Sham-immunized Group Research Articles

Protection against Congenital CMV Infection Conferred by MVA-Vectored Subunit Vaccines Extends to a Second Pregnancy after Maternal Challenge with a Heterologous, Novel Strain Variant.

Maternal reinfection of immune women with novel human cytomegalovirus (HCMV) strains acquired during pregnancy can result in symptomatic congenital CMV (cCMV) infection. Novel animal model strategies are needed to explore vaccine-mediated protections against maternal reinfection. To investigate this in the guinea pig cytomegalovirus (GPCMV) model, a strictly in vivo-passaged workpool of a novel strain, the CIDMTR strain (dose, 1 × 107 pfu) was used to infect dams that had been challenged in a previous pregnancy with the 22122 strain, following either sham-immunization (vector only) or vaccination with MVA-vectored gB, gH/gL, or pentameric complex (PC) vaccines. Maternal DNAemia cleared by day 21 in the glycoprotein-vaccinated dams, but not in the sham-immunized dams. Mean pup birth weights were 72.85 ± 10.2, 80.0 ± 6.9, 81.4 ± 14.1, and 89.38 ± 8.4 g in sham-immunized, gB, gH/gL, and PC groups, respectively (p < 0.01 for control v. PC). Pup mortality in the sham-immunized group was 6/12 (50%), but reduced to 3/35 (8.6%) in combined vaccine groups (p = 0.0048). Vertical CIDMTR transmission occurred in 6/12 pups (50%) in the sham-vaccinated group, compared to 2/34 pups (6%) in the vaccine groups (p = 0.002). We conclude that guinea pigs immunized with vectored vaccines expressing 22122 strain-specific glycoproteins are protected after a reinfection with a novel, heterologous clinical isolate (CIDMTR) in a second pregnancy.

A Bivalent Protein r-PAbxpB Comprising PA Domain IV and Exosporium Protein BxpB Confers Protection Against B. anthracis Spores and Toxin.

Anthrax vaccines primarily relying only on protective antigen (PA), the cell binding component in anthrax toxins provide incomplete protection when challenged with spores of virulent encapsulated Bacillus anthracis strains. Alternatively, formaldehyde inactivated spores (FIS) or recombinant spore components generate anti-spore immune responses that inhibit the early stages of infection and augment the PA protective efficacy. In the present study domain IV of PA was spliced with exosporium antigen BxpB via a flexible G4S linker to generate a single functional antigen r-PAbxpB that was further assessed for its protective efficacy against anthrax toxins and spore infection. Immunization of mice with r-PAbxpB elicited significantly high titer antibodies comprising IgG1:IgG2a isotypes in 1:1 ratio, balanced up-regulation of both Th1 (IL2, IL12, IFN-γ) and Th2 (IL4, IL5, IL10) cytokines and high frequencies of CD4+ and CD8+ T cell subsets. The anti-r-PAbxpB antibodies significantly enhanced spore phagocytosis, and killing within macrophages; inhibited their germination to vegetative cells and completely neutralized the anthrax toxins as evidenced by the 100% protection in passive transfer studies. Active immunization with r-PAbxpB provided 100 and 83.3% protection in mice I.P. challenged with 5 × LD100 LD of toxins and 5 × 104 cfu/ml Ames spores, respectively while the sham immunized group succumbed to infection in 48 h. Therefore, the ability of r-PAbxpB to generate protective immune responses against both spores and toxin and provide significant protection suggests it as an efficient vaccine candidate against B. anthracis infection.

Oral Therapeutic Vaccination withStreptococcus sobrinusRecombinant Enolase Confers Protection against Dental Caries in Rats

Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved. Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall, these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental caries in humans.

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.

Immunogenicity and protective immunity induced by synthetic peptides associated with putative immunodominant regions of Streptococcus mutans glucan-binding protein B.

Glucan-binding protein B (GbpB) from Streptococcus mutans has been shown to induce protective immunity to dental caries in experimental models. Having recently sequenced the gbpB gene, our objective in this study was to identify immunogenic regions within the GbpB sequence for use in subunit vaccines. Potential regions of immunogenicity were sought by use of a matrix-based algorithm (EpiMatrix) to estimate the binding characteristics of peptides derived from the GbpB sequence by using a database of known major histocompatibility complex class II binding alleles. Screening the entire sequence revealed several peptides with estimated high binding probabilities. Two N-terminal 20-mer peptides (SYI and QGQ) subtending two of these regions were synthesized. A preliminary experiment, in which these peptides were synthesized in the multiple antigenic peptide format and were used to subcutaneously immunize Sprague-Dawley rats twice at a 21-day interval, revealed that the SYI peptide induced a higher percentage of responses to the inciting peptide as well as to intact GbpB, as measured by enzyme-linked immunosorbent assay. The effect of immunization with the SYI peptide construct on the cariogenicity of S. mutans was then investigated by immunizing weanling Sprague-Dawley rats twice at a 9-day interval with SYI or with phosphate-buffered saline. All rats were then orally infected with S. mutans strain SJ. After a 78-day infection period, the SYI-immunized groups had significant reductions in dental caries on both smooth and occlusal surfaces compared with the sham-immunized group. Thus, these experiments indicated that at least one linear sequence, derived from the N-terminal third of GbpB, was sufficiently immunogenic to induce a protective immune response in this experimental rat model for dental caries.

Schistosoma mansoni: Immunization with cercarial glycocalyx preparation increases the adult worm burden

Cercariae are covered by a thick glycocalyx (GCX) that activates complement by the alternative pathway and is recognized by antibodies in the serum of infected animals (Standen 1952; Samuelson and Caulfield 1985). Further, it contains antigens reactive with protective monoclonal antibodies produced in rats and mice (Grzych et al., 1982; Ham et al. 1984). Recently, Veira et al. (1986) found that cercarial transformation fluid, which contained GCX and other cercarial components, suppressed the in vitro proliferative response of human peripheral blood lymphocytes to both schistosome antigens and mitogens. Despite this evidence that GCX is immunologically relevant in schistosome infections, its effect as a primary immunogen has not been studied. We have recently characterized GCX prepared with sodium dodecyl sulfate (SDS) or guanidine hydrochloride (GuHCl) as solubilizing agents (Caulfield et al. 1987). Both preparations were rich in carbohydrate, 40% for GuHCl-GCX and 80% for SDS-GCX, with fucose as the major sugar. Here we injected mice with pooled fractions of either preparation of GCX, using phosphate-buffered saline (PBS), Maalox, or Bacille Calmette-Guerin (BCG) as carriers and/or adjuvants. We assessed the effect of immunization with GCX by comparing the numbers of adult worms recovered (worm burden) be mesenteric vein perfusion (Stirewah et al. 1951; Warren and Peters 1967) in GCXand sham-immunized mice 6 weeks postcercarial challenge. The number of adult worms recovered from mice was dramatically different between GuHCl-GCX and sham-immunized groups. As shown in Table I, animals injected with GuHCl-GCX in either PBS or Maalox had increased adult worm burdens in ah four experiments. The data were compared statistically using a stratified Wilcoxon’s test (Lagakos 1982; Table I). The statistical significance of the difference between the GuHCl-GCX-immunized and sham-immunized controls was 0.02 < P < 0.01. Thus, GuHCl-GCX when administered with PBS or Maalox significantly increased the number of surviving adult worms at 6 weeks postinfection when compared to shamimmunized controls. When BCG was administered with GuHCI-GCX, we found that the adult worm burdens were the same as in mice which received BCG alone. Mice immunized with SDS-GCX gave very different results from mice immunized with GuHCl-GCX. In two separate experiments with SDS-GCX, the adult worm burdens of SDS-GCX-immunized and shamimmunized controls were not significantly different when PBS or Maalox were used as adjuvants. Thus, immunization with SDS-GCX did not affect the number of surviving adult worms when compared to control-immunized mice. Several immunological mechanisms could explain the increase in numbers of adult worms recovered in mice immunized with GuHCl-GCX. Immunization with GuHCl-GCX may have suppressed cellular immunity, as suggested by Veira et al. (1986). Other studies have also demonstrated that schistosome materials suppressed the proliferative response of peripheral blood lymphocytes in vitro (Dessaint et al. 1977; Colley et al. 1979; Camus et al. 1981). Significantly, egg antigens, some of which cross-react with the cercarial surface (Ham et al. 1984), have been implicated in the suppression of the cell-mediated immune response (Boros et al. 1975; Colley 1975; Naggar and Colley 1982). Alternatively, GuHCl-GCX-immunized mice may have produced blocking antibodies which would bind to and protect invading larval parasites. Blocking antibodies to cercarial tegumental epitopes have been demonstrated in the rat model, both in vitro and in vivo (Grzych et al. 1982; Khahfe et al. 1986). Lastly, immunization with GuHCl-GCX may have induced a state of tolerance with respect to the carbohydrate antigens in GCX, analogous to that induced

Protective efficacy of active and passive immunizations against experimental infection with Bacteroides gingivalis in ligated hamsters.

The protective efficacy of immunization against Bacteroides gingivalis infection was examined in hamsters. Whole cells or extracted hemagglutinin of B. gingivalis 381 was injected with incomplete Freund adjuvant into the inguinal regions of hamsters. Two weeks after the rats received a booster injection, cotton threads were tied coronally to the gingival margins of the mandibular first molars, and then a streptomycin-resistant B. gingivalis 381R' strain was inoculated into the rats' oral cavities. The subcutaneous immunizations resulted in slight reductions in the numbers of B. gingivalis on the ligature threads compared with the sham-immunized group. Peroral administration of whole cells of B. gingivalis to hamsters elicited salivary immunoglobulin responses, but no reduction of B. gingivalis 381R' colonization was found in this group. Repeated passive immunizations with rabbit antiserum to B. gingivalis into the oral cavities of the hamsters resulted in a reduction in the number of organisms in the periodontal region.

Effects of systemic anti-plaque antibody administration on inflamed periodontal tissue in rats.

The purpose of the present study was to investigate the response of inflamed rat gingiva to passive immunization with anti-plaque antibodies. Thirty 3-month-old rats were divided into 3 groups, immunized (n=10), sham-immunized (n=10), and control (n= 10), and were fed keyes 2000 diet until 6 months of age. In the immunized and sham-immunized groups, anti-plaque antibodies and sham-antibodies were administered intravenously via the tail vein for 5 consecutive days prior to the day of sacrifice. The control group received phosphate-buffered-saline (PBS) in a equal amount. At 3 and 6 months of age, blood was collected. Serum IgG was quantitatively determined by single radial immunodiffusion (SRID) ; and the serum IgG anti-plaque antibody by enzyme-linked immunosorbent assay (ELISA). Polymorphonuclear leukocytes (PMN leukocytes) and mononuclear cells (MN cells) in the interproximal gingival epithelium and in the subepithelial connective tissue from the first to third molars were counted microscopically and the cell-infiltrated areas and the distances from CEJ to the alveolar bone margin were calculated in the same gingival propria. The number of anaerobic bacteria colonizing the teeth was also estimated at the termination of experiment.Serum IgG levels were almost the same among the three groups at 3 and 6 months of age. The serum IgG anti-plaque antibody titer of the immunized group was 3.1 and 5.8 times higher than those of the sham-immunized and control groups, respectively, at 6 months of age (P<0.001). No significant differences were detected among the 3 groups with regard to the number of infiltrating PMN leukocytes and MN cells, relative percent of cell-infiltrated area or distance from CEJ to the alveolar bone margin.This tendency could not suggest that the administration of antiplaque antibodies has a possible inhibitory effect on the inflamed gingival tissue in rats.

Ａｃｔｉｎｏｍｙｃｅｓ ｖｉｓｃｏｓｕｓより抽出した菌体表層抗原免疫による実験的歯周疾患の予防

The purpose of this study was to investigate the effect of immunization with the cell surface antigen extracted from Actinomyces viscosus on periodontal disease of hamsters, which was induced by inoculation with A. viscosus K-TL +.Cell surface substances were released from A. viscosus K-TL+ cells by stirring them in 0.05M Tris-HCl buffer, pH 7.4, containing 1M NaCl. These were purified by ammonium sulfate precipitations, gel filtration and ion-exchange chromatography, and fibril-rich substance was obtained. This prepaation was used as an immunogen.Golden hamsters were divided into three groups, immunized, shamimmunized and diet-control groups, and they were immunized in their pouches at 4 and 5 weeks of age. At six weeks of age, they were infected with A. viscosus K-TL+, and the establishment of the strain was examined during the experiment. At the end of the experiment, 8 weeks after the last immunization, the hamsters were killed after sampling their saliva and sera, and the mandibulars were evaluated for their alveoral bone loss and root surface caries.In the immunized group, the mean recovery of A. viscosus K-TL+ was less, and the occurrence of alveolar bone loss and root surface caries considerbly decreased comparing with the sham-immunized group. Both saliva and sera obtained from hamsters in the immunized group also showed more elevated levels of antibody against cell surface antigens than in the sham-immunized group.The results suggested that immunization with the cell surface antigen might be effective in prevening periodontal diseases induced by A. viscosus.

Effects of local immunization of hamsters with glucosyltransferase antigens from Streptococcus sanguis on dental caries caused by Streptococcus mutans

Glucosyltransferase (GTF) from Strep. sanguis strain 34 was tested for antigenic relatedness to GTF from Strep. mutans and for the effect of immunization with this GTF on experimental dental caries caused by Strep. mutans. The sera and salivas of hamsters immunized in the vicinity of the salivary glands with Strep. sanguis strain 34 GTF contained high levels of antibody and GTF-inhibitory activity to the homologous GTF as well as low but significant levels of antibody activity to GTF from Strep. mutans strains E49 (serotype a) and strain 6715 (serotype g). Likewise, sera or salivas from hamsters immunized with either Strep. mutans GTF showed significant reaction with Strep. sanguis GTF in the enzyme-linked immunosorbent assay (ELISA). However, groups of hamsters locally immunized with GTF from Strep. sanguis strain 34, followed by infection with cariogenic Strep. mutans strain E49, showed no protective effect on molar tooth surfaces as measured by extent of infection, caries or lesions compared with sham-immunized groups. Control groups immunized with Strep. mutans strain 6715 GTF and infected with Strep. mutans strain E49 organisms had significantly reduced caries and numbers of lesions ( p < 0.02) compared with sham-immunized groups. Therefore, the ability of GTF antigens to elicit a protective immune response among strains of Strep. mutans did not extend to the use of GTF from Strep. sanguis strain 34 as antigen under the conditions of these experiments.

Effect of oral administration of glucosyltransferase antigens on experimental dental caries.

The effect of oral administration of soluble antigen preparations containing glucosyltransferase on dental caries in hamsters was studied. Immunization was accomplished by feeding glucosyltransferase for 21 to 27 consecutive days. This immunization regimen resulted in the formation of salivary antibody, which was detected by functional inhibition of enzymatic activity and by a modified enzyme-linked immunosorbent assay. A serum response also occurred in two of the three experiments performed. After infection with cariogenic Streptococcus mutans strain 6715, glucosyltransferase-fed hamsters had significantly fewer S. mutans cells recoverable from molar surfaces on six of nine occasions, compared with buffer-fed control groups. Hamsters orally immunized with glucosyltransferase also always had lower mean caries scores and mean numbers of lesions than comparably infected sham-immunized groups. The results of this study suggest that significant protection from experimental dental caries can be accomplished by oral administration of soluble antigen preparations containing glucosyltransferase.

Effects of local immunization with glucosyltransferase fractions from Streptococcus mutans on dental caries in hamsters caused by homologous and heterologous serotypes of Streptococcus mutans.

Seven serotypes of Streptococcus mutans have been identified. The biochemical, genetic, and serological characteristics of these serotypes have indicated that certain serotypes are quite similar, whereas others are quite distinct. The effect of local immunization with glucosyltransferase (GTF) enzymes from serotypes a, c, or g on infection and disease caused by homologous or heterologous cariogenic S. mutans is reported. Organisms with either similar (a and g) or different (c and g) biochemical and serological characteristics were selected for heterologous challenge. NIH white hamsters were injected four times at weekly intervals with GTF prepared by 6 M guanidine-hydrochloride elution from water-insoluble glucan of serotypes a, c, or g, which resulted in enzyme (homologous) inhibitory activity in sera and salivas. After infection of GTF-immunized and sham-immunized groups of hamsters with cariogenic S. mutans of the same serotype as the injected antigen (homologous infection) or with S. mutans of a different serotype from the injected antigen (heterologous infection), the numbers of streptomycin-labeled S. mutans, caries, and lesions were determined. Immunization with GTF preparations from each of the three serotypes resulted in statistically significant reductions in the extent of infection and disease and number of lesions caused by infections with homologous cariogenic S. mutans. Statistically significant reductions in these three parameters were also observed in groups immunized with enzyme from serotype a (strain E49) and challenged with cariogenic serotype g (strain 6715) organisms; or immunized with enzyme from serotype c (strain Ingbritt) and challenged with cariogenic serotype g (strain 6715) organisms; or immunized with enzyme from serotype g (strain 6715) and challenged with cariogenic serotype c (strain Ingbritt) organisms. These studies suggest that soluble antigen preparations containing GTF from one serotype may elicit a protective immune response against infection with cariogenic S. mutans from many or possibly all serotypes.