuvants and antigen dosages elicited strong antibody responses particularly with Quil A. Vaccinated groups showed significant reduction of fecal egg excretion and abomasal helminth burdens. Highest protection of lambs against challenge was achieved with aluminum hydroxide and 200 μg/dose rHc23 with a reduction of over 70% of the abomasal burden and over 80% of fecal egg output. Results suggest that rHc23 could be a valuable recombinant candidate for vaccination against haemonchosis. No clear relationship was found between antibody levels and protection this pointing towards involvement of both humoral and cellular components in the protective response elicited by rHc23.

Dietary soya allergen β-conglycinin induces intestinal inflammatory reactions, serum-specific antibody response and growth reduction in a carnivorous fish species, turbot Scophthalmus maximus L.

This study was conducted to investigate the effects of dietary β-conglycinin on the growth performance, digestion, gut morphology and immune responses of juvenile turbot (Scophthalmus maximus L.). Four diets were formulated to contain 0%, 2%, 4% and 8% purified β-conglycinin. Triplicate groups of 30 fish were fed to apparent satiation twice daily for 12 weeks. Fish fed 4% and 8% dietary β-conglycinin showed significantly reduced specific growth rate, feed efficiency ratio, apparent digestibility coefficient of nutrients and whole-body lipid contents, as well as a profound infiltration of mixed leucocytes in the lamina propria and a significant decrease in the absorptive surface of distal intestine. The expression of pro-inflammatory cytokines, TNF-α and IL-1β, in the distal intestine was significantly upregulated by 4% dietary β-conglycinin, whereas a significantly lower expression level of IgM and anti-inflammatory cytokine TGF-β1 was observed in fish fed 8% dietary β-conglycinin. Serum lysozyme and alternative complement pathway activity were first significantly enhanced by 2% dietary β-conglycinin and then rapidly declined by 4% and 8% dietary β-conglycinin. Respiratory burst activity of head kidney macrophages and serum superoxide dismutase activity were significantly suppressed by 4% and 8% dietary β-conglycinin. Dietary β-conglycinin (2–8%) significantly increased the level of specific antibody against β-conglycinin in serum. Collectively, these results suggested that higher levels of dietary β-conglycinin (4–8%) induced a variety of non-specific and specific immune responses and intestinal mucosal lesions in turbot, resulting in inferior feed utilization and poor growth performance.

Intrauterine Neospora caninum inoculation of heifers and cows using contaminated semen with different numbers of tachyzoites

Objective To investigate the potential of different Neospora caninum tachyzoite doses to infect heifers (experiment 1) and cows (experiment 2) when administered in utero by artificial insemination via contaminated semen. Methods In experiment 1, five groups of 5, 7, 8, 9, and 5 cyclic heifers were hormonally synchronized and artificially inseminated with semen containing 0 (A, controls), 10 2 (B), 5 × 10 3 (C), 5 × 10 4 (D), and 5 × 10 5 (E) live N. caninum NC-1 isolate-tachyzoites, respectively. Experimental infection was followed for 100 days. Parasitaemia and specific serum IgG, and interferon-gamma (IFN-γ) responses were studied. In experiment 2, four groups of 9, 10, 9, and 9 adult multiparous cows with confirmed infertility problems of diverse aethiology were hormonally synchronized and artificially inseminated with semen containing 0 (a, controls), 10 2 (b), 5 × 10 3 (c), and 5 × 10 5 (d) live N. caninum NC-1 isolate-tachyzoites, respectively. Experimental infection was followed for 63 days. Parasitaemia and specific serum IgG responses were studied. Results In experiment 1, parasitaemia was detected in 1, 2, and 3 heifers from groups B, C, and D, respectively, between 9 and 23 days after insemination. Persistent specific serum antibody responses were detected in 2 and 3 heifers from groups D and E, respectively. Transient specific serum antibody responses were detected in 2, 1 and 1 heifers from groups C, D, and E, respectively. In addition, 1 heifer from group B showed a serum-specific antibody level higher than cut off value at 21 days post-insemination. Heifers seroconverted between 23 and 47 days after insemination. Specific IFN-γ levels were detected in 1, 4, 6, and 3 heifers from groups B, C, D, and E, respectively, between 9 and 55 days after insemination. Pregnancy rate in the control group (60%) was higher than those observed in inoculated heifers (0–42.9%). Pregnancy rates in inoculated heifers were lower when the tachyzoite dose was increased (B 42.9%, C 12.5%, D 11.1%, and E 0%). In experiment 2, no Neospora DNA in blood nor specific serum IgG to N. caninum were detected in any of the cows studied, except in one cow inoculated with 5 × 10 5 tachyzoites (group d) which showed a relative index × 100 (RIPC) values of 9.4, 18.9, and 18.1 at 42, 56, and 63 days after insemination, respectively. Conclusions This study provides evidence that the intrauterine infection via contaminated semen using 5 × 10 4 and 5 × 10 5 tachyzoites caused persistent serum-specific antibody responses in some heifers. On the basis of serological data, a dose–response effect was also observed. In addition, N. caninum would be a probable cause of early foetal death in inoculated heifers. In contrast, results obtained in a similar experiment with cows showing confirmed infertility indicate that higher doses, such as of 5 × 10 5 tachyzoites, were necessary to induce seroconversion in at least one animal.

Systemic cytokine response in humans with chiclero’s ulcers

In this work, we report the existence of enhanced concentrations of IL-2 (Th1 cytokine), TNF-alpha (mainly a Th1 cytokine), IL-12p40, a strong delayed type hypersensitivity response, and the absence of a serum-specific antibody response (IgG or IgE) in patients with active chiclero's ulcers. There was a low serum concentration of IL-6 (a Th2 cytokine) that was further reduced once patients healed following antimonial therapy (i.e., 4 months after treatment started). There was an absence of serum IL-4 (a Th2 cytokine) in patients before and after healing. The serum content of nitric oxide, transforming growth factor-beta1, and IL-12p70 in patients with progressive chiclero's ulcers was not different from that of controls. Patients had an alteration in blood-cell counts and showed a poor T cell proliferation after in vitro stimulation with Leishmania mexicana gp63. We concluded that patients with progressive chiclero's ulcers develop a Th1 type of response at this stage of the disease which correlates with increases in circulating eosinophils and B cells and a reduction of circulating CD8(+) T lymphocytes.

Lack of specific antibody response in common variable immunodeficiency (CVID) associated with failure in production of antigen-specific memory T cells. MRC Immunodeficiency Group.

Several T cell defects have been described in the antibody deficiency disease, CVID, but there have been few data on the generation of responses of specific T cell populations to primary neoantigens. We have now used immunization with the neoantigens, keyhole limpet haemocyanin (KLH) and DNP-Ficoll, to evaluate immune responses in CVID patients and normal donors. B and T cell responses were examined 2 and 4 weeks post-immunization. Sera were examined for IgM and IgG anti-KLH responses by ELISA and for anti-DNP-Ficoll activity by haemagglutination. The frequency of KLH-responsive T cells was measured by DNA synthesis in a limiting dilution culture system. Low density cells enriched for dendritic cells were pulsed with KLH and cultured with different numbers of autologous T cells. T cells from normal donors and from patients showed a low frequency of antigen-specific precursor T cells (< or = 1:200,000). After KLH immunization the frequency increased in normal donors (1:60,000 and 1:30,000 at 2 and 4 weeks, respectively), while in CVID patients it did not change from the pre-immunization level. The defect may extend to a dysfunction of antigen-specific cells, rather than being solely due to the reduced numbers of cells, since mean responses of 'positive' wells were also reduced. The serum-specific antibody response paralleled the T cell data, in that all normal donors but none of the CVID patients generated IgG KLH-specific antibodies. CVID patients did produce IgM antibodies against the T-independent DNP-Ficoll, but at a lower level than normal controls. These data show that both T and B cells from CVID patients have defective responses to specific antigen, implicating both lineages in the antibody deficiency.

Enhanced antigen-specific delayed-type hypersensitivity and immunoglobulin G2b responses after oral administration of viable Lactobacillus casei YIT9029 in Wistar and Brown Norway rats.

In this study, the effects of orally administered viable Lactobacillus casei Shirota strain YIT9029 on the immunity parameters of Wistar and Brown Norway rats were examined. For this purpose, we used the Trichinella spiralis host resistance model. Two weeks before and during T. spiralis infection, rats were fed 10(9) viable L. casei bacteria 5 days per week. The T. spiralis-specific delayed-type hypersensitivity (DTH) response was significantly enhanced in both Wistar and Brown Norway rats given L. casei. In both rat strains fed L. casei, serum T. spiralis-specific immunoglobulin G2b (IgG2b) concentrations were also significantly increased. In the model, no significant effects of L. casei on larval counts or inflammatory reactions in the tongue musculature, body weights, or lymphoid organ weights were observed. Serum specific antibody responses, other than IgG2b, were not changed by feeding of L. casei. In contrast to L. casei, it was shown that orally administered Bifidobacterium breve or Bifidobacterium bifidum had no influence on the measured infection and immunity indices in the rat infection model. Since the rat DTH response is considered to be a manifestation of Th1 cell-mediated immunity and the IgG2b isotype has been associated with Th1 activity, it was concluded that Th1 cells could play an active role in the immunomodulatory effects of orally administered L. casei. Furthermore, our data do not indicate that the effect of oral supplementation with L. casei is dependent on the genetic background of the host.

Effects of ginseng treatment on neutrophil chemiluminescence and immunoglobulin G subclasses in a rat model of chronic Pseudomonas aeruginosa pneumonia.

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is almost impossible to eradicate with antibiotic treatment. In the present study, the effects of treatment with the Chinese herbal medicine ginseng on blood polymorphonuclear leukocyte (PMN) chemiluminescence and serum specific antibody responses were studied in a rat model of chronic P. aeruginosa pneumonia mimicking CF. An aqueous extract of ginseng was administered by subcutaneous injection at a dosage of 25 mg/kg of body weight/day for 2 weeks. Saline was used as a control. Two weeks after the start of ginseng treatment, significantly increased PMN chemiluminescence (P </= 0. 001) and a decreased level in serum of immunoglobulin G (IgG) against P. aeruginosa (P < 0.05) were found. Furthermore, a higher IgG2a level (P < 0.04) but lower IgG1 level (P < 0.04) were found in the ginseng-treated infected group than in the control group. In the ginseng-treated group the macroscopic lung pathology was milder (P = 0.0003) and the percent PMNs in the cells collected by bronchoalveolar lavage was lower (P = 0.0006) than in the control group. However, the alveolar macrophage (AM) chemiluminescence values were not significantly different in the two groups infected with P. aeruginosa. The differences between the ginseng-treated noninfected rats and the control group (without P. aeruginosa lung infection) for the PMN chemiluminescence and AM chemiluminescence were not significant. These results suggest that ginseng treatment leads to an activation of PMNs and modulation of the IgG response to P. aeruginosa, enhancing the bacterial clearance and thereby reducing the formation of immune complexes, resulting in a milder lung pathology. The changes in IgG1 and IgG2a subclasses indicate a possible shift from a Th-2-like to a Th-1-like response. These findings indicate that the therapeutic effects of ginseng may be related to activation of a Th-1 type of cellular immunity and down-regulation of humoral immunity.

Immune response of Clun Forest sheep to vaccination with membrane glycoproteins from Haemonchus contortus.

Clun Forest lambs were injected with a fraction containing integral membrane glycoproteins derived from the intestinal microvilli of Haemonchus contortus in two equal doses of 0.5, 5, 50 or 500 micrograms protein and then challenged with 10,000 infective larvae. The time-course of serum specific antibody responses were determined. Compared to the adjuvant control group, the animals injected with 1000 micrograms protein were better than 84% protected and those injected with 100 micrograms, better than 95% protected by all criteria. For these two groups parasite egg output was reduced 99% on average over the patent period. Two of the animals injected with 10 micrograms protein were partially protected, with 86% reduction in egg output. Two animals in the group injected with 1 microgram protein also showed partial protection. Antibody level correlated with protection.