Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women.

To determine which mucosal immunization routes may be optimal for induction of antibodies in the rectum and female genital tract, groups of women were immunized a total of three times either orally, rectally, or vaginally with a cholera vaccine containing killed Vibrio cholerae cells and the recombinant cholera toxin B (CTB) subunit. Systemic and mucosal antibody responses were assessed at 2-week intervals by quantitation of CTB-specific antibodies in serum and in secretions collected directly from mucosal surfaces of the oral cavity, rectum, cervix, and vagina with absorbent wicks. The three immunization routes increased levels of specific immunoglobulin G (IgG) in serum and specific IgA in saliva to similar extents. Rectal immunization was superior to other routes for inducing high levels of specific IgA and IgG in rectal secretions but was least effective for generating antibodies in female genital tract secretions. Only vaginal immunization significantly increased both specific IgA and specific IgG in both the cervix and the vagina. In addition, local production of CTB-specific IgG in the genital tract could be demonstrated only in vaginally immunized women. Vaginal immunization did not generate antibodies in the rectum, however. Thus, generation of optimal immune responses to sexually transmitted organisms in both the rectal and the genital mucosae of women may require local immunization at both of these sites.     

重组幽门螺杆菌尿素酶B亚单位疫苗鼻腔免疫的实验研究

目的:探讨基因工程疫苗Hp重组尿素酶B亚单位(rUreB)鼻腔接种的免疫效果。方法:以rUreB不同剂量或加不同佐剂滴鼻免疫BALB/c小鼠。末次免疫7 d后,收集血清及胃黏膜、小肠黏膜、鼻黏膜及气管黏膜冲洗液,用ELISA法检测抗rUreB特异性抗体。结果:rUreB鼻腔免疫后各实验组血清特异性IgG及各黏膜冲洗液中特异性IgA的水平均明显增高,与对照组相比较差异显著(P<0.01)。20μg剂量组与10μg剂量组相比较,仅血清特异性IgG水平增高,其它黏膜特异性IgA的水平未见增高。大肠杆菌不耐热肠毒素B亚单位(LTB)的佐剂效果较霍乱毒素B亚单位(CTB)强,卡泊波可增强鼻腔接种疫苗在胃黏膜洗液中的抗体应答水平。结论:CTB、LTB、卡泊波均可作为rUreB鼻腔黏膜接种的佐剂。HprUreB鼻黏膜接种,不仅可诱导血清特异性抗体反应,而且能引起多个黏膜部位的免疫应答,是一种方便、有效、廉价的免疫途径。     

Intranasal Immunization with Pneumococcal Polysaccharide Conjugate Vaccines with Nontoxic Mutants of Escherichia coli Heat-Labile Enterotoxins as Adjuvants Protects Mice against Invasive Pneumococcal Infections

Host defenses against Streptococcus pneumoniae depend largely on phagocytosis following opsonization by polysaccharide-specific immunoglobulin G (IgG) antibodies and complement. Since colonization of the respiratory mucosa is the first step in pneumococcal pathogenesis, mucosal immune responses may play a significant role. In addition to inducing systemic immune responses, mucosal vaccination with an effective adjuvant has the advantage of inducing mucosal IgA antibodies. The heat-labile enterotoxin (LT) of Escherichia coli is a well-studied mucosal adjuvant, and adjuvant activity of nontoxic LT mutants has been demonstrated for several protein antigens. We investigated the immunogenicity of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 in mice after intranasal (i.n.) immunization by using as an adjuvant the nontoxic LT mutant LT-K63 or LT-R72, which has minimal residual toxicity. Pneumococcal serotype-specific antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA), and vaccine-induced protection was evaluated by i.n. challenge with virulent pneumococci of the homologous serotype. When administered with LT mutants, i.n. immunization with both conjugates induced systemic and mucosal immune responses, and serum IgG antibody levels were significantly higher than after subcutaneous immunization. All mice immunized i.n. with PNC-1 and LT mutants were protected against bacteremia and cleared the pneumococci from the lung 24 h after i.n. challenge; pneumococcal density correlated significantly with serum IgG antibody levels. Similarly, the survival of mice immunized i.n. with PNC-3 and LT mutants was significantly prolonged. These results demonstrate that i.n. vaccination with PNC and potent adjuvants can protect mice against invasive and lethal pneumococcal infections, indicating that mucosal vaccination with PNC may be an alternative vaccination strategy for humans.     

Generation of female genital tract antibody responses by local or central (common) mucosal immunization

Genital antibody responses were compared in female mice immunized intravaginally (i.vag.) or intranasally (i.n.) with a bacterial protein antigen (AgI/II of Streptococcus mutans) coupled to the B subunit of cholera toxin. Serum and salivary antibodies were also evaluated as measures of disseminated mucosal and systemic responses. Although i.vag. immunization induced local vaginal immunoglobulin A (IgA) and IgG antibody responses, these were not disseminated to a remote secretion, the saliva, and only modest levels of serum antibodies were generated. In contrast, i.n. immunization was substantially more effective at inducing IgA and IgG antibody responses in the genital tract and in the circulation, as well as at inducing IgA antibodies in the saliva. Moreover, mucosal and systemic antibodies induced by i.n. immunization persisted for at least 12 months. Analysis of the molecular form of genital IgA indicated that the majority of both total IgA and specific IgA antibody was polymeric, and likely derived from the common mucosal immune system.     

Effect of oral immunization with recombinant urease on murine Helicobacter felis gastritis.

The ability of oral immunization to interfere with the establishment of infection with Helicobacter felis was examined. Groups of Swiss Webster mice were immunized orally with 250 micrograms of Helicobacter pylori recombinant urease (rUrease) and 10 micrograms of cholera toxin (CT) adjuvant, 1 mg of H. felis sonicate antigens and CT, or phosphate-buffered saline (PBS) and CT. Oral immunization with rUrease resulted in markedly elevated serum immunoglobulin G (IgG), serum IgA, and intestinal IgA antibody responses. Challenge with live H. felis further stimulated the urease-specific intestinal IgA and serum IgG and IgA antibody levels in mice previously immunized with rUrease but activated primarily the serum IgG compartment of PBS-treated and H. felis-immunized mice. Intestinal IgA and serum IgG and IgA anti-urease antibody responses were highest in rUrease-immunized mice at the termination of the experiment. Mice immunized with rUrease were significantly protected (P < or = 0.0476) against infection when challenged with H. felis 2 or 6 weeks post-oral immunization in comparison with PBS-treated mice. Whereas H. felis-infected mice displayed multifocal gastric mucosal lymphoid follicles consisting of CD45R+ B cells surrounded by clusters of Thy1.2+ T cells, gastric tissue from rUrease-immunized mice contained few CD45R+ B cells and infrequent mucosal follicles. These observations show that oral immunization with rUrease confers protection against H. felis infection and suggest that gastric tissue may function as an effector organ of the mucosal immune system which reflects the extent of local antigenic stimulation.     

Specific immune responses and enhancement of murine pulmonary clearance of Moraxella catarrhalis by intranasal immunization with a detoxified lipooligosaccharide conjugate vaccine

Moraxella catarrhalis is an important human mucosal pathogen. This study investigated the effect of intranasal immunization with a detoxified-lipooligosaccharide-cross-reactive mutant of diphtheria toxin (dLOS-CRM) vaccine candidate on pulmonary clearance following an aerosol challenge of mice with M. catarrhalis. Intranasal immunization with dLOS-CRM plus cholera toxin induced a significantly dose-dependent increase of immunoglobulin A (IgA) and IgG in the nasal wash, lung lavage fluid, saliva, and fecal extract. In addition, serum IgG, IgM, and IgA against LOS of M. catarrhalis were detected. LOS-specific antibody-forming cells were found in the nasal passages, spleens, nasally associated lymphoid tissues, cervical lymph nodes, lungs, and Peyer's patches using an enzyme-linked immunospot assay. The dLOS-CRM vaccine induced a significant bacterial clearance (70 to 90%) of both homologous and heterologous strains in the lungs compared to that observed in the controls (P < 0.01). Intriguingly, intranasal immunization with dLOS-CRM showed a higher level of bacterial clearance compared with subcutaneous injections with dLOS-CRM. These data indicate that dLOS-CRM induces specific mucosal and systemic immunity through intranasal immunization and also provides effective bacterial clearance. On the basis of these results, we believe that dLOS-CRM should undergo continued testing to determine whether it would induce protective immune response in humans.     

Intranasal coadministration of the Cry1Ac protoxin with amoebal lysates increases protection against Naegleria fowleri meningoencephalitis

Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 x 10(4) live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines.     

Intranasal Immunization with Pneumococcal Polysaccharide Conjugate Vaccines Protects Mice against Invasive Pneumococcal Infections

Host defenses against Streptococcus pneumoniae depend largely on opsonophagocytosis mediated by antibodies and complement. Since pneumococcus is a respiratory pathogen, mucosal immune responses may play a significant role in the defense against pneumococcal infections. Thus, mucosal vaccination may be an alternative approach to current immunization strategies, but effective adjuvants are required. Protein antigens induce significant mucosal immunoglobulin A (IgA) and systemic IgG responses when administered intranasally (i. n.) with the glyceride-polysorbate based adjuvant RhinoVax (RV) both in experimental animals and humans. The immunogenicity and efficacy of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 was studied in mice after i.n. immunization with RV. Antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA) and compared to antibody titers induced by parenteral immunization. The PNCs induced significant systemic IgG and IgA antibodies after i.n. immunization only when given with RV and, for serotype 1, serum IgG titers were comparable to titers induced by subcutaneous immunization. In addition, i.n. immunization with PNC-1 in RV elicited detectable mucosal IgA. These results demonstrate that RV is a potent mucosal adjuvant for polysaccharides conjugated to proteins. A majority of the PNC-1-immunized mice were protected against both bacteremia and pneumonia after i.n. challenge with a lethal dose of serotype 1 pneumococci, and protection correlated significantly with the serum IgG titers. Similarly, the survival of mice immunized i.n. with PNC-3 in RV was significantly prolonged. These results indicate that mucosal vaccination with PNC and adjuvants may be an alternative strategy for prevention against pneumococcal infections.     

Clostridium difficile toxin A carboxyl-terminus peptide lacking ADP-ribosyltransferase activity acts as a mucosal adjuvant

The receptor binding domains of the most potent mucosal adjuvants, bacterial toxins and plant lectins, are organized in repeat units to recognize specific sugar residues. The lectin-like structure of the C-terminal region of Clostridium difficile toxin A prompted us to investigate the mucosal adjuvant properties of a nontoxigenic peptide corresponding to amino acids 2394 to 2706 (TxA(C314)). We compared TxA(C314) adjuvant activity to those of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin subunit B (EtxB) coadministered orally or nasotracheally with poor peptide antigens (keyhole limpet hemocyanin [KLH] and hen egg lysozyme [HEL]). Levels of anti-KLH-specific serum immunoglobulin G (IgG) and IgA as well as that of mucosal IgA were significantly higher in animals immunized orally with TxA(C314) plus KLH than with KLH alone, CT plus KLH, or EtxB plus KLH. Following intranasal immunization with TxA(C314) plus HEL, levels of serum- and mucosa-specific antibodies were comparable to those induced by coadministering HEL with CT or EtxB. The TxA(C314) adjuvant effect following oral, but not intranasal, immunization was dose dependent. The analysis of the subclasses of anti-KLH-specific IgG isotypes and the cytokines released from splenocytes of immunized mice challenged in vitro with KLH indicates the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch. We conclude that TxA(C314) enhances immune responses against mucosa-coadministered foreign antigens and represents a promising mucosal adjuvant, especially because its ability to stimulate mixed Th1/Th2 responses with a strong a Th1 component is extremely worthwhile against intracellular pathogens.     

Recombinant cholera toxin B subunit is not an effective mucosal adjuvant for oral immunization of mice against Helicobacter felis.

Cholera toxin is a potent oral mucosal adjuvant for enteric immunization. Several studies suggest that commercial cholera toxin B subunit (cCTB; purified from holotoxin) may be an effective non-toxic alternative for oral immunization. The present study was performed, using an infectious disease model, to determine if the oral mucosal adjuvanticity of CTB is dependent on contaminating holotoxin. Mice were orally immunized with Helicobacter felis sonicate and either cholera holotoxin, cCTB or recombinant cholera toxin B subunit (rCTB). Serum immunoglobulin G (IgG) and intestinal immunoglobulin A (IgA) antibody responses were determined and the mice were challenged with live H. felis to determine the degree of protective immunity induced. All orally immunized mice responded with serum IgG antibody titres regardless of the adjuvant used. However, only mice immunized with either holotoxin or the cCTB responded with an intestinal mucosal IgA response. Consistent with the production of mucosal antibodies, mice immunized with either holotoxin or cCTB as adjuvants were protected from challenge while mice receiving H. felis sonicate and rCTB all became infected. cCTB induced the accumulation of cAMP in mouse thymocytes at a level equal to 0.1% of that induced by holotoxin, whereas rCTB was devoid of any activity. These results indicate that CTB possesses no intrinsic mucosal adjuvant activity when administered orally. Therefore, when used as an oral adjuvant, CTB should also include small, non-toxic doses of cholera toxin.     

Induction of specific immunoglobulin A in the small intestine, colon-rectum, and vagina measured by a new method for collection of secretions from local mucosal surfaces.

In order study patterns of local antibody responses following mucosal immunization of mice via different routes, a method for collection of secretions directly from mucosal surfaces was developed. Mice were immunized on days 0, 10, 17, and 24 by administration of cholera toxin into the oral cavity, stomach, colon-rectum, or vagina. At sacrifice on day 32, absorbent wicks were placed in the oral cavity and, via an applicator tube, into the vagina and distal colon-rectum and along the entire small intestine after flushing of luminal contents. Protein was quantitatively extracted from wicks, and specific anti-cholera toxin immunoglobulin A (IgA) and IgG were measured by enzyme-linked immunosorbent assay. Concentrations of specific IgA in secretions at various mucosal sites were dramatically influenced by the route of immunization. Oral immunization effectively induced IgA in saliva, and the intragastric route was optimal for induction of IgA in the small intestine. High levels of specific IgA appeared on the colonic-rectal mucosal surface only after rectal delivery of antigen. Oral, gastric, and rectal immunizations also produced distant responses in the vagina. Following vaginal immunization, however, neither local nor distant IgA responses were detected. These results suggest that vaccines intended for protection of colonic-rectal and vaginal mucosal surfaces might best be administered by the rectal route.     

Mucosal vaccination with recombinantly attenuated staphylococcal enterotoxin B and protection in a murine model

Previous work in our laboratory revealed that mice parenterally vaccinated with recombinantly attenuated staphylococcal enterotoxin (SE) or toxic shock syndrome toxin 1 develop protective antibodies against a lethal intraperitoneal (i.p.) toxin challenge. This study investigated the efficacy of nasal and oral immunizations with an SEB vaccine (SEBv) toward an i.p. or mucosal (via an aerosol) toxin challenge. Both vaccination routes, with the immunoadjuvant cholera toxin (CT), elicited comparable SEB-specific immunoglobulin A (IgA) and IgG levels in saliva. Nasal or oral inoculations also generated SEB-specific IgA, IgG, and IgM in the serum, but the nasal route yielded higher specific IgG titers. SEBv alone, when given nasally or orally, did not induce any detectable SEB-specific antibody. Mice vaccinated mucosally were protected against a 50% lethal dose of wild-type SEB given i.p. or mucosally, thus demonstrating that nasal or oral administration of this SEBv, with CT, elicits systemic and mucosal antibodies to SEB that protect against SEB-induced lethal shock.     

Antibody Responses in the Lower Respiratory Tract and Male Urogenital Tract in Humans after Nasal and Oral Vaccination with Cholera Toxin B Subunit

Nasal vaccine delivery is superior to oral delivery in inducing specific immunoglobulin A (IgA) and IgG antibody responses in the upper respiratory tract. Although an antibody response in the nasal passages is important in protecting against primary colonization with lung pathogens, antibodies in the lungs are usually required as well. We immunized 15 male volunteers twice nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum, bronchoalveolar lavage (BAL) fluid, and urine before and 2 weeks after immunization. Nasal immunization induced fivefold increases in the levels of specific IgA antibodies in BAL fluid of most volunteers, whereas there were no significant specific IgA responses after oral immunization. The specific IgG antibody level increased eightfold in BAL fluid in the nasally vaccinated subjects, and the major part of IgG had most probably been transferred from serum. Since the specific IgG response in serum was lower in the individuals vaccinated orally, the IgG response in BAL fluid in this group was also lower and not significant. In conclusion, nasal immunization is also preferable to the oral route when vaccinating against lower respiratory tract infections, and a systemic immune response is considerably more important in the lower than in the upper respiratory tract. Moreover, both nasal and oral immunizations were able to stimulate 6- to 10-fold specific IgA and IgG responses in urine in about half of the individuals, which indicates that distant mucosal vaccination might be used to prevent adhesion of pathogens to the urogenital tract.     

Immunoprophylaxis of Chlamydia trachomatis lymphogranuloma venereum pneumonitis in mice by oral immunization.

Groups of BALB/c mice were orally immunized with chlamydiae and challenged intranasally to determine whether oral immunization offers protection against pulmonary disease and to characterize the nature and kinetics of the chlamydial antibody response in the lung and other mucosal sites. Protection by oral immunization from chlamydial lung disease was demonstrated by lack of replication of the organism and the lack of chlamydial antigen in lung tissue. The chlamydial immunoglobulin A (IgA) antibody response was present at all body sites, reaching peak levels in the lung as well as in the serum. Classical IgA booster effect kinetics was observed after intranasal challenge, especially in the lung. Specific IgG antibody was detected at all body sites but at lower levels. Furthermore, animals immunized orally had no pneumonic process, as determined by histopathology. These studies also suggest that passively acquired specific serum IgG antibody may not significantly influence the course of mucosal replication of the organism. These observations indicate that oral immunization activating the gut-associated lymphoid tissue system gave total protection against chlamydial lung disease, suggesting migration of immunologically competent cells from the intestine to the lung.     

Systemic and mucosal immune responses in mice after mucosal immunization with group B streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate vaccine

Group B streptococci (GBS) colonize the female genital and rectal tracts and can cause invasive infection in susceptible newborns. An optimally effective GBS vaccine should induce mucosal and systemic immunity. In this study, we investigate the local and systemic immune responses to GBS type III capsular polysaccharide (CPS) after mucosal vaccination of mice via intranasal, peroral, rectal, and vaginal routes, with GBS type III CPS conjugated with recombinant cholera toxin B subunit (GBS III CPS-rCTB). Cholera toxin (CT) was added as an adjuvant. Immunoglobulin G (IgG) and IgA antibodies to the CPS were tested in serum, lungs, and intestinal, rectal, and vaginal extracts by enzyme-linked immunosorbent assay. The conjugated CPS administered by intranasal, peroral, rectal, and vaginal routes was much more effective at inducing both mucosal and systemic antibody responses to GBS III CPS than was unconjugated CPS. The CPS-specific immune responses in various organs were dependent on the route of immunization. Generally, the highest levels of IgA and IgG were generated in the regions or sites of the conjugate exposure. Thus, intranasal vaccination elicited the highest anti-CPS IgA and IgG antibody levels in the lungs, whereas peroral administration in the intestinal site and vaginal vaccination elicited the highest antibody levels in the vagina. Rectal vaccination was superior to the other routes in inducing high antibody levels in the rectum. The four routes of mucosal vaccination also induced distant antibody responses to CPS. Rectal vaccination induced high specific IgA levels in the vagina and intestine, and oral administration induced high specific IgA levels in the lungs and rectum. All four routes of vaccination with the conjugate elicited similarly high levels of anti-CPS IgG in serum. Intranasal vaccination with different doses of the conjugate (10, 30, and 80 microg of CPS) did not have a significant influence on the anti-CPS specific antibody responses. Intranasal immunization induced better antibody responses when one dose of the conjugate was divided and given on three consecutive days compared to administration of the full dose on one occasion. In conclusion, rectal and vaginal vaccination may be the best way of stimulating anti-CPS immune responses in the rectal and vaginal tracts, while high levels of anti-CPS antibodies in the lungs can be achieved after intranasal administration. The vaccination regimen thus might influence the mucosal immune response to CPS. This conjugate may serve as an effective mucosal vaccine for preventing mucosal colonization and invasive infection caused by GBS.     

Production of secretory immunoglobulin A against Shiga toxin-binding subunits in mice by mucosal immunization

The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.     

LOS诱导的特异性抗体分泌细胞的ELISPOT法检测

目的 :动态测定卡他性莫拉氏菌 (Moraxellacatarrhalis,M .cat)脱毒脂寡糖 (dLOS)蛋白质结合疫苗诱导的抗体分泌细胞的应答状态。方法 :以M .catdLOS蛋白质结合疫苗滴鼻免疫BALB/c小鼠。应用酶联免疫斑点试验 (ELISPOT)检测免疫小鼠不同免疫诱导部位和免疫效应部位 ,包括 :鼻相关淋巴组织 (NALT)、脾脏、颈部淋巴结、鼻内容物、肺脏和派伊尔氏结的特异性抗体分泌细胞 ,并同时测定血清、鼻冲洗液、肺泡灌洗液、唾液及粪便提取液中特异性IgA、IgG和IgM的水平。结果 :M .catdLOS蛋白质结合疫苗免疫小鼠的NALT、脾脏、颈部淋巴结、鼻内容物、肺脏和派伊尔氏结中 ,均测出分泌LOS特异抗体的抗体分泌细胞 ,以鼻内容物中IgA分泌细胞的数目最多 ,其次是在NALT和肺脏中 ,这与特异性抗体测定的结果相一致。结论 :M .catdLOS蛋白质结合疫苗经滴鼻免疫 ,能刺激产生LOS特异的黏膜和全身抗体分泌细胞的应答。ELISPOT试验具有快速、灵敏、特异的优点 ,为动态分析单个抗体分泌细胞应答规律提供了新方法。     

Mucosal immunity in the genital tract: prospects for vaccines against sexually transmitted diseases--a review.

PROBLEM: Consistent with the absence of protective immunity resulting from previous infection with Neisseria gonorrhoeae, the genital mucosal immune response in human gonorrhea is weak: only low levels of immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies are detectable against gonococci, and inflammatory cytokine responses are poor. METHOD OF STUDY: Mucosal immunization strategies designed to induce persisting genital antibody responses might afford protection against infection, if appropriate conserved antigens can also be identified. RESULTS: Intragastric or intranasal immunization with bacterial antigens expressed as recombinant chimeric proteins with cholera toxin A2/B subunits induced persisting IgA antibodies in genital and other secretions, and circulating IgG antibodies. CONCLUSION: Although gonococci may avoid inducing or even suppress immune responses during natural infection, alternative approaches to vaccine development may be successful. However, inadequate understanding of the origins of antibodies in the genital tract, and their effector mechanisms, will need to be rectified to make this possible.     

Salivary, nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the Cholera toxin B subunit.

Previous attempts to induce mucosal antibodies in rhesus monkeys by enteric immunization have resulted in only modest and short-lived responses, dominated by immunoglobulin M (IgM) antibodies in the plasma. In this study, two groups of rhesus monkeys were immunized intranasally three times at 2-week intervals with a bacterial protein antigen (AgI/II) either chemically coupled to or mixed with the B subunit of cholera toxin (CT), a known potent mucosal immunogen and carrier for other immunogens. Cells secreting antibodies, predominantly of the IgA isotype, to AgI/II and to CT were detected in the peripheral blood 1 week after each immunization, indicating the dissemination of IgA-secreting precursor cells through the mucosal immune system. IgG and, to a lesser extent, IgA antibodies to both proteins were induced in the plasma commencing after the second immunization. Plasma IgE concentrations and IgE antibody levels were not consistently raised during the immunization period. IgA antibodies were found in nasal and vaginal washes. Nasal IgG but not IgA antibodies showed a significant positive correlation with plasma IgG antibody levels, suggesting that they were largely derived by transudation from the circulation. Analysis of the molecular form of vaginal IgA indicated that both secretory and monomeric forms of IgA were present in various proportions. Furthermore, neither IgG nor IgA antibodies in vaginal washes were correlated with plasma antibody responses, suggesting the contribution of locally synthesized antibodies of both isotypes. Comparison of the responses between the two groups of animals showed only sporadic significant differences, indicating that intranasal immunization with AgI/II either coupled to or mixed with the B subunit of CT was equally effective at inducing generalized IgA antibody responses in the mucosal immune system and predominantly IgG antibodies in the plasma.     

Transcutaneous immunization induces mucosal and systemic immunity: a potent method for targeting immunity to the female reproductive tract

Female BALB/c mice were immunized with tetanus toxoid (TT) admixed with cholera toxin by direct application to shaved skin (Transcutaneous immunization, TCI). Tetanus toxoid-specific IgG and IgA in serum, saliva, vaginal lavage and fecal pellets were assayed by ELISA. Tetanus toxoid specific antibody-secreting cell (ASC) numbers were also determined by immunohistochemistry in sections of vagina, uterus, salivary gland and small intestine of immunized mice. TCI elicited significant levels of TT-specific IgG in serum, saliva and vaginal lavage, with the greatest increases over background seen in saliva (80-400 fold) and vaginal lavage (2-87 fold). TCI induced only modest levels of IgA in any of the samples tested (range 2-7 fold increase). In the absence of cholera toxin, application of TT alone did not result in detectable TT-specific antibodies in mucosal secretions. ASCs were found in all tissues following TCI. Cells were most frequent in uterus and vaginal tissues with ASC numbers less frequent in small intestine and salivary gland. This suggests that local production, rather than transudation from serum, is a major contributor of antibody in reproductive tract secretions. Further studies focussed on the role of sex hormones and immune induction following TCI. Animals immunized at the stage of oestrus cycle at which estrogen is abundant (Estrus), showed significantly lower levels of TT-specific IgG in vaginal lavage samples. Collectively, these data confirm the findings of Glenn and colleagues (1998), who showed TCI using cholera toxin can elicit high levels of serum IgG to both the toxin and co-administered antigen and further demonstrates that this route of immunization is particularly effective at eliciting humoral immunity in saliva and in the female reproductive tract.