Somatic antigens of adult Dicrocoelium dendriticum recognised by bile antibodies of naturally infected cattle

Bile samples, from slaughtered cattle harbouring between 120 and 280 adult lancet flukes, were used to investigate the range of somatic proteins inducing local antibody responses in naturally infected animals. Lancet fluke infections induced local (bile) antibody responses against Tris-buffered saline (TBS) soluble, sodium dodecyl sulphate (SDS) soluble and 2-mercaptoethanol (2-Me) soluble somatic proteins of adult Dicrocoelium dendriticum. IgA antibody isotypes predominated in the response against buffer-soluble somatic antigens, whereas SDS-soluble and 2-Me-soluble proteins induced similar level of both IgA and IgG1 antibodies. Analysis of the antigens recognised by particular isotype-specific bile antibodies suggests that different antigens preferentially induce isotype restricted antibody responses. The bile antibody response was highly species specific, only one antigen from somatic protein extracts of Fasciola hepatica being precipitated by bile samples showing the highest reactivity against D. dendriticum.     

Exogenous estrogen may exacerbate thrombophilia, impair bone healing and contribute to development of chronic facial pain

Humoral and cellular responses to Schistosoma bovis antigens have been evaluated over a period of 11 weeks in mice exposed to S. bovis cercariae and data analysed in the context of the parasitic parameters (worm and egg loads) recorded at days 30, 60 and 80 of the ongoing infection. Results revealed a decrease of worm burden, particularly marked for female worms, between day 60 and day 80 of infection suggesting a higher susceptibility of female schistosomes to attrition mechanisms. The B-cell response, studied by measuring the production of different isotypes, was directed against different stage specific antigens, with a predominance of IgG1 antibodies associated with a significant increase of IgA and IgE antibodies after egg deposition. The T-cell response, assessed after in vitro stimulation of splenocytes, showed a predominant production of Th-2 cytokines (IL-4, IL-5 and IL-10) occurring after egg laying. Interestingly in contrast to S. mansoni infection the Th-2 polarization did not seem to be exclusively triggered by egg-associated antigens since significant amounts of IL-10 were produced after stimulation with adult worm antigen preparation (SWAP) before the beginning of egg deposition.     

Community-acquired pneumonia in adults: initial antibiotic therapy

One of the current goals in vaccine development is the noninvasive administration of protective antigens via mucosal surfaces. In this context, the gut-associated lymphoid tissues have already been extensively explored. Vaccination via the nasal route has only recently been the focus of intensive investigation, and no live vector specifically designed for the respiratory mucosa is yet available. In this study we show that intranasal administration of the recombinant Bordetella pertussis BPGR60, producing the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm28GST) protective antigen fused to filamentous hemagglutinin, induces priming in mice for the production of serum antibodies. In addition to significant levels of anti-Sm28GST immunoglobulin A (IgA) antibodies, high levels of anti-Sm28GST serum antibodies were obtained after intranasal boost with the purified antigen or infection with S. mansoni following intranasal priming with BPGR60. These antibodies were of the IgG1, IgG2a, and IgG2b isotypes, suggesting a mixed immune response. No priming was observed in animals that had received nonrecombinant B. pertussis or purified Sm28GST, indicating specific priming by BPGR60. This priming was also evident in immune protection against S. mansoni challenge. Significant protection against worm burden and egg output was obtained in mice primed with BPGR60 and intranasally boosted with purified Sm28GST. A lower but still significant degree of protection against egg output was also obtained in mice infected with a single dose of BPGR60. These results indicate that intranasal administration of recombinant B. pertussis can prime for serum antibody responses against a foreign antigen and for heterologous protection.     

Relationship of impairment of schistosome 28-kilodalton glutathione S-transferase (GST) activity to expression of immunity to Schistosoma mattheei in calves vaccinated with recombinant Schistosoma bovis 28-kilodalton GST

Sera from calves vaccinated with the recombinant Schistosoma bovis-derived 28-kDa glutathione S-transferase (28GST) and subsequently naturally or experimentally exposed to Schistosoma mattheei were studied for their content of specific immunoglobulin G (IgG) and IgA antibodies to recombinant S. bovis 28GST as well as for their capacity to inhibit the enzymatic activity of the antigen. The results were analyzed in regard to the presence (natural infection) or absence (experimental infection) of a protective effect(s) (reductions in worm burden, egg load, fecal egg counts, and excretion of viable eggs) toward S. mattheei challenge. Under such conditions, no differences in the IgG- and IgA-specific antibodies to recombinant S. bovis 28GST or in the ability to block the catalytic function of the antigen between the two groups were recorded. Nevertheless, correlation analysis between the specific antibody responses to recombinant S. bovis 28GST and the inhibition of GST activity suggested an association with IgG in experimentally infected vaccinated animals, while in naturally infected vaccinated calves, the inhibitory activity appeared to be linked to a greater degree with IgA. These results suggest that in contrast to schistosomiasis in humans, IgG antibodies in calves with schistosomiasis may exhibit inhibitory functions toward GST enzymatic activity or have a modulatory effect on IgA antibody properties. Furthermore, sera from animals immunized with recombinant S. bovis 28GST recognized the native S. mattheei 28GST and achieved comparable levels of inhibition of activity of recombinant S. bovis 28GST and S. matthei 28GST, indicating the presence of cross-reactive epitopes on these two molecules.     

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.     

Intranasal immunization confers protection against murine Pneumocystis carinii lung infection

To evaluate the feasibility of mucosal immunization against Pneumocystis carinii (Pc) experimental infection, female BALB/c mice were intranasally immunized three times with soluble Pc antigens plus cholera toxin fraction B (Pc-CTB); control groups received either Pc antigen, CTB, or phosphate-buffered saline (PBS) alone. Two weeks after the last immunization, five animals from each group were sacrificed, and cellular and humoral immune responses were evaluated. The remaining five mice were CD4 depleted using a monoclonal antibody against mouse CD4 and inoculated with viable Pc. Significantly higher specific lymphoproliferative responses from tracheobronchial lymph node cells, immunoglobulin M (IgM) and IgG antibody levels in serum, and bronchoalveolar lavage (BAL)-derived IgA antibody concentrations were observed in the Pc-CTB group of mice relative to control groups (P < 0.01). Five weeks after challenge, no Pc organisms were observed in the lung smears of the Pc-CTB group, while the animals receiving antigen, adjuvant, or PBS had progressively higher numbers of Pc microorganisms. By Western blot analysis, a strongly reactive 55- to 60-kDa antigen was recognized by BAL IgA and by serum IgG. In summary, mucosal immunization elicited specific cellular and humoral immune responses and protected against Pc lung infection after immunosuppression.     

Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally co-administered bovine serum albumin

We examined the mucosal adjuvant activity of recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB by intranasal or oral co-administration of bovine serum albumin (BSA). Intranasal administration stimulated a high level of BSA-specific serum IgG antibody response and BSA-specific IgA antibody responses in the nasal and pulmonary lavages. Oral administration induced a moderate level of BSA-specific serum IgG antibody and a low level of BSA-specific IgA antibody in the large intestinal washes. These results show that CTB alone can act as an intranasal or oral delivery carrier; it also has strong adjuvant properties for stimulating serum IgG and mucosal IgA immune responses to unrelated, non-coupled antigens after intranasal or oral co-immunization.     

Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant

Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.     

Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyer's patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.     

Isotype-specific antibody responses to foot-and-mouth disease virus in sera and secretions of "carrier' and "non-carrier' cattle

Isotype-specific antibody responses to foot-and-mouth disease virus (FMDV) were measured in the sera and upper respiratory tract secretions of vaccinated and susceptible cattle challenged with FMDV by direct contact or by intranasal inoculation. A comparison was made between cattle that eliminated FMDV and those that developed and maintained a persistent infection. Serological and mucosal antibody responses were detected in all animals after challenge. IgA and IgM were detected before the development of IgG1 and IgG2 responses. IgM was not detected in vaccinated cattle. Challenge with FMDV elicited a prolonged biphasic secretory antibody response in FMDV "carrier' animals only. The response was detected as FMDV-specific IgA in both mucosal secretions and serum samples, which gained statistical significance (P < 0.05) by 5 weeks after challenge. This observation could represent the basis of a test to differentiate vaccinated and/or recovered convalescent cattle from FMDV "carriers'.     

Oral immunization with recombinant Norwalk virus-like particles induces a systemic and mucosal immune response in mice

Recombinant Norwalk virus-like particles (rNV VLPs) produced in insect cells were evaluated as an oral immunogen in CD1 and BALB/c mice by monitoring rNV-specific serum total and subclass immunoglobulin G (IgG) and intestinal IgA responses. Dose and kinetics of response were evaluated in the presence and absence of the mucosal adjuvant cholera toxin (CT). rNV-specific serum IgG and intestinal IgA were detected in the absence of CT, and the number of responders was not significantly different from that of mice administered VLPs with CT at most doses. The use of CT was associated with induction of higher levels of IgG in serum; this effect was greater at higher doses of VLPs. IgG in serum was detected in the majority of animals by 9 days postimmunization (dpi), and intestinal IgA responses were detected by 24 dpi. In the absence of CT, IgG2b was the dominant IgG subclass response in both mouse strains. Thus, nonreplicating rNV VLPs are immunogenic when administered orally in the absence of any delivery system or mucosal adjuvant. These studies demonstrate that rNV VLPs are an excellent model to study the oral delivery of antigen, and they are a potential mucosal vaccine for NV infections.     

IL-12 is a potent neonatal vaccine adjuvant

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-gamma and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.     

gamma/delta T cell-deficient mice have impaired mucosal immunoglobulin A responses

Mucosal tissues of mice are enriched in T cells that express the gamma/delta T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in gamma/delta T cell receptor-deficient (TCRdelta-/-) mice versus control mice of the same genetic background. The frequency of intestinal immunoglobulin (Ig) A plasma cells as well as IgA levels in serum, bile, saliva, and fecal samples were markedly reduced in TCRdelta-/- mice. The TCRdelta-/- mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toxoid plus cholera toxin as adjuvant. Conversely, the antigen-specific IgM and IgG antibody responses were comparable to orally immunized control mice. Direct assessment of the cells forming antibodies against the tetanus toxoid and cholera toxin antigens indicated that significantly lower numbers of IgA antibody-producing cells were present in the intestinal lamina propria and Peyer's patches of TCRdelta-/- mice compared with the orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of gamma/delta T cells suggests a specialized role for gamma/delta cells in mucosal immunity.     

Antibody response to pneumococcal polysaccharide vaccine in young, adult and old mice

The anti-pneumococcal antibody response was studied in young (5-week-old) and adult (10-week-old) BALB/c and CBA/J mice and in adult (9-10-week-old) and old (12-, 18- and 24-month-old) AB6F1 and B6D2F1 mice after s.c. immunization with a 23-valent pneumococcal polysaccharide vaccine. Both young and adult mice showed a significant IgM antibody response to the vaccine 6 days after immunization with 1-11 micrograms antigen. There were significant immune responses to serotypes 1, 2, 4 and 7F in contrast to small responses to serotypes 14, 19F and 23F after immunization with the vaccine. One month after immunization, there were only marginal differences in IgM anti-pneumococcal antibody levels to the vaccine (anti-PPS) between immunized and unimmunized BALB/c mice, whereas in CBA/J mice the anti-PPS remained higher in immunized than in unimmunized mice. Immunization of old mice induced a significant IgM antibody response 6 days after immunization, but the anti-PPS thereafter decreased rapidly towards preimmunization values in AB6F1 mice. A significant IgG anti-PPS was not detected in any of the mice studied. The IgA anti-PPS tended to vary over time with no consistent pattern. It is important to carefully consider age and strain of the mice used when studying the immune response to pneumococcal polysaccharide antigens.     

T-Cell-independent immunoglobulin G responses in vivo are elicited by live-virus infection but not by immunization with viral proteins or virus-like particles

Immunoglobulin G (IgG) responses to viruses are generally assumed to be T-cell dependent (TD). Recently, however, polyomavirus (PyV) infection of T-cell-deficient (T-cell receptor beta chain [TCR-beta] -/- or TCR-betaxdelta -/-) mice was shown to elicit a protective, T-cell-independent (TI) antiviral IgM and IgG response. A repetitive, highly organized antigenic structure common to many TI antigens is postulated to be important in the induction of antibody responses in the absence of helper T cells. To test whether the repetitive structure of viral antigens is essential and/or sufficient for the induction of TI antibodies, we compared the abilities of three forms of PyV antigens to induce IgM and IgG responses in T-cell-deficient mice: soluble capsid antigens (VP1), repetitive virus-like particles (VLPs), and live PyV. Immunization with each of the viral antigens resulted in IgM production. VLPs and PyV elicited 10-fold-higher IgM titers than VP1, indicating that the highly organized, repetitive antigens are more efficient in IgM induction. Antigen-specific TI IgG responses, however, were detected only in mice infected with live PyV, not in VP1- or VLP-immunized mice. These results suggest that the highly organized, repetitive nature of the viral antigens is insufficient to account for their ability to elicit TI IgG response and that signals generated by live-virus infection may be essential for the switch to IgG production in the absence of T cells. Germinal centers were not observed in T-cell-deficient PyV-infected mice, indicating that the germinal center pathway of B-cell differentiation is TD even in the context of a virus infection.     

Vaccine potential of a recombinant glutathione S-transferase cloned from Schistosoma haematobium in primates experimentally infected with an homologous challenge

Patas monkeys were twice immunized with a Schistosoma haematobium-derived recombinant glutathione S-transferase (Sh28GST) then challenged with an homologous calibrated challenge. BCG and Freund's Complete Adjuvant (FCA) were used as adjuvants in two distinct protocols. Specific IgG and IgA antibody responses were intense and homogeneous in the animals receiving Sh28GST in the presence of FCA, whereas BCG could only induce moderate and heterogeneous antibody titres. No significant effect on worm burdens was evidenced 36 weeks post-infection in either group of Sh28GST-immunized animals compared to their matched controls receiving an irrelevant protein. Although not significant, 50% reductions in the numbers of eggs located in all tissues (FCA group) and in the urogenital system (BCG group) were noted. Moreover, the total number of excreted eggs was dramatically diminished by 60% and 77% in the BCG and FCA groups, respectively. These reductions reached 75% and 80% in the urines of vaccinated monkeys. Bladder pathology was also reduced in the animals displaying the lowest urinary egg excretions. There was no clear positive or negative correlate between antibody responses and individual levels of protection. Taken as a whole, our results show that Sh28GST was capable of significantly reducing S. haematobium worm fecundity in experimentally infected primates. Although FCA induced higher levels of protection, the efficacy of BCG as an adjuvant appeared sufficient to justify consideration of the future application of this new formulation as a vaccine against human urogenital schistosomosis.     

HIV gp120-specific cell-mediated immune responses in mice after oral immunization with recombinant Salmonella

Salmonella is of great interest as a potential human immunodeficiency virus vaccine vector because of its ability to elicit potent mucosal and systemic immune responses when administered orally. To determine whether such a vaccine could elicit an immune response in mice, plasmids expressing HIV gp120-LAI were introduced into attenuated S. typhimurium. Three serial doses of 10(10) recombinant organisms were administered orally to BALB/c mice at 2-week intervals. Immunized mice but not control mice demonstrated proliferative T cell responses to gp120-LAI, comparable in magnitude to the proliferative responses to Salmonella antigens. Immunized mice had detectable serum and intestinal Salmonella-specific IgA and serum Salmonella-specific IgG. However, no gp120-specific antibody was detected in either serum or intestinal washes. These results indicate that live recombinant Salmonella-based vaccine constructs can induce HIV-specific cellular immune responses in vivo.     

Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route

GRA4, a dense granule protein of Toxoplasma gondii elicits both mucosal and systemic immune responses following oral infection of mice with cysts. We studied the antigenicity and immunogenicity of truncated and soluble forms of GRA4 expressed as glutathione S-transferase fusion proteins in Escherichia coli. Protein C (amino-acids 297-345) was particularly well recognized by serum IgG antibodies, milk IgA antibodies and intestinal IgA antibodies from T. gondii infected mice and by serum IgG antibodies from T. gondii infected humans and T. gondii infected sheep. One major B epitope was localized within the last 11 C-terminal residues of GRA4. A second epitope, recognized with lower frequency, was mapped within the region 318-334. In contrast, the N domain of GRA4 (amino acids 25-276) was poorly recognized. Oral immunization of C57BL/6 mice with N, C or NC (amino acids 25-276 fused to 297-345) in association with cholera toxin induced a significant production of serum anti-GRA4 IgG antibodies but a weak and inconsistent intestinal anti-GRA4 IgG antibody response and afforded partial resistance to oral infection with T. gondii. These results provide new molecular and immunological understanding of GRA4 and indicate that it is a potential candidate for oral vaccination against T. gondii.     

A nontoxic adjuvant for mucosal immunity to pneumococcal surface protein A

In this study, we demonstrated that pneumococcal surface protein A (PspA) nasally administered with a nontoxic A subunit mutant of cholera toxin (mCT) S61F elicited a protective immune response. Immunization with PspA and mCT elicited higher levels of PspA-specific IgG and IgA Abs in serum and of IgG and IgA anti-PspA Ab-forming cells in spleens, cervical lymph nodes (CLN), and lung tissue when compared to nonimmunized mice. Furthermore, significant PspA-specific IgA Abs were induced in saliva and nasal secretions. These responses were dependent on the use of mCT as a mucosal adjuvant. The PspA-specific Ab responses induced by mCT S61F were comparable with those induced by native CT (nCT). Analysis of cytokine responses showed that nasal PspA plus mCT S61F enhanced the induction of PspA-specific CD4+ T cells producing IL-4 but not IFN-gamma in CLN at both the protein and mRNA levels. Importantly, significant numbers of mice intranasally immunized with PspA plus mCT S61F were protected from lethal challenge with capsular serotype 3 Streptococcus pneumoniae A66. These results show that intranasal administration of PspA together with mCT S61F is an effective mucosal vaccine against pneumococcal infection and induces CD4+ Th2-type cells, which provide help for both mucosal and systemic Ab responses.