Porphyromonas gingivalis virulence in mice: induction of immunity to bacterial components.

Selected cell envelope components of Porphyromonas gingivalis were tested in a BALB/c mouse model in an attempt to elucidate further the outer membrane components of this putative oral pathogen that might be considered as virulence factors in host tissue destruction. Lipopolysaccharide (LPS), outer membrane, and outer membrane vesicles of P. gingivalis W50, ATCC 53977, and ATCC 33277 were selected to examine an immunological approach for interference with progressing tissue destruction. Mice were actively immunized with heat-killed (H-K) or Formalin-killed (F-K) whole cells or with the outer membrane fraction, LPS, or outer membrane vesicles of the invasive strain P. gingivalis W50. The induction of invasive spreading lesions with tissue destruction and lethality were compared among different immunization groups in normal, dexamethasone-treated (dexamethasone alters neutrophil function at the inflammatory site), and galactosamine-sensitized (galactosamine sensitization increases endotoxin sensitivity) mice after challenge infection with the homologous strain (W50) and heterologous strains (ATCC 53977 and ATCC 33277). Enzyme-linked immunosorbent assay analyses revealed significantly elevated immunoglobulin G and M antibody responses after immunization with H-K or F-K cells or the outer membrane fraction compared with those of nonimmunized mice. The killed whole-cell vaccines provided significantly greater protection against challenge infection in normal mice (decreased lesion size and death) than did either the outer membrane fraction or LPS immunization. The lesion development observed in dexamethasone-pretreated mice was significantly enhanced compared with that of normal mice after challenge with P. gingivalis. Immunization with P. gingivalis W50 provided less protection against heterologous challenge infection with P. gingivalis ATCC 53977; however, some species-specific antigens were recognized and induced protective immunity. Only viable P. gingivalis induced a spreading lesion in normal, dexamethasone-treated, or galactosamine-sensitized mice; F-K or H-K bacteria did not induce lesions. The F-K and outer membrane vesicle immunization offered greater protection from lesion induction than did the H-K immunogen after challenge infection simultaneous with galactosamine sensitization. The H-K cell challenge with galactosamine sensitization produced 100% mortality without lesion induction, suggesting that LPS or LPS-associated outer membrane molecules were functioning like endotoxin. Likewise, P. gingivalis W50 LPS (1 micrograms per animal) administered intravenously produced 80% mortality in galactosamine-sensitized mice. In contrast to the effects of immunization on lesion development, immunization with H-K or F-K cells or LPS provided no protection against intravenous challenge with LPS; 100% of the mice died from acute endotoxin toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of immunization on experimental Bacteroides gingivalis infection in a murine model.

BALB/c mice were immunized with an invasive (A7A1-28) or noninvasive (381) Bacteroides gingivalis strain, Bacteroides intermedius, or Ringer solution. All immunized mice were subsequently challenged with the invasive B. gingivalis strain and examined for septicemia or secondary spread of the infection or both. Mice immunized with the invasive B. gingivalis strain localized the infection to the challenge site. Mice immunized with the noninvasive B. gingivalis strain, B. intermedius, or Ringer solution developed spreading infections. These data suggest that immunization with an invasive B. gingivalis strain can alter the course of subsequent infections.     

Experimental Porphyromonas gingivalis infection in nonimmune athymic BALB/c mice.

The purpose of this report was to study the role of T lymphocytes following injection of Porphyromonas gingivalis in a mouse abscess model. Three invasive P. gingivalis isolates (ATCC 53977, W83, and AJW4) were injected into athymic BALB/c mice and their heterozygous (nu/+) littermates. The athymic BALB/c (nu/nu) mice were able to localize the invasive P. gingivalis isolates at the injection site. By comparison, the heterozygous BALB/c (nu/+) littermates developed hemorrhagic secondary lesions within 24 h after subcutaneous injection of the same invasive P. gingivalis isolates. These results suggest that naive T lymphocytes may contribute to the pathology associated with P. gingivalis infection.     

Comparison of protective efficacy of subcutaneous versus intranasal immunization of mice with a Brucella melitensis lipopolysaccharide subunit vaccine

Groups of mice were immunized either subcutaneously or intranasally with purified Brucella melitensis lipopolysaccharide (LPS) or with LPS as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (LPS-GBOMP). Control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally with virulent B. melitensis strain 16M 4 weeks after the second dose of vaccine. Sera, spleens, lungs, and livers of mice were harvested 8 weeks after challenge. The bacterial loads in the organs were determined by culture on brucella agar plates. Protective efficacy was determined by comparing the clearance of bacteria from organs of immunized mice with the clearance of bacteria from organs of control mice. At 8 weeks postchallenge there was significant protection from disseminated infection of spleens and livers of mice intranasally immunized with either vaccine compared to infection of control mice (P < 0.01). There was no significant difference in clearance of bacteria from the lungs of immunized mice and control mice. However, mice immunized subcutaneously with either LPS or LPS-GBOMP vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). These results show that intranasal immunization of mice with either vaccine provided significant protection against disseminated infection of the spleen and liver but subcutaneous immunization of mice with the vaccines conferred significant protection against infection of the spleen, liver, and lungs.     

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F. nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria, F. nucleatum had no effect on the T cell production of cytokines induced by P. gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F. nucleatum alone had high levels of serum anti-F. nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P. gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-F. nucleatum antibodies in mice injected with F. nucleatum followed by P. gingivalis were the same as in mice immunized with F. nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F. nucleatum produced equal levels of both anti-P. gingivalis and anti-F. nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F. nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F. nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P. gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.     

Influence of immunization on Porphyromonas gingivalis colonization and invasion in the mouse chamber model.

The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.     

Orally administered microencapsulated Bordetella pertussis fimbriae protect mice from B. pertussis respiratory infection.

Fimbriae from Bordetella pertussis have been encapsulated in poly(lactide-co-glycolide) microparticles of a size appropriate for uptake by the immune inductive tissues of the gastrointestinal tract. Mice were immunized by oral gavage with a single dose of 10 micrograms of microencapsulated fimbriae. The resulting immune responses were compared with those resulting from intraperitoneal injection of mice with equivalent amounts of fimbriae absorbed onto alhydrogel. The examination of serum and mucosal secretions, collected over a 6-week period, for specific antifimbrial antibodies clearly demonstrated that only orally immunized animals mounted measurable immune responses in external secretions. Six weeks after immunization, all immunized animals were protected against intranasal challenge with live B. pertussis.     

Effect of preexisting immunity to Salmonella on the immune response to recombinant Salmonella enterica serovar typhimurium expressing a Porphyromonas gingivalis hemagglutinin

Recombinant Salmonella strains expressing foreign heterologous genes have been extensively studied as live oral vaccine delivery vectors. We have investigated the mucosal and systemic immune responses following oral immunization with a recombinant Salmonella enterica serovar Typhimurium expressing the hemagglutinin HagB from Porphyromonas gingivalis, a suspected etiological agent of adult periodontal disease. We have previously shown a primary mucosal and systemic response following oral immunization with chi4072/pDMD1 and recall responses following boosting at 14 weeks after primary immunization. In this study, we examined the effects of earlier boosting as well as the effects of deliberately induced immunity to the Salmonella carrier strain on subsequent immune responses. Mice boosted at week 7 following immunization, a point which corresponded to the peak of the primary response, generally showed lower responses than those boosted at week 14. When mice were preimmunized with the Salmonella carrier alone and then immunized with the recombinant strain 7 or 14 weeks later, significant reductions were seen for serum immunoglobulin G (IgG) antibodies at week 14 and for salivary IgA at week 7. No reductions were seen in serum IgA or vaginal wash IgA antibodies. Mice appear to be refractory to boosting with orally administered salmonellae at 7 weeks. Deliberate immunization with the carrier strain did not appreciably affect recall responses at 14 weeks, with the exception of the serum IgG responses, nor did it affect colonization of the Peyer's patches.     

Liposome potentiation of humoral immune response to lipopolysaccharide and O-polysaccharide antigens of Brucella abortus.

Liposomes were evaluated for their effectiveness as vaccine carriers in the potentiation of the mouse humoral response to the lipopolysaccharide (LPS) and O-polysaccharide (OPS) antigens of Brucella abortus. LPS and OPS were extracted from a pathogenic strain of B. abortus and were encapsulated within multilamellar vesicles. Groups of mice, immunized with liposome-encapsulated and free LPS or OPS, were bled weekly and the specific IgM and IgG levels in the sera were determined by an indirect fluorogenic enzyme-linked immunosorbent assay. Humoral response to these antigens were found to be dose-dependent. Mice immunized with LPS and OPS encapsulated within liposomes were found to have significantly higher IgG levels than mice immunized with free LPS and OPS. In addition, the antibody levels in mice that were immunized with liposome-encapsulated LPS and OPS were more sustained and remained at elevated levels--even after 5 weeks post immunization. As expected, OPS was found to be less immunogenic than LPS, but multiple injections of OPS encapsulated within liposomes greatly improved the immunogenicity. These results indicate that the humoral response to LPS and OPS of B. abortus can be enhanced when these antigens are encapsulated within liposomes.     

Occurrence of antigen-specific B cells following oral or parenteral immunization with Porphyromonas gingivalis fimbriae.

The induction and distribution of antigen-specific antibody-secreting cells in various tissues were assessed in BALB/c mice immunized with the purified fimbrial protein of the Porphyromonas gingivalis strain 381. Groups of mice were immunized by gastric intubation of liposomes containing fimbriae and GM-53 on days 0, 1, 27, and 28. Additional groups of mice were immunized with P. gingivalis fimbriae and adjuvant GM-53 in Freund's incomplete adjuvant by subcutaneous injection on days 0 and 28. In the latter group of mice, levels of serum IgM anti-fimbria antibodies were first detected on day 7, while high levels of serum IgG anti-fimbria antibodies were seen after secondary immunization. Fimbria-specific spot-forming cells (SFC) were detected in the spleen, circulating blood mononuclear cells (CBMC), and brachial lymph nodes of immunized mice by ELISPOT. Fimbria-specific IgM SFC appeared by day 5 and antigen-specific IgG SFC were seen later in subcutaneously immunized mice. Mice immunized orally exhibited serum anti-fimbria IgG and IgA antibodies after boosting. Although numerical analysis revealed that the numbers of fimbria-specific SFC were generally lower than in subcutaneously immunized mice, significant numbers of antigen-specific IgA SFC were seen in lamina propria and mesenteric lymph nodes of orally immunized mice. In contrast, antigen-specific IgM and IgG SFC were observed mainly in CBMC. The route of immunization with fimbriae and GM-53 also influenced the total numbers of immunoglobulin-secreting cells. Thus, subcutaneous immunization enhanced the total number of IgM and IgG SFC, including fimbria-specific antibody-secreting cells in CBMC and the spleen. (ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of IgE antibody production after exposure to ozone in mice

The effect of ozone exposure on IgE antibody production with aerosolized ovalbumin (OA) administration was investigated in Balb/c mice. Mice were continuously exposed to 0.8 ppm ozone for 1, 2, or 4 weeks, respectively, and subsequently aerosolized OA was administered through the respiratory tract for 6 min with a nebulizer. The mice were then immunized intraperitoneally 1 week later with OA. IgE antibody production was suppressed in ozone-exposed mice. However, no significant difference in the primary IgE antibody production by intraperitoneal immunization alone with OA was observed between ozone-exposed mice and nonexposed mice. In order to elucidate the suppressive mechanism of IgE antibody production, hapten-carrier antigenic system was used. It is shown that the induction of helper T cell function was suppressed if the aerosolized carrier protein was administered before intraperitoneal immunization in the mice exposed to ozone. These results suggest that ozone exposure has the effect on the stage of administration of inhaled antigen and the quite insignificant effect on the IgE antibody production after intraperitoneal immunization with OA.     

Effects of whole-body irradiation on antibody affinity.

Mice exposed to a sublethal dose of X-rays were immunized with alum-precipitated DNP-KLH (dinitrophenyl-keyhole limpet haemocyanin) and B. pertussis either before or after irradiation. The primary anti-DNP antibody response was evaluated during 8 weeks after immunization by the equilibrium dialysis technique using ammonium sulphate- precipitated serum globulins and the ligand 3H-labelled xi-DNP-L-Lysine. The serum concentrations of antibody sites in mice immunized 1-5 days before or 2 h-8 weeks after 450 rad were below the values in unirradiated controls at all bleeding times. Antibody affinity, however, was found to be up to 20 fold higher in irradiated mice than in control mice when antigen was injected before, or 3-8 weeks after, irradiation. Spleen cells from mice exposed to 450 rad 1-9 weeks before killing were stimulated in vitro with PHA, ConA, or LPS. Recovery profiles of mitotic responsiveness suggest that enhancement of antibody affinity in irradiated mice could result from relative lack of suppressor T Cells.     

Immunization of Mice Against Encephalomyocarditis Virus II. Intraperitoneal and Respiratory Immunization with Ultraviolet-Inactivated Vaccine: Effect of Bordetella pertussis Extract on the Immune Response

Mice were immunized by intraperitoneal (ip) or respiratory administration of ultraviolet-inactivated virus alone or with Bordetella pertussis extract (BPE) as an adjuvant. The effect of immunization was tested by determination of antibody titers and by survival of a lethal challenge with 200 LD(50) of a virulent (large-plaque variant) strain of EMC virus. For plain vaccine the ip 50% effective dose (ED(50)) was 37 hemagglutination units (HAU; ca. 4 x 10(6) plaque-forming unit equivalents); with adjuvant the ip ED(50) was reduced to 20 HAU. After respiratory immunization by intratracheal injection, an ED(50) value of 100 HAU was found, which was not affected by BPE. After ip vaccination the primary immune response was enhanced by BPE, but the challenge response, measured 3 weeks after challenge, was unaffected. Respiratory immunization induced a primary response which was not influenced by BPE, but here the challenge response was enhanced by the adjuvant. After secondary treatment (challenge or booster vaccination) serum antibodies and protection against challenge persisted for at least 1 year.     

Active immunization by a dengue virus-induced cytokine.

Dengue type 2 virus (DV)-induced cytotoxic factor (CF) is capable of reproducing various pathological lesions in mice that are seen in human dengue. The present study was undertaken to investigate the protective effect of active immunization of mice with CF. Mice were immunized with 5 microgram of CF and prevention of CF-induced increase in capillary permeability and damage to the blood-brain barrier were studied at weekly intervals, up to 48 weeks, by challenging with 3 microgram of CF. Maximum protection against increase in capillary permeability and damage to the blood-brain barrier was observed in week 4 after immunization. A breakthrough in the protection occurred with higher doses of CF in a dose-dependent manner. Challenge with a lethal intracerebral (i.c.) dose of DV showed significantly prolonged mean survival time and delayed onset of symptoms of sickness in the immunized mice compared with the normal mice, but the titre of the virus in the brain was similar in the two groups. On i.p. challenge with the virus the protection against damage to the blood-brain barrier was 86 +/- 7% at week 4 and 17 +/- 4% at week 26 after immunization. Sera obtained from the immunized mice showed the presence of CF-specific antibodies by ELISA, Western blot, and by neutralization of the cytotoxic activity of CF in vitro. The present study describes successful prevention of a cytokine-induced pathology by specific active immunization.     

Toxicity of lipopolysaccharide and of soluble extracts of Salmonella typhimurium in mice immunized with a live attenuated aroA salmonella vaccine.

Mice immunized intravenously 10 days earlier (but not those immunized 2 months earlier) with an attenuated Salmonella typhimurium SL3261 aroA live vaccine and tested for delayed-type hypersensitivity by injection of crude Salmonella extracts in the footpad can die within 24 to 48 h of an unexplained allergic reaction. The lethal reaction could be prevented by prior administration of anti-tumor necrosis factor alpha serum. Injection of lipopolysaccharide (LPS) (either purified phenol-water-extracted [Westphal] LPS or protein-rich trichloracetic acid-extracted [Boivin] LPS) was also lethal for mice immunized 10 days before. An LPS-rich crude Salmonella extract was more toxic than one which contained less LPS, suggesting that LPS may have been involved in the lethal reactions to crude antigens. Mild alkaline hydrolysis removes O-linked acyl groups from lipid A and eliminates many toxic effects of LPS; however, both Boivin LPS and Westphal LPS remained toxic for immunized mice after alkaline hydrolysis. In contrast, alkaline hydrolysis of crude whole Salmonella extracts (which caused marked protein degradation) reduced the lethal toxicity of the extracts, especially for an LPS-rich preparation. Mice immunized orally with the live vaccine did not show hypersensitivity to either LPS or crude extracts. The results suggest that the lethal reaction to crude Salmonella antigens in mice immunized 10 days earlier is complex, that tumor necrosis factor alpha is involved, and that allergic reactions to crude antigens (but not to LPS alone) can be reduced by mild alkaline hydrolysis.     

Enhancement of immune responses by an attenuated Salmonella enterica serovar Typhimurium strain secreting an Escherichia coli heat-labile enterotoxin B subunit protein as an adjuvant for a live Salmonella vaccine candidate

A plasmid harboring eltB, the gene encoding heat-labile enterotoxin (LTB), was constructed by insertion of eltB into an Asd(+) β-lactamase signal plasmid (pMMP65). This was introduced into the Δlon ΔcpxR Δasd Salmonella enterica serovar Typhimurium strain and designated the LTB adjuvant strain. LTB protein production and secretion from the strain were demonstrated with an immunoblot assay and enzyme-linked immunosorbent assay. The LTB strain was evaluated for enhancement of immunity and protection efficacy induced by a previously constructed live Salmonella vaccine candidate. In addition, immunization strategies using the LTB strain were optimized for effective salmonellosis protection. Seventy female BALB/c mice were divided into seven groups (A to G; n = 10 mice per group). Mice were primed at 6 weeks of age and boosted at 9 weeks of age. All mice were orally challenged with a virulent wild-type strain at week 3 postbooster. Serum IgG and IgA titers from mice immunized with the LTB strain alone or with a mixture of the LTB strain and the vaccine candidate were significantly increased. The secretory IgA titers from mice immunized with the LTB strain alone or with the mixture were at least 2.2 times greater than those of control mice. In addition, all group E mice (primed with the vaccine-LTB mixture and boosted with the vaccine candidate) were free of clinical signs of salmonellosis and survived a virulent challenge. In contrast, death due to the challenge was 100% in control mice, 80% in group A mice (single immunization with the vaccine candidate), 60% in group B mice (primed and boosted with the vaccine candidate), 40% in group C mice (single immunization with the LTB strain), 30% in group D mice (primed and boosted with the LTB strain), and 30% in group F mice (primed and boosted with the vaccine-LTB mixture). These results suggest that vaccination with the LTB strain, especially when added at the prime stage only, effectively enhances immune responses and protection against salmonellosis.     

Oral immunization against influenza virus

Summary Mice and ferrets were immunized by oral, intranasal or intraperitoneal route with attenuated or inactivated influenza A₂/Aichi/68. Production of antibodies in groups immunized orally was lower than in groups immunized intranasally or intraperitoneally when attenuated virus was administered. When inactivated virus was inoculated the serum antibody response was high following intraperitoneal injection, low following intranasal injection and nil following oral administration.Deaths occurred between the 3rd and the 11th day after intranasal immunization with partially attenuated virus, showing that immunization by the intranasal route necessitates more attenuated strains than by oral or by intraperitoneal routes. Higher doses of vaccine were required by oral than intranasal or intraperitoneal route to confer an equivalent degree of protection.This work was partially supported by a grant from National Research Council (915).     

Immunization with the RgpA-Kgp proteinase-adhesin complexes of Porphyromonas gingivalis protects against periodontal bone loss in the rat periodontitis model

A major virulence factor of Porphyromonas gingivalis is the extracellular noncovalently associated complexes of Arg-X- and Lys-X-specific cysteine proteinases and adhesins designated the RgpA-Kgp complexes. In this study we investigated the ability of RgpA-Kgp as an immunogen to protect against P. gingivalis-induced periodontal bone loss in the rat. Specific-pathogen-free Sprague-Dawley rats were immunized with either formalin-killed whole P. gingivalis ATCC 33277 cells with incomplete Freund's adjuvant, RgpA-Kgp with incomplete Freund's adjuvant, or incomplete Freund's adjuvant alone. The animals were then challenged by oral inoculation with live P. gingivalis ATCC 33277 cells. Marked periodontal bone loss was observed in animals immunized with incomplete Freund's adjuvant alone; this bone loss was significantly (P < 0.05) greater than that detected in animals immunized with formalin-killed whole cells or RgpA-Kgp or in unchallenged animals. There was no significant difference in periodontal bone loss between animals immunized with formalin-killed whole cells and those immunized with RgpA-Kgp. The bone loss in these animals was also not significantly different from that in unchallenged animals. DNA probe analysis of subgingival plaque samples showed that 100% of the animals immunized with incomplete Freund's adjuvant alone and challenged with P. gingivalis ATCC 33277 were positive for the bacterium. However, P. gingivalis ATCC 33277 could not be detected in subgingival plaque samples from animals immunized with formalin-killed whole cells or with RgpA-Kgp. Immunization with formalin-killed whole cells or RgpA-Kgp induced a high-titer serum immunoglobulin G2a response. Western blot analysis of RgpA-Kgp using pooled protective antisera taken from rats immunized with RgpA-Kgp revealed immunodominant bands at 44, 39, and 27 kDa. In conclusion, immunization with RgpA-Kgp restricted colonization by P. gingivalis and periodontal bone loss in the rat.     

Changes of cytokines in mice immunized with recombinant Bb-EmII/3-Em14-3-3 vaccine of Echinococcus multilocularis

AIM: To study the effects of recombinant Bb-EmII/3-Em14-3-3 vaccine on cytokine secretion in mice challenged with protoscoleces of Echinococcus multilocularis. METHODS: BALB/c mice were immunized with rBb-EmII/3-Em14-3-3 vaccine by subcutaneous injection, intramuscular injection, nasal mucosa inoculation or oral administration. After 12 weeks of immunization,all the mice were challenged with 50 protoscoleces of Echinococcus multilocularis by intraperitoneal injection. 18 weeks later, the mice were sacrificed and the splenocytes were stimulated with EmAg and ConA or LPS in vitro, then IL-12, IL-10, IFN-gamma and TNF-alpha in the culture supernatant were measured by ELISA. RESULTS: The level of IFN-gamma, IL-12 and TNF-alpha were greatly higher than that of PBS control group (P<0.05 or P<0.01), and the level of IL-10 was lower. The level of IFN-gamma, IL-12, TNF-alpha and IL-10 in each group with EmAg and ConA or LPS stimulation was greatly higher than that in the group without stimulation(P<0.05 or P<0.01). CONCLUSION: Th1 response is induced in mice challenged with Echinococcus multilocularis by rBb-EmII/3-Em14-3-3 vaccine, and the vaccine may enhance protective response against the challenge with protoscoleces of Echinococcus multilocularis.     

几种不同基因免疫途径的比较研究

以乙肝表面抗原基因疫苗为模型, 采用肌肉注射、腹腔注射、表皮划痕三种免疫途径和不同剂量的裸露质粒ＤＮＡ免疫小鼠, 以ＥＬＩＳＡ方法检测小鼠血清中抗ＨＢｓＡｇ 抗体变化。实验结果表明表皮划痕方式免疫小鼠免疫反应阳性率高,且在相同剂量下其抗体滴度水平最高; 肌肉注射方式免疫小鼠, 抗体反应可维持较长时间; 腹腔注射方式免疫小鼠, 抗体反应产生快, 但维持时间较短。