Induction of secretory immunity with bioadhesive poly (D,L-lactide-co-glycolide) microparticles containing Streptococcus sobrinus glucosyltransferase

The effect of mucosal delivery of Streptococcus sobrinus glucosyltransferase (GTF) in bioadhesive poly (D,L-lactide-co-glycolide) (PLGA) microparticles on induction of salivary IgA and serum IgG antibody responses was measured in Sprague-Dawley rats. Preparations of GTF/PLGA/gelatin microparticles, or PLGA/gelatin microparticles or GTF in alum, were administered four times at weekly intervals by intranasal or intragastric routes. Two subcutaneous injections of GTF in PLGA/gelatin microparticles or in alum were given to separate groups of rats. Significant elevations in salivary IgA antibody levels to S. sobrinus GTF were observed only in the groups immunized intranasally 28 days after immunizations were begun. Five of six rats given the GTF microparticles intranasally had positive salivary IgA antibody responses to GTF, and the mean salivary IgA antibody level of this group was 30-fold higher than any other mucosally or systemically immunized group. Salivary IgA responses in the GTF-microparticle group remained significantly higher than all other mucosally immunized groups for at least 10 weeks after the primary immunization. All rats in this group demonstrated aspects of anamnesis following a more limited secondary course of intranasal administration. Intranasal administration of GTF in microparticles also induced a serum IgG response to GTF in some rats. After secondary intranasal GTF microparticle administration, several rats had sustained serum IgG antibody levels that were within the range of sera from rats subcutaneously injected with GTF in microparticles or in alum. Thus intranasal delivery of GTF-containing bioadhesive microparticles induced the highest and longest lasting salivary immune response of any mucosal or systemic route or vehicle tested and could be expected to be a useful method for induction of mucosal immunity.     

Induction of secretory immunity with bioadhesive poly ( D,L‐lactide‐co‐glycolide) microparticles containing Streptococcus sobrinus glucosyltransferase

The effect of mucosal delivery of Streptococcus sobrinus glucosyltransferase (GTF) in bioadhesive poly ( d,l ‐lactide‐co‐glycolide) (PLGA) microparticles on induction of salivary IgA and serum IgG antibody responses was measured in Sprague‐Dawley rats. Preparations of GTF/PLGA/gelatin microparticles, or PLGA/gelatin microparticles or GTF in alum, were administered four times at weekly intervals by intranasal or intragastric routes. Two subcutaneous injections of GTF in PLGA/gelatin microparticles or in alum were given to separate groups of rats. Significant elevations in salivary IgA antibody levels to S. sobrinus GTF were observed only in the groups immunized intranasally 28 days after immunizations were begun. Five of six rats given the GTF microparticles intranasally had positive salivary IgA antibody responses to GTF, and the mean salivary IgA antibody level of this group was 30‐fold higher than any other mucosally or systemically immunized group. Salivary IgA responses in the GTF‐microparticle group remained significantly higher than all other mucosally immunized groups for at least 10 weeks after the primary immunization. All rats in this group demonstrated aspects of anamnesis following a more limited secondary course of intranasal administration. Intranasal administration of GTF in microparticles also induced a serum IgG response to GTF in some rats. After secondary intranasal GTF microparticle administration, several rats had sustained serum IgG antibody levels that were within the range of sera from rats subcutaneously injected with GTF in microparticles or in alum. Thus intranasal delivery of GTF‐containing bioadhesive microparticles induced the highest and longest lasting salivary immune response of any mucosal or systemic route or vehicle tested and could be expected to be a useful method for induction of mucosal immunity.     

牙龈卟啉单胞菌FimA蛋白和白细胞介素-15共表达质粒免疫小鼠的实验研究

目的观察重组质粒pIRES- fimA:IL15经滴鼻免疫BABL/c小鼠后诱导产生的血清和唾液抗体反应及白细胞介素- 15（IL- 15）对sIgA反应的调节作用。方法重组质粒pIRES- fimA:IL15与pIRES- fimA经滴鼻免疫和肌肉注射免疫BABL/c小鼠,以间接ELISA方法检测血清中IgG和唾液中sIgA抗体水平。结果重组质粒pIRES- fimA:IL15与pIRES- fimA经滴鼻免疫可诱导产生唾液sIgA反应,且该反应明显高于肌肉注射组,血清IgG水平与肌肉注射组无统计学差异。pIRES- fimA:IL15经滴鼻免疫产生的唾液sIgA滴度显著高于pIRES- fimA组,且有统计学差异（P<0.05）。结论滴鼻免疫可以作为抗牙龈卟啉单胞菌DNA疫苗有效的黏膜免疫途径,诱导循环和口腔局部抗体反应;IL- 15可以作为细胞因子佐剂通过滴鼻途径增强该疫苗诱导的sIgA反应强度。     

DNA防龋疫苗不同途径免疫BALB/c小鼠的实验研究

目的：观察编码pac结构基因A—P片段的重组质粒pCIA—P以不同途径免疫BALB／c小鼠后的特异性免疫反应。方法：重组质粒pCIA—P经鼻腔滴注和颌下腺区皮下腺注射两种途径免疫小鼠，ELISA法检测血清和唾液中特异性抗体水平动态变化。结果：滴鼻法和皮下注射法免疫后唾液IgA型特异性抗体和血清IgG型特异性抗体均明显升高，并持续数周。且滴鼻法比皮下注射法诱导唾液IgA型特异性抗体要早。结论：重组质粒pCIA—P是一种有效的免疫原，滴鼻法和颌下区皮下注射法接种防龋DNA疫苗都可有效诱导机体全身及局部黏膜免疫应答。而滴鼻法较皮下注射法能更早诱导局部黏膜免疫应答。     

变形链球菌防龋基因疫苗pcDNA3-gtfB免疫途径筛选的实验研究

目的:利用含有葡糖基转移酶抗原基因的重组质粒pcDNA3-gtfB作为基因疫苗,筛选有效的免疫途径。方法:重组质粒pcDNA3-gtfB通过股四头肌注射、鼻腔灌注和颌下腺周注射免疫Wistar大鼠,采用ELISA法测定血清 IgG、唾液IgA的动态变化。结果:经股四头肌注射免疫后产生的血清IgG抗体水平明显高于其它两组血清IgG抗体水平(P<0101),且鼻腔灌注组和颌下腺周注射组间的血清IgG抗体水平无显著性差异(P>0105)。各组唾液S-IgA 抗体水平均存在显著差异(P<0101),腺周注射组产生的唾液S-IgA抗体水平最高(P<0101)。结论:基因疫苗通过腺周注射免疫途径能最有效激发特异性唾液S-IgA抗体的产生,可望成为一种有效的防龋基因疫苗免疫途径,为下一步抗龋动物实验提供了实验基础。     

The effects of transforming growth factor- and interleukins 2,5 and 6 on immunoglobulin production in cultured rat salivary gland tissues

The effects of transforming growth factor-β, alone, and in combination with selected interleukins on immunoglobulin production were investigated using an in vitro rat tissue fragment culture system with either parotid, submandibular or sublingual gland tissue. In the majority of culture groups, TGF-β alone, or in combination with either interleukin 2 (IL-2), IL-5, IL-6 or IL-5 and IL-6 together, significantly increased immunoglobulin A (IgA) levels over those obtained in untreated cultures. The levels of IgG and IgM were generally not affected, with the exception of one sublingual and two submandibular groups(s), where cytokine administration up-regulated either IgG or IgM production. These data indicate that transforming growth factor-β in combination with IL-2, IL-5, IL-6 or IL-5 and IL-6 can exert a stimulatory effect on IgA production in vitro, supporting a potential regulatory role for these cytokines in salivary gland IgA responses.     

Correlation of salivary and serum IgG, IgA levels with total protein in oral submucous fibrosis

Oral submucous fibrosis (OSMF) is a disabling, potentially malignant condition of the oral cavity. The aetiology of OSMF is multifactorial but remains obscure. Although arecanut is considered to be the most important causative agent, responses observed in individuals using arecanut vary in relation to quantity and duration. It is considered that an immunological process is responsible for the pathogenesis of disease. We correlated salivary immunoglobulin A (IgA), salivary immunoglobulin G (IgG) and serum immunoglobulin A (IgA), serum immunoglobulin G (IgG), levels by turbidometric immunoassay. We estimated the levels of total serum protein (TSP) and haemoglobin (Hb) to determine the role of nutritional deficiency. The study population comprised 30 cases of OSMF and 10 controls. Five milliliters of blood and 2 ml of saliva were collected. Quantitative analysis of serum and salivary IgG, IgA was done by turbidometric immunoassay. TSP and Hb were estimated by Biuret and cyanmethaemoglobin methods, respectively. All patients showed significant (P < 0.01) increase in serum and salivary IgG, IgA levels as compared to controls. TSP patients showed significant (P < 0.01) decrease as compared to controls. Results of Hb in patients were not significant. The estimation of immunoglobulin levels is important to support the concept of autoimmune basis. Estimation of TSP and Hb suggests that nutrition has a definite role in OSMF.     

Impaired secretory immunity in dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa is a congenital disorder characterized by blistering of the skin and oral mucosa. This study investigated the hypothesis that children with dystrophic epidermolysis bullosa have impaired oral secretory immunity. Immunoglobulin A (IgA), secretory IgA and IgG concentrations, and IgA and secretory IgA antibody levels to Candida albicans, Lactobacillus casei and Streptococcus mutans were measured in whole saliva from 22 children with dystrophic epidermolysis bullosa and 22 matched controls. Salivary total IgA and total IgG concentrations were significantly raised in dystrophic epidermolysis bullosa due to serum leakage from oral blistering, but the converse was seen with secretory IgA. This suggestion of a mucosal immune defect was supported by decreased secretory IgA antibody responses to all three microorganisms tested. This apparent defect in secretory immunity in dystrophic epidermolysis bullosa may be due to mucosal involvement and damage resulting in impaired antigen sampling in mucosal associated lymphoid tissue or to impaired transport of secretory IgA across the salivary gland mucosa.     

Salivary gland delivery of pDNA-cationic lipoplexes elicits systemic immune responses

OBJECTIVE: To test the ability of two cationic lipoplexes, Vaxfectin and GAP-DLRIE/DOPE, to facilitate transfection and elicit immune responses from plasmid DNAs (pDNAs) after retrograde instillation into salivary glands. METHODS: Two pDNA expression vectors encoding either the influenza NP protein or human growth hormone (hGH) were complexed with the cationic lipid transfection reagents, GAP-DLRIE/DOPE or Vaxfectin, and delivered to the submandibular glands of rats. Samples from rats receiving the influenza NP protein pDNA and cationic lipoplexes were analyzed for anti-influenza NP-specific IgG1, IgG2a, and IgG2b in serum, and IgA in saliva, by an enzyme- linked immunosorbent assay (ELISA). Cytotoxic T-cell lymphocyte (CTL) assays were also performed. Transgene protein expression (hGH) was determined from extracts of submandibular glands of rats receiving hGH lipoplexes. RESULTS: Serum antibodies (IgG) against the NP protein developed and were highest in all rats vaccinated with GAP-DLRIE/DOPE or Vaxfectin. The major serum IgG subclass stimulated by this route of immunization was IgG2b, followed by IgG2a. CTL assay results showed statistically significantly higher percentage killing in the Vaxfectin group than controls (P < 0.05). No rats developed IgA antibodies to NP protein in saliva. Animals receiving plasmid encoding hGH and either lipoplex expressed significantly higher amounts of hGH compared with those receiving the hGH plasmid and water. Although hGH expression was higher in the animals receiving pDNA/Vaxfectin (approximately 30-fold > pDNA/water), the difference with those receiving pDNA/GAP-DLRIE/DOPE (approximately 10-fold > pDNA/water) was not significant. CONCLUSIONS: Retrograde instillation of pDNA complexed with Vaxfectin into the salivary glands can stimulate cytotoxic and humoral responses to the influenza NP protein antigen. Optimization of the conditions required to stimulate humoral and secretory antibody formation may facilitate use of this tissue for specific clinical applications of pDNA immunization.     

Oral immunization of humans with dehydrated liposomes containing Streptococcus mutans glucosyltransferase induces salivary immunoglobulin A2 antibody responses

Seven healthy adult volunteers each ingested an enteric-coated capsule containing 500 fig of Streptococcus mutans glucosyltransferase (GTF) in dehydrated liposomes for 3 consecutive days. The immunization regimen was repeated 28 days later. Parotid saliva and plasma were collected prior to and at a weekly interval for 8 weeks following the first immunization for analysis of anti-GTF activity by enzyme-linked immunosorbent assay. The levels of immunoglobulin A, (IgA), and IgA: anti-GTF activities increased in the parotid saliva from 5 of 7 individuals after immunization. Increases in the mean level of IgA, and lgA: anti-GTF responses peaked on day 35 (77% and 175% increase over baseline, respectively), although variation was noted in the kinetics and subclass of responses between individuals. No salivary IgG or IgM responses were observed. Low plasma IgM, IgG and IgA anti-GTF responses were seen in immunized subjects. Oral immunization with a dehydrated liposome-protein vaccine was effective in inducing a secretory IgA antibody response, which was primarily of the IgA: subclass. These results provide the first evidence for the use an oral dehydrated liposome-protein vaccine in humans.     

Oral immunization of humans with dehydrated liposomes containing Streptococcus mutans glucosyltransferase induces salivary immunoglobulin A2 antibody responses

Seven healthy adult volunteers each ingested an enteric-coated capsule containing 500 fig of Streptococcus mutans glucosyltransferase (GTF) in dehydrated liposomes for 3 consecutive days. The immunization regimen was repeated 28 days later. Parotid saliva and plasma were collected prior to and at a weekly interval for 8 weeks following the first immunization for analysis of anti-GTF activity by enzyme-linked immunosorbent assay. The levels of immunoglobulin A, (IgA), and IgA: anti-GTF activities increased in the parotid saliva from 5 of 7 individuals after immunization. Increases in the mean level of IgA, and lgA: anti-GTF responses peaked on day 35 (77% and 175% increase over baseline, respectively), although variation was noted in the kinetics and subclass of responses between individuals. No salivary IgG or IgM responses were observed. Low plasma IgM, IgG and IgA anti-GTF responses were seen in immunized subjects. Oral immunization with a dehydrated liposome-protein vaccine was effective in inducing a secretory IgA antibody response, which was primarily of the IgA: subclass. These results provide the first evidence for the use an oral dehydrated liposome-protein vaccine in humans.     

*编码Pac结构基因的DNA疫苗免疫动物的实验研究

目的：本项研究将已构建的编码pac结构基因A－P片段的重组质粒pCIA-P免疫Wistar大鼠，观察重组表面蛋白抗原PAc在大鼠体内不同组织中的原位表达以及免疫定菌鼠后的防龋效果。方法：重组质粒pCIA-P经股四头肌肌肉注射和颌下腺区皮下注射两种途径免疫大鼠，以免疫组化技术观察重组蛋白PAc在免疫部位的表达。采用股四头肌肌肉注射，颌下腺区皮下注射和颊粘膜下注射3种方法免疫定菌鼠，ELISA法检测血清和唾液中特异性抗体水平，Keyes计分法评估免疫定菌鼠后鼠磨牙的患龋情况。结果：大鼠股四头肌细胞中可见PAc蛋白不均匀的受限表达，双侧颌下腺组织均可检测到PAc蛋白的阳性染色，在导管内表达呈强阳性。用重组质粒pCIA-P经颌下腺区皮下注射和颊粘膜免疫的方法可显著增加唾液中抗PAc-IgA水平和血清中特异性抗PAc-IgG水平，重组质粒免疫定菌鼠后能显著降低定菌鼠龋损计分。特别是颌下腺区皮下和颊粘膜注射免疫组，大鼠牙本质龋的破坏程度明显低于其他组，结论：重组质粒pCIA-P是一种有效的免疫原，粘膜免疫是较理想的DNA防龋疫苗的接种途径。     

Salivary immunoglobulin A production against a synthetic oligopeptide antigen of Actinobacillus actinomycetemcomitans fimbriae

Actinobacillus actinomycetemcomitans is an important pathogen in human periodontal diseases. We attempted to produce effective antibodies against A. actinomycetemcomitans fimbrial antigen, which was shown to be an attachment factor. An oligopeptide based on the amino acid sequence of A. actinomycetemcomitans fimbriae was synthesized and conjugated with branched lysine polymer resin beads. Mice were immunized with the synthetic antigen together with one or more of Freund's incomplete adjuvant, the Ribi adjuvant system, liposomes, mouse interleukin 4 expression plasmids, and cholera toxin. After immunization, salivary immunoglobulin A (IgA) and serum IgG levels against the synthetic oligopeptide antigen were determined by enzyme-linked immunosorbent assay. The serum IgG responses against the fimbrial antigen were significantly higher in animals immunized intramuscularly with the Ribi adjuvant system. A high salivary IgA response to the fimbriae was induced by intranasal mucosal immunization with cholera toxin and mouse interleukin 4 expression plasmids.     

Intranasal immunization of mice with recombinant protein antigen of serotype c Streptococcus mutans and cholera toxin B subunit

The cholera toxin subunit and the recombinant cell-surface antigen (molecular mass of 190,000 Da) were administered intranasally to BALB/c mice. After 30 days, the mice were immunized intranasally with the recombinant protein antigen alone. High serum IgG and salivary IgA responses to the protein antigen were induced by the intranasal immunization.     

Studies on the induction of the humoral immune responses to Bacteroides gingivalis fimbrial antigen in mice

Serum and salivary antibody responses to Bacteroides gingivalis fimbriae administered either orally or subcutaneously (s.c.) with or without an adjuvant in various strains of mice were examined in this study. Following results were obtained. 1) Oral administration of B. gingivalis fimbriae with GM-53 as an adjuvant in liposomes, but not in Tris-HCl buffer, definitely enhanced the fimbriae-specific IgG responses, mainly IgG1 followed by IgG2b, IgG2a and IgG3 in serum and IgA response in saliva of BALB/c mice. On the other hand, s.c. injection of fimbriae with GM-53 or MDP-Lys (L18) also raised the fimbriae-specific IgG followed by IgA and IgM responses in serum, and both IgA and IgG responses in saliva of BALB/c mice. Oral immunization was less effective than s.c. injection in terms of the production of serum antibody in the mice. However, the level of salivary antibody of mice injected s.c. was similar to that of mice immunized orally. 2) High anti-fimbriae antibodies in serum were maintained in BALB/c mice immunized orally with fimbriae and GM-53 in liposomes for approximately 7 months after the primary immunizations. Oral administration also induced and held the fimbriae-specific IgA response in saliva for at least 6 months after the primary immunizations. The levels of fimbriae-specific IgA in saliva after the second boosters on days 123 and 124 were higher than those after the primary ones on days 27 and 28. 3) Among various strains of mice immunized orally with fimbriae and GM-53 in liposomes, BALB/c and DBA/2 mice (H-2d) significantly produced high levels of both serum IgG and salivary IgA antibodies specific for fimbriae. Furthermore, B10.D2 mice (H-2d) were responders followed by B10.BR (H-2k), while C57BL/10 mice (B10, H-2b) were low responders to the fimbriae. These results show that the combined use of fimbriae together with an adjuvant results in a sharply increased IgA antibody response in saliva and a predominantly stimulated IgG antibody in serum, and it was suggested that these responses are restricted by H-2 haplotype.     

靶向牙周炎DNA疫苗免疫原性及免疫效能的实验研究

目的：检测靶向牙周炎DNA疫苗pCTLA4-FimA的免疫反应性,并评价其免疫保护能力。方法：分3组免疫BALB/c小鼠：pCTLA4-FimA鼻黏膜免疫组,非靶向牙周炎DNA疫苗pFimA鼻黏膜免疫组,pCI载体鼻黏膜免疫组。酶联免疫吸附实验检测血清及唾液中特异性抗体水平。建立小鼠牙周炎模型,检测牙槽骨水平吸收程度。结果：与pFimA免疫组相比,pCTLA4-FimA免疫组诱导了显著增强的血清特异性IgG抗体和唾液特异性IgA抗体水平（P〈0.01）。与免疫前相比,3实验组牙槽骨均有吸收,其中pCTLA4-FimA免疫组牙槽骨水平吸收程度最低。结论：靶向牙周炎DNA疫苗pCTLA4-FimA能有效诱导机体的免疫反应,并抑制牙周炎的发展,效果优于非靶向牙周炎DNA疫苗pFimA。     

A DNA vaccine encoding a cell-surface protein antigen of Streptococcus mutans protects gnotobiotic rats from caries

A cell-surface protein antigen (PAc) of Streptococcus mutans is considered a virulence factor because it may mediate initial attachment of Streptococcus mutans to tooth surfaces. Thus, inhibiting PAc is predicted to provide protection against caries. To develop vaccines against dental caries, we constructed a DNA vaccine, pCIA-P, which encodes two high-conservative regions of PAc. Expression of the recombinant protein was obtained in eukaryotic cells in vitro and in vivo. In this report, we provide evidence that fewer caries lesions, and high levels of PAc-specific salivary IgA antibody and serum IgG antibody, were observed in gnotobiotic rats following targeted salivary gland (TSG) administration of pCIA-P. This study shows that the recombinant DNA vaccine pCIA-P could induce protective anti-caries immune responses and that TSG immunization is a promising strategy for the inhibition of dental caries.     

Influence of microparticle formulation on immunogenicity of SYI, a synthetic peptide derived from Streptococcus mutans GbpB

Subcutaneous immunization with SYI, a peptide construct based on Streptococcus mutans glucan binding protein B (GbpB) residues 113-132, significantly reduces experimental dental caries. Since mucosal immunization may be preferred for human vaccine applications, the present objective was to determine what formulation of SYI combined with polylactide-coglycolide microparticles could give rise to significant levels of salivary IgA antibody reactive with the native GbpB protein. A comparison of the SYI construct, loaded into or mixed with polylactide-coglycolide revealed the SYI-loaded microparticles to induce significant and sustainable levels of salivary and nasal wash IgA antibody to the peptide and the native protein. SYI mixed with unloaded microparticles was less effective in mucosal antibody response induction. These studies indicate that mucosal immunization with the SYI construct can induce salivary IgA antibody to a pathogenesis-associated component of S. mutans if delivered within polylactide-coglycolide microparticles, suggesting that this approach could successfully induce protective salivary immunity to dental caries caused by S. mutans.     

DNA防龋疫苗联合蛋白质疫苗诱导小鼠免疫应答的研究

目的:研究以一种新型免疫策略即追加相应蛋白质抗原来加强免疫DNA疫苗的免疫效果。方法:重组质粒pCIA-P经鼻腔滴注途径免疫小鼠,并以其相应抗原rPAc蛋白或rPAc蛋白及黏膜佐剂rCTB经鼻腔滴注加强免疫,ELISA法检测血清和唾液中特异性抗体水平。结果:以rPAc蛋白或rPAc蛋白及黏膜佐剂rCTB加强免疫可显著提高唾液中IgA型特异性抗体和血清IgG型特异性抗体水平。并且以rPAc蛋白和黏膜佐剂rCTB加强免疫组产生了最高水平的唾液IgA型特异性抗体和血清IgG型特异性抗体。结论:DNA防龋疫苗联合蛋白质疫苗可有效增强DNA防龋疫苗免疫效果。     

Remote glucosyltransferase-microparticle vaccine delivery induces protective immunity in the oral cavity

Intranasally administered dental caries vaccines show significant promise for human application. Alternate mucosal routes may be required, however, to induce caries-protective salivary IgA antibody in children with respiratory diseases. Since rectal mucosa contains inductive lymphoid tissue, we hypothesized that the rectal route could be used to induce salivary immunity to mutans streptococcal glucosyltransferase (GTF), resulting in protective immunity to experimental dental caries. We first explored the ability of glucosyltransferase, incorporated into polylactide-co-glycolide (PLGA) microparticles (MP), and administered rectally together with mucosal adjuvant, to induce a salivary IgA antibody response. Groups of Sprague-Dawley rats (6/group) were immunized rectally on days 0, 7, 14 and 21 with a) GTF-MP alone, b) GTF-MP with cholera toxin, c) GTF-MP with detoxified mutant Escherichia coli toxin (dLT), or d) sham immunized with PLGA and cholera toxin. An additional group was immunized intranasally with GTF-MP alone. Saliva and nasal washes of all intranasally immunized rats contained IgA antibody to glucosyltransferase on day 28. Salivary IgA antibody was also detected in 7/12 rats rectally immunized with GTF-MP and cholera toxin or dLT, although responses were lower than those obtained by intranasal immunization. Most fecal extracts from rectally delivered GTF-MP plus cholera toxin or dLT rats contained IgA antibody to GTF-MP. Low levels of fecal IgA antibody were detected in 3/6 intranasally immunized rats and 2/6 rats rectally immunized with GTF-MP alone. We then examined the extent to which salivary IgA antibody induced by the rectal route could be protective. At 25, 31 and 38 days of age, two groups of female Sprague-Dawley rats (13/group) were rectally immunized with GTF-MP and cholera toxin or with empty microparticles and cholera toxin (sham group). A third group was intranasally immunized with GTF-MP alone. After demonstrating salivary IgA responses to GTF in most GTF-immunized rats, all animals were infected with streptomycin-resistant Streptococcus sobrinus and placed on diet 2000. After 79 days of infection, total caries on molar surfaces were lower in both rectally (7.9 +/- 1.0) and intranasally (7.1 +/- 0.9; P < 0.0.03) immunized groups compared with the sham-immunized group (11.9 +/- 1.6). Smooth surface caries were significantly lower (P < 0.05) in both rectally and intranasally immunized groups. These results support the interconnectedness of the mucosal immune system and indicate that rectal immunization with GTF-MP, together with adjuvant, or intranasal immunization with GTF-MP alone, can induce protective levels of salivary antibody in rats.