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Oral colonization and immune responses to Streptococcus gordonii: Potential use as a vector to induce antibodies against respiratory pathogens
Abstract
Mucosal immunization should be an excellent method of preventing respiratory infections because the local immunoglobulin A antibodies can neutralize the invading pathogens at the site of entry. Because Streptococcus gordonii, a normal inhabitant of the human oral cavity, can naturally elicit a mucosal immune response, it has been a prime candidate for investigations as a live oral vaccine vector for immunization against respiratory infections.
Antigens from a number of respiratory bacteria, such as Bordetella pertussis, and one virus have been expressed extracellularly or on the cell surface of S. gordonii. The antigens expressed were single or multiple proteins from one or more pathogens. The recombinant S. gordonii expressing surface-localized heterologous antigens could colonize and persist in the oral cavity of mice and rats. Oral colonization induced a mucosal immunoglobulin A response and, in some instances, also a systemic immunoglobulin G response to the heterologous antigens. When given parenterally, the heterologous antigens generated a systemic immunoglobulin G response. These findings indicate that antigens expressed by S. gordonii are immunogenic. A new approach to the use of S. gordonii as a vaccine vector is to modulate immune responses by co-expressing cytokines with the antigen.
The ability to express antigens from respiratory pathogens and induce immune responses during oral colonization suggests that S. gordonii may be developed into a live vector for oral immunization against respiratory infections. The major challenge ahead is to find ways to achieve a high level of immune response following oral colonization.
... The response of the host to bacterial colonization of the mouth may provide a degree of host immune protection if these autochthonous organisms carry antigens that cross-react with antigens of pathogens that are important in virulence. Such antigens could be proteins involved in the adherence of pathogens to tissues (20). Conversely, tolerance of the host to such antigens that contribute to the virulence of pathogens could compromise immune protection. ...
... Conversely, tolerance of the host to such antigens that contribute to the virulence of pathogens could compromise immune protection. In addition, commensal gram-positive bacteria, in particular lactobacilli and oral streptococci, have been proposed as viable vectors of protective antigens in vaccines (1,20,30). In order for such vaccines to be effective, it is important that the organisms colonize the host for a sufficient period of time and stimulate a protective immune response. ...
... Study population. Nine healthy, full-term, breast-fed infants-referred to here as infants 1,7,8,12,14,18,20,21, and 24-were selected for this study. They comprised five males and four females. ...
The mouths of three human infants were examined from birth to age 2 years to detect colonization of Actinomyces naeslundii genospecies 1 and 2. These bacteria did not colonize until after tooth eruption. The diversity of posteruption isolates was determined by ribotyping. Using immunoblotting and enzyme-linked immunosorbent assay, we determined the reactivity of secretory immunoglobulin A (SIgA) antibodies in saliva samples collected from each infant before and after colonization against cell wall proteins from their own A. naeslundii strains and carbohydrates from standard A. naeslundii genospecies 1 and 2 strains. A. naeslundii genospecies 1 and 2 carbohydrate-reactive SIgA antibodies were not detected in any saliva sample. However, SIgA antibodies reactive with cell wall proteins were present in saliva before these bacteria colonized the mouth. These antibodies could be almost completely removed by absorption with A. odontolyticus, a species known to colonize the human mouth shortly after birth. However, after colonization by A. naeslundii genospecies 1 and 2, specific antibodies were induced that could not be removed by absorption with A. odontolyticus. Cluster analysis of the patterns of reactivity of postcolonization salivary antibodies from each infant with antigens from their own strains showed that not only could these antibodies discriminate among strains but antibodies in saliva samples collected at different times showed different reactivity patterns. Overall, these data suggest that, although much of the salivary SIgA antibodies reactive with A. naeslundii genospecies 1 and 2 are directed against genus-specific or more broadly cross-reactive antigens, species, genospecies, and possibly strain-specific antibodies are induced in response to colonization.
... Streptococcus gordonii is a commensal bacterium found in the oral cavities of humans. The organism is considered to be an attractive vector as a live-oral-vaccine vehicle (14,23). A number of heterologous antigens have been expressed in this organism as either secreted proteins (15,27) or cell wall-anchored proteins (16,17,19,20,25,26). ...
... The oral colonization of 4-week-old BALB/c mice (female; n ϭ 3) with S. gordonii SecCR1 and S. gordonii SL3 was carried out using methods described previously (16). S. gordonii SL3 is a kanamycin-resistant strain but lacks the anti-CR1 gene (14). To induce the expression of the anti-CR1 scFv, the animals were given intraperitoneal injections of 100 g tetracycline in 100 l of PBS on days 1, 8, 16, and 22 (18). ...
... To induce the expression of the anti-CR1 scFv, the animals were given intraperitoneal injections of 100 g tetracycline in 100 l of PBS on days 1, 8, 16, and 22 (18). The animals were euthanized on day 29, at which time microbiological swabs were obtained from the nasal cavity, oral cavity, and pharynx (14,16). The swabs were inoculated onto brain heart infusion agar plates containing kanamycin. ...
Streptococcus gordonii, an oral commensal organism, is a candidate vector for oral-vaccine development. Previous studies have shown that recombinant
S. gordonii expressing heterologous antigens was weakly immunogenic when delivered intranasally. In this study, antigen was specifically
targeted to antigen-presenting cells (APC) in order to potentiate antigen-APC interactions and increase the humoral immune
response to the antigen. To achieve this goal, a single-chain variable-fragment (scFv) antibody against complement receptor
1 (CR1) was constructed. Anti-CR1 scFv purified from Escherichia coli was able to bind to mouse mixed lymphocytes and bone marrow-derived dendritic cells. The in vivo function of the anti-CR1
scFv protein was assessed by immunizing mice intranasally with soluble scFv and determining the immune response against the
hemagglutinin (HA) peptide located on the carboxy terminus of the scFv. The serum anti-HA immunoglobulin G (IgG) immune response
was dose dependent; as little as 100 ng of anti-CR1 scFv induced a significant IgG immune response, while such a response
was minimal when the animals were given an unrelated scFv. The anti-CR1 scFv was expressed in S. gordonii as a secreted protein, which was functional, as it bound to dendritic cells. Mice orally colonized by the anti-CR1-secreting
S. gordonii produced an anti-HA IgG immune response, indicating that such an approach can be used to increase the immune response to
antigens produced by this bacterium.
... These pathogenic typically use mucosal surfaces to initiate infections and pose a risk of regaining their pathogenic potential, making them unsuitable for individuals with weakened immune systems, especially children [39]. However, using non-pathogenic bacteria, such as lactic acid-producing bacteria, mitigates this risk [40]. Examples of such engineered bacteria include Lactococcus lactis, Salmonella typhi, and E. coli, which are used as drug carriers ...
... LAB are proficient at colonizing the digestive and reproductive systems in both animals and humans and functioning as immune modulators that aid in diseases treatment and prevention, thereby making them suitable candidates for oral vaccines. Additionally, LAB have been explored for delivering digestive enzymes in humans [40,41]. ...
Helicobacter pylori was described in 1979. This bacterium, which thrives in the harsh conditions of the stomach, is typically acquired during childhood and can remain colonized for life. Approximately, 90% of the global population is affected, and H. pylori is linked to various conditions, including gastritis, peptic ulcers, lymphoproliferative gastric lymphoma, and even gastric cancer. Currently, antibiotics are the primary treatment method, but the associated challenges of antibiotic use have led to the consideration of oral vaccination as a viable preventive measure against this infection. However, the stomach’s harsh environment characterized by its acidic conditions and numerous proteolytic enzymes poses significant obstacles to the development and effectiveness of oral vaccines. To address these challenges, researchers have proposed and evaluated several delivery systems. One of the most promising options is the use of probiotics. Among the various probiotics, Lactococcus lactis stands out as a suitable candidate for oral vaccine delivery against H. pylori due to the advancements in genetic engineering that have been applied to it. This review article discusses the limitations of current treatment strategies and rationalizes the shift toward vaccination, particularly oral vaccination for this infection. It also explores the advantages and challenges of using probiotic bacteria, with a focus on L. lactis as a delivery system. Ultimately, despite the existing challenges, L. lactis continues to be recognized as a promising delivery system. Nonetheless, further research is essential to fully assess its effectiveness and address the challenges associated with this approach.
... In addition, it has been shown that in vitro biofilm formation correlates to in vivo colonization in mice for S. gordonii [16] and S. pneumoniae [17]. Given the findings in biofilm formation, we performed a competitive assay between the parent and msrAB mutant in a mouse model of oral colonization [16,18]. We also included the msrA mutant for comparison. ...
... Animals were given plenty of foods and water as well as normal environmental enrichment devices (toys) to alleviate sufferings. Oral colonization was tested as described previously [14,18]. BALB/c mice (female, 4 weeks old, n = 9 per group) were fed kanamycin (500 μg/ml) in drinking water for 2 days prior to colonization. ...
The ability of Streptococcus gordonii to cope with oxidative stress is important for survival and persistence in dental plaque. In this study, we used mutational, phenotypic, and biochemical approaches to characterize the role of a methionine sulfoxide reductase (MsrAB) and proteins encoded by genes in the msrAB operon and an adjacent operon in oxidative stress tolerance in S. gordonii. The results showed that MsrAB and four other proteins encoded in the operons are needed for protection from H2O2 and methionine sulfoxide. These five proteins formed a reducing pathway that was needed for oxidative stress tolerance, biofilm formation, and oral colonization in mice. In the pathway, MsrAB was the enzyme that repaired oxidatively damaged proteins, and the two thioredoxin-like lipoproteins (SdbB and Sgo_1177) and two CcdA proteins were proteins that maintained the catalytic cycle of MsrAB. Consistent with the role in oxidative stress tolerance, the production of MsrAB, SdbB, and Sgo_11777 was induced in aerobic growth and planktonic cells.
... On the contrary, it has been suggested that S. gordonii can modulate the virulence properties of S. mutans by interfering with the comCDE quorum-sensing system, which regulates biofilm formation, bacteriocin production and genetic competence (Wang & Kuramitsu, 2005). Because of its pioneer colonizing properties and because of its potential to elicit immune responses in infants and children, it has been proposed to use engineered strains of S. gordonii for live vaccine delivery after mucosal colonization (Fischetti et al., 1996; Lee, 2003). Ideally, such antigens would be exported or displayed on the cell surface of S. gordonii (Lee, 2003). ...
... Because of its pioneer colonizing properties and because of its potential to elicit immune responses in infants and children, it has been proposed to use engineered strains of S. gordonii for live vaccine delivery after mucosal colonization (Fischetti et al., 1996; Lee, 2003). Ideally, such antigens would be exported or displayed on the cell surface of S. gordonii (Lee, 2003). Although S. gordonii belongs to the GRAS (generally regarded as safe) organisms, it has been implicated in causing infective endocarditis (Douglas et al., 1993). ...
... Although there is a potential for taxonomic misclassification of the S. gordonii resembling OTU88, this OTU is highly widespread among individuals and body sites. Furthermore, S. gordonii is an important dental colonizer [11], with the potential to enter the blood stream and colonize the aortic valve [22]. This may indicate that OTU88 is a cosmopolitan colonizer, with the potential to colonize a wide range of body sites. ...
... Pointing towards a mutualistic interaction is the fact that the related S. gordonii can produce hydrogen peroxide-potentially preventing the colonization by pathogens [25]. Furthermore, despite the high numbers and prevalence, relatively few incidences of infections have been reported for S. gordonii [11]. The fact that this bacterium together with other low-pathogenicity streptococci can colonize the aortic valve without causing instant fatal disease may also support its low virulence [7]. ...
Understanding the transmission of the human microbiota from mother to child is of major importance. Although we are gaining knowledge using 16S rRNA gene analyses, the resolution of this gene is not sufficient to determine transmission patterns. We therefore developed an Illumina deep sequencing approach targeting the 16-23S rRNA Internal Transcribed Spacer (ITS) for high-resolution microbiota analyses. Using this approach, we analyzed the composition and potential mother to child transmission patterns of the microbiota (milk and stool) in a longitudinal cohort of 20 mother/child pairs. Our results show overlap in the infant stool microbiota with both mother's milk and stool, and that the overlap with stool increases with age. We found an Operational Taxonomic Unit resembling Streptococcus gordonii as the most widespread colonizer of both mothers and their children. In conclusion, the increased resolution of 16-23S rRNA ITS deep sequencing revealed new knowledge about potential transmission patterns of human-associated bacteria.
... On the contrary, it has been suggested that S. gordonii can modulate the virulence properties of S. mutans by interfering with the comCDE quorum-sensing system, which regulates biofilm formation, bacteriocin production and genetic competence (Wang & Kuramitsu, 2005). Because of its pioneer colonizing properties and because of its potential to elicit immune responses in infants and children, it has been proposed to use engineered strains of S. gordonii for live vaccine delivery after mucosal colonization (Fischetti et al., 1996;Lee, 2003). Ideally, such antigens would be exported or displayed on the cell surface of S. gordonii (Lee, 2003). ...
... Because of its pioneer colonizing properties and because of its potential to elicit immune responses in infants and children, it has been proposed to use engineered strains of S. gordonii for live vaccine delivery after mucosal colonization (Fischetti et al., 1996;Lee, 2003). Ideally, such antigens would be exported or displayed on the cell surface of S. gordonii (Lee, 2003). Although S. gordonii belongs to the GRAS (generally regarded as safe) organisms, it has been implicated in causing infective endocarditis (Douglas et al., 1993). ...
Streptococcus gordonii OMZ1039, isolated from supragingival dental plaque, was found to harbour a prophage, PH15, whose excision could be induced by mitomycin treatment. Phage PH15 belongs to the Siphoviridae. The complete genome sequence of PH15 was determined. The genome was 39 136 bp in size and contained 61 ORFs. The genome of PH15 was most similar in the structural module to the temperate bacteriophages MM1 and phiNIH1.1 from Streptococcus pneumoniae and Streptococcus pyogenes, respectively. In strain OMZ1039, PH15 was found to reside as a prophage in the cysteinyl-tRNA gene. A plasmid, harbouring the attP site and the integrase gene downstream of a constitutive promoter, was capable of site-specific integration into the genomes of different oral streptococcal species. The phage endolysin was purified after expression in Escherichia coli and found to inhibit growth of all S. gordonii strains tested and several different streptococcal species, including the pathogens Streptococcus mutans, S. pyogenes and Streptococcus agalactiae.
... Unfortunately, they could recover their pathogenic potential and are not totally safe for use in humans, especially in children and immunosuppressed patients. In controversy, the foodgrade LAB constitute safer candidates as live mucosal vaccines [Lee, 2003; Seegers, 2002; Ståhl et al., 1997]: they have been used for centuries in the fermentation and preservation of food and are considered as 'generally recognized as safe' organisms. Mucosal administration of recombinant LAB has been shown to elicit successfully both systemic and mucosal immunity Grangette et al., 2001 Grangette et al., , 2004 Lee, 2003; Maassen et al., 1999; Norton et al., 1994; Robinson et al., 1997]. ...
... In controversy, the foodgrade LAB constitute safer candidates as live mucosal vaccines [Lee, 2003; Seegers, 2002; Ståhl et al., 1997]: they have been used for centuries in the fermentation and preservation of food and are considered as 'generally recognized as safe' organisms. Mucosal administration of recombinant LAB has been shown to elicit successfully both systemic and mucosal immunity Grangette et al., 2001 Grangette et al., , 2004 Lee, 2003; Maassen et al., 1999; Norton et al., 1994; Robinson et al., 1997]. L. lactis , the most widely used LAB in the production of fermented milk products, is considered as the model LAB because many genetic tools have been developed and its genome has been completely sequenced [Bolotin et al., 2001]. ...
The noninvasive and food-grade Gram-positive bacterium Lactococcus lactis is well adapted to deliver medical proteins to the mucosal immune system. In the last decade, the potential of live recombinant lactococci to deliver such proteins to the mucosal immune system has been investigated. This approach offers several advantages over the traditional systemic injection, such as easy administration and the ability to elicit both systemic and mucosal immune responses. This paper reviews the current research and advances made with recombinant L. lactis as live vector for the in situ delivery of biologically active interleukin-12, a potent pleiotropic cytokine with adjuvant properties when co-delivered with vaccinal antigens, at mucosal surfaces. Three well-illustrated examples demonstrate the high potential of interleukin-12-secreting lactococci strains for future prophylactic and therapeutic uses.
... The interplay between the CiaRH and Com systems enhances both in vitro biofilm formation and in vivo colonization by the ΔsdbA mutant. S. gordonii is associated with oral health, and it has potential biotechnological value as a live vaccine vector, or possibly even as a probiotic [51]. The enhanced biofilm formation and colonization of the ΔsdbA mutant suggests that it could be particularly useful for such applications. ...
Streptococcus gordonii is a commensal inhabitant of human oral biofilms. Previously, we identified an enzyme called SdbA that played an important role in biofilm formation by S. gordonii. SdbA is thiol-disulfide oxidoreductase that catalyzes disulfide bonds in secreted proteins. Surprisingly, inactivation of SdbA results in enhanced biofilm formation. In this study we investigated the basis for biofilm formation by the ΔsdbA mutant. The results revealed that biofilm formation was mediated by the interaction between the CiaRH and ComDE two-component signalling systems. Although it did not affect biofilm formation by the S. gordonii parent strain, CiaRH was upregulated in the ΔsdbA mutant and it was essential for the enhanced biofilm phenotype. The biofilm phenotype was reversed by inactivation of CiaRH or by the addition of competence stimulating peptide, the production of which is blocked by CiaRH activity. Competition assays showed that the enhanced biofilm phenotype also corresponded to increased oral colonization in mice. Thus, the interaction between SdbA, CiaRH and ComDE affects biofilm formation both in vitro and in vivo.
... Streptococcus gordonii is a potential live vaccine vehicle (Lee, 2003;Medaglini et al., 1997). This commensal bacterium is one of the pioneer organisms in the human oral cavity, colonizing the oral mucosa and tooth surface. ...
... Several oral flora, including Streptococcus (S.) gordonii have been known as an initiator and promoter of dental plaque formation in human medicine (5,9,11). S. gordonii is generally nonpathogenic but highly immunogenic and associated with bacterial endocarditis by platelet adhesion (14,15). This case report shows S. gordonii infection in the subcutaneous of face by canine tooth fistula in a Slow loris monkey. ...
A 1-year-old female Slow loris was presented with swelling of left nasal bridge. On physical examination, fracture of left upper canine tooth was found. Cytologic and radiographic examination on the lesion revealed bacterial infection and pulpitis, respectively. The bacteria were identified as Streptococcus gordonii by culture and molecular diagnosis. After removal of damaged tooth and administration of antibiotics, the abscess was resolved.
... Other bacterial species that have been studied for heterologous antigen delivery include Streptococcus gordonii (Lee 2003;Oggioni et al., 1995), Vibrio cholerae (Kaper and Levine 1990;Silva et al., 2008), Mycobacterium bovis (BCG) (Bastos et al., 2009;Nasser Eddine and Kaufmann, 2005), Yersinia enterocolitica (Leibiger et al., 2008), and Shigella flexnery (Barry et al., 2006;Sizemore et al., 1995). Relatively new species that have been investigated for use as vaccine vectors include Pseudomonas aeruginosa (Epaulard et al., 2006), Bacillus subtilis (Duc et al., 2003;Isticato et al., 2001;Paccez et al., 2007), and Mycobacterium smegmatis (Lü et al., 2009). ...
Genetically attenuated microorganisms, pathogens, and some commensal bacteria can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system, while still offering good levels of safety. A key feature of these live vectors is their capacity to stimulate mucosal as well as humoral and/or cellular systemic immunity. This enables the use of different forms of vaccination to prevent pathogen colonization of mucosal tissues, the front door for many infectious agents. Furthermore, delivery of DNA vaccines and immune system stimulatory molecules, such as cytokines, can be achieved using these special carriers, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response. More recently, the unique features and versatility of these vectors have also been exploited to develop anti-cancer vaccines, where tumor-associated antigens, cytokines, and DNA or RNA molecules are delivered. Different strategies and genetic tools are constantly being developed, increasing the antigenic potential of agents delivered by these systems, opening fresh perspectives for the deployment of vehicles for new purposes. Here we summarize the main characteristics of the different types of live bacterial vectors and discuss new applications of these delivery systems in the field of vaccinology.
... There are two types of bacterial vectors used to Recently LAB secreting elafin have been tested in deliver compounds at the mucosal level: attenuated chronic and acute colitis models and it was found that pathogens and non-pathogenic bacteria. Gram-positive inflamed epithelium was protected from increased commensal or food-grade bacteria constitute good intestinal permeability and from the release of cytokines alternatives to pathogenic bacteria [33]. The reason and chemokines by LAB secreting elafin [39]. ...
Intestinal microbiota is a key component of both the metabolism and immunity. These can be helpful in healthcare, especially for the management of digestive diseases and food-borne illnesses. Through genetic engineering it is possible to fully express biologically active copies of such powerful molecules from food and commensal bacteria. Genetically engineered probiotics can be used to treat inflammatory bowel disorders such as Crohn's disease, ulcerative colitis, as well as other disorders that result from an overactive immune system. Genetically modified (GM) probiotics are also used as food additives to control the release of human growth factors by the modified bacteria to fight against injury and inflammation in the gut. For example the use of a plant sugar called xylan to stimulate the genetically modified human gut probiotic bacterium Bacteroides ovatus to produce specific proteins that can repair damaged cells and dampen down the immune system in the intestine that causes inflammation and disease. Studies have confirmed that administration of xylan with the genetically engineered probiotic bacteria resulted in a significant improvement of colitis, reduced weight loss, improved stool consistency, reduced rectal bleeding and accelerated healing of damaged colonic cells. GM probiotics posses potential in clinical applications like delivery of antigens for vaccines and thus are more readily accepted. This would provide a safer method of vaccination than the use of attenuated pathogens e.g. GM, Lactococcus lactis, produces IL-10 in the mouse intestine. The safety of such organisms that produce very powerful bioactive substances is of major concern as excess production of these substances in a healthy individual may be detrimental. INTRODUCTION Probiotics have many potential therapeutic uses,
... In principle, two types of bacterial vectors can be used to deliver compounds at the mucosal level: attenuated pathogens and non-pathogenic bacteria. Gram-positive commensal or food-grade bacteria constitute attractive good alternatives to pathogenic bacteria [79,81]. Particularly, the food-grade LAB are attractive candidates because they have been used for centuries in the fermentation and preservation of food, and are considered to be safe organisms with a GRAS (Generally Recognized As Safe) status. ...
The human gut is one of the most complex ecosystems, composed of 1013-1014 microorganisms which play an important role in human health. In addition, some food products contain live bacteria which transit through our gastrointestinal tract and could exert beneficial effects on our health (known as probiotic effect). Among the numerous proposed health benefits attributed to commensal and probiotic bacteria, their capacity to interact with the host immune system is now well demonstrated. Currently, the use of recombinant lactic acid bacteria to deliver compounds of health interest is gaining importance as an extension of the probiotic concept. This review summarizes some of the recent findings and perspectives in the study of the crosstalk of both commensal and probiotic bacteria with the human host as well as the latest studies in recombinant commensal and probiotic bacteria. Our aim is to highlight the potential roles of recombinant bacteria in this ecosystem.
... Streptococcus gordonii [31] Lactococcus lactis [5] Lactobacillus spp. [32] Staphylococcus spp. ...
Today, sufficient consistent data are available to reinforce the interest of the use of Lactic Acic Bacteria (LAB), particularly lactococci and lactobacilli strains, to develop novel mucosal vaccination strategies. LAB are Gram positive bacteria exploited since the highest Antiquity by humans to produce fermented foods. They are thus good candidates to develop novel oral vectors and constitute attractive alternatives to vaccinal strategies based on attenuated pathogens which could induce healthy risks. Here, we summarized the most recent researches performed on the use of LAB as live vaccine delivery vectors.
... Streptococcus gordonii is a Gram-positive commensal bacterium of the human oral cavity. The bacterium has garnered interest as a potential live oral vaccine delivery vector due to its commensal nature and its ease in genetic manipulation (Lee, 2003;Oggioni et al., 1999). Although S. gordonii is a commensal bacterium, it is readily recognized by professional antigen-presenting cells, such as monocytes and dendritic cells (DCs), inducing both the production of cytokines and the upregulation of surface proteins indicative of cellular maturation (Chan et al., 2007;Ciabattini et al., 2006;Corinti et al., 1999). ...
Streptococcus gordonii, a normal inhabitant of the human oral cavity, is a potential live vaccine vehicle. Several pathogen-associated molecular patterns from S. gordonii that are recognized by antigen-presenting cells have recently been identified. In this study, we have identified that the cell-wall-anchored proteins SspA and SspB are immunostimulatory components of S. gordonii. SspA and SspB are members of the antigen I/II family of proteins widely expressed by viridans oral streptococci. The results showed that the mutant (OB219) lacking SspA and SspB had a reduced ability to induce cytokine/chemokine production in epithelial cells and bone-marrow-derived dendritic cells as compared with the parent strain (DL1). Purified SspA induced interleukin-6 and monocyte chemotatic protein-1 production from human lung epithelial A549 cells. The induction could be inhibited by a function-blocking anti-β1 integrin mAb and the purified SspA could bind to β1 integrin precoated on microtitre plates, suggesting that the induction was effected by SspA-β1 integrin interactions. The role of SspA and SspB in innate immunity was further demonstrated in a mouse intranasal challenge experiment, which showed that the clearance of OB219, the recruitment of neutrophils (as indicated by myeloperoxidase activity), and chemokine and cytokine production in the lungs of OB219-inoculated mice were delayed or reduced as compared with the DL1-inoculated mice. In addition to the above, S. gordonii OB219 was more sensitive to polymyxin, nisin and histatin-5 than DL1, suggesting that SspA and SspB also play a role in susceptibility to cationic antimicrobial peptides. Collectively, the results indicate that SspA and SspB are immunostimulatory components of S. gordonii and play an important role in modulating the host's innate immunity.
... Unfortunately, such organisms can potentially recover their pathogenic potential and are not totally safe for use in humans, especially in children and immunosuppressed patients. This risk could be avoided by using non-pathogenic bacteria, such as LAB [23,24]. Besides their GRAS category, some LAB have the capacity to colonize the gastrointestinal tract or the genital mucosa of animals and humans, can act as immunomodulators and have proved useful in the treatment and prevention of diseases, making them excellent candidates for oral vaccine development [2,17]. ...
... Streptococcus gordonii, a commensal bacterium of the human oral cavity, has gained interest as a live oral vaccine vehicle (Lee, 2003;Medaglini et al., 1997). One of the major limitations facing the development of Streptococcus gordonii into a successful live vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. ...
One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.
... Streptococcus gordonii is a Gram-positive commensal bacterium of the human oral cavity. The bacterium has garnered interest as a potential live oral vaccine delivery vector due to its commensal nature and its ease in genetic manipulation (Lee, 2003;Oggioni et al., 1999). A number of heterologous antigens have been expressed in this organism; however, there are difficulties in stimulating a strong protective immune response against the recombinant antigens (Lee et al., 1999(Lee et al., , 2002a(Lee et al., ,b, 2004Medaglini et al., 1995Medaglini et al., , 2001Ricci et al., 2000;Sharma et al., 1996Sharma et al., , 1997. ...
Streptococcus gordonii, a potential mucosal vaccine delivery vector, is proficient at colonizing murine oral mucosa; however, it often fails to elicit significant antibody titers against its vaccine antigen payloads. The poor response may be due to an inability of S. gordonii to elicit cytokines needed to suppress mucosal tolerance; exogenously supplied cytokines, such as TNF, could overcome this effect. To test this, murine bone marrow-derived dendritic cells (BM-DCs) were stimulated with UV-killed S. gordonii PM14, that surface expresses a fragment of the immunodominant S1 subunit of pertussis toxin. Peptidoglycan (PGN), lipoteichoic acid (LTA), lipoprotein (LP), and DNA were also isolated from the bacteria, and used to stimulate BM-DCs. Stimulation with TNF, S. gordonii, PGN, LTA, or LP all resulted in increased surface expression of MHCII, CD80, and CD86, compared to unstimulated BM-DCs. Stimulation with S. gordonii elicited IL-6, IL-10, and IL-12p70 production from the BM-DCs, while stimulation with the bacterial components induced some or all of the three cytokines. When BM-DCs were simultaneously stimulated with S. gordonii and TNF, a marginal increase in surface marker upregulation was observed, and the two stimuli synergized to elicit substantially greater quantities of IL-6, IL-10, and IL-12p70. Synergy between TNF and the purified bacterial components was also observed. The effect of TNF was abolished when BM-DCs were obtained from mice deficient for either TNFR1 or TNFR2, and cytokine induction by S. gordonii was entirely dependent on functional MyD88. Synergistic IL-10 induction by S. gordonii and TNF was not observed in TLR-2(-/-) BM-DCs, and TNF was found to cause TLR-2 upregulation, providing at least a partial mechanism for the observed synergy. When S. gordonii and TNF were used to immunize mice, a more robust anti-S. gordonii IgG response was elicited as compared to immunization with S. gordonii alone. However, the addition of TNF did not result in stronger responses against the antigenic insert (S1 fragment) in immunized mice. These findings collectively demonstrate that TNF is able to prime BM-DCs to better respond to S. gordonii, through a mechanism at least partially involving TLR-2 upregulation.
... Several bacteria, such as Salmonella spp. (12), Mycobacterium bovis BCG (25), Streptococcus gordonii (17,28), Lactobacillus plantarum (8), and Staphylococcus spp. (6,11), have received attention as live vaccine delivery vehicles. ...
A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface
protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with
cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA2) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA2 was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin
G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.
... In addition to the enteric pathogens discussed above, other bacteria being considered as live vectors include Yersinia enterocolitica (55), Y. pseudotuberculosis (115), Bordetella bronchiseptica, and B. pertussis (129). The mucosal commensals such as Streptococcus gordonii (85) and the lactic acid bacteria (54,100) such as the Lactococcus and Lactobacillus spp. have also been shown to raise immunity to foreign antigens in animal models. ...
Live attenuated bacteria are attractive vaccine vectors that can elicit host immune responses to foreign antigens. Bacterial vectors may mimic natural infection and therefore interact with the mucosal, humoral, and cellular compartments of the immune system. A plethora of foreign (heterologous) antigens derived from bacterial, fungal, viral, or parasitic organisms have been expressed within bacterial vectors. Delivery of con- traceptive and tumor antigens, cytokines, and adjuvants has also been explored. The result is a type of "vaccine factory" (29) in which the bacterial vector produces a foreign protein. Since bacterial vectors replicate within the host, it is hoped that they provide sustained exposure to the antigen, potentially augmenting the type and level of immune response. In addi- tion, bacteria naturally possess immunostimulatory molecules such as lipopolysaccharide (LPS) and flagella that can stimu- late immune responses (56). Internationally, there are three live bacterial vaccines commercially available and in clinical use: Salmonella enterica serovar Typhi Ty21a, Vibrio cholerae CVD 103-HgR, and Mycobacterium bovis BCG. These strains plus many other attenuated strains have been used as vectors to express heterologous antigens. Live bacterial vector vaccines have been evaluated in numerous animal and human studies, but there are currently no licensed live bacterial vector vac- cines being used clinically. The most compelling rationales for the development of live bacterial vectors are the possibility of oral delivery and pro- tection at mucosal surfaces. This review will cover attenuated bacterial enteric pathogens used as vectors to express foreign (heterologous) antigens, with emphasis on those that have reached clinical study, including Salmonella, Shigella, V. chol- erae, and Listeria. This review will not cover bacterial vectors used to deliver DNA vaccines or subunit and conjugate vac- cines.
... In recent years, there has been an increasing interest in the use of commensal bacteria as live vector vaccines. Streptococcus gordonii has been suggested to be a potential live oral vaccine delivery candidate for use to prevent diseases (Medaglini et al. 1995;Lee 2003). To pursue the goal of a live oral vaccine against childhood diseases, we recently surface expressed in S. gordonii DL-1 the diphtheria toxin fragment A (DTA) as a fusion protein to the P1 antigen (SpaP) encoded by the spaP gene originated from Streptococcus mutans (Lee et al. 2004). ...
The nontoxic full-length diphtheria toxin (DTX), fragment A (DTA), and fragment B (DTB) were each genetically fused to the major surface protein antigen P1 (SpaP) of Streptococcus mutans. Repeated attempts to express the recombinant DTX and DTB in the live oral vaccine candidate Streptococcus gordonii were unsuccessful, whereas DTA could be readily expressed in this bacterium. However, the recombinant DTX, DTB, and DTA could be expressed in the related oral bacterium S. mutans. Western blotting and enzyme-linked immunosorbant assay (ELISA) using anti-DTX and anti-P1 antibodies demonstrated the expression of the three fusion proteins in S. mutans. Mouse antisera raised against the recombinant S. mutans recognized the native DTX in Western immunoblotting. The antibodies raised against S. mutans expressing the recombinant DTX and DTA neutralized the cytotoxicity of the native toxin in a Vero cell assay, but the neutralization titers were relatively low. The potential of using S. gordonii as a live vaccine against diphtheria faces major challenges in the expression of DTX in this organism and in the induction of high-titer toxin-neutralizing antibodies.
... A live oral vaccine that can be maintained within the population, that can be easily transmitted from mother to child, and that can elicit a protective immune response would be an optimal solution. The oral commensal bacterium Streptococcus gordonii has been suggested as a suitable live oral vaccine vehicle, particularly, against childhood diseases (Lee 2003;Medaglini et al. 1995). ...
Previous work aimed at developing a live oral vaccine expressing pertussis toxin S1 fragment on the surface of the bacterium Streptococcus gordonii elicited a lower than expected antibody response, perhaps because of low antigen expression. In this study, in-frame promoter fusions were constructed to investigate whether an increase in antigen production by the streptococcal vaccine strain results in a better antibody response. The promoters tested were (i) the Streptococcus mutans sucrose-inducible fructosyltransferase (ftf) promoter and (ii) the Bacillus subtilis/Escherichia coli chimeric tetracycline-inducible xyl/tetO promoter. Each of these two promoters was placed upstream of the spaP/s1 fusion gene to drive its expression. The constructs were introduced into S. gordonii DL1 and S. mutans 834. The inducibility of the promoters was confirmed through the determination of SpaP/S1 production via Western blottings. Induced production of SpaP/S1 was observed in S. gordonii and S. mutans with each of the promoters, but the level of expression was the highest in S. mutans, using the xyl/tetO promoter. Thus, S. mutans carrying the xyl/tetO/spaP/s1 construct (S. mutans PM14) was used in oral colonization studies in BALB/c mice. Streptococccus mutans PM14 was able to colonize the animals for the 14-week duration of experimentation. A mucosal IgA response was observed in all the treatment groups but was highest in mice receiving tetracycline induction. In the mouse model of Bordetella pertussis respiratory infection, animals colonized with S. mutans PM14 showed a decreased in B. pertussis lung colony count (P = 0.03) on day 3 compared with control mice colonized by the parent S. mutans 834.
Background and Objective: Associations between probiotics as dietary supplements for health enhancement and illness management and human health include a long history. Currently, probiotics represent a potential category of microorganisms used in the development of oral vaccines for the treatment of allergies, infectious diseases, and cancers. The vaccine promises as safe therapeutic options, their capacity to elicit mucosal and systemic immune responses, and their cost-effectiveness resulting from the absence of complex purification processes have been addressed.
Results and Conclusion: Despite the advantages of probiotics as oral vaccines, their uses still include problems such as inadequate targeted colonization, diminished immune response in populations with low hygiene standards, reliance on individual microbiota, poor stability, limited efficacy, and absence of targeted immunogenicity. To address these problems, probiotics can be engineered using gene editing technologies, particularly CRISPR/Cas system. Concerns are reported regarding the safety of genetic alterations and deficiencies in efficient delivery mechanisms linked to the use of modified probiotics as oral vaccines. Further studies are needed to assess problems associated with accurate genetic alteration and efficient delivery methods to achieve the ultimate goal of further effective and safer vaccinations.
The dynamic and polymicrobial oral microbiome is a direct precursor of diseases such as dental caries and periodontitis, two of the most prevalent microbially induced disorders worldwide. Distinct microenvironments at oral barriers harbour unique microbial communities, which are regulated through sophisticated signalling systems and by host and environmental factors. The collective function of microbial communities is a major driver of homeostasis or dysbiosis and ultimately health or disease. Despite different aetiologies, periodontitis and caries are each driven by a feedforward loop between the microbiota and host factors (inflammation and dietary sugars, respectively) that favours the emergence and persistence of dysbiosis. In this Review, we discuss current knowledge and emerging mechanisms governing oral polymicrobial synergy and dysbiosis that have both enhanced our understanding of pathogenic mechanisms and aided the design of innovative therapeutic approaches for oral diseases.
Today, sufficient data are available to support the use of lactic acid bacteria (LAB), notably lactococci and lactobacilli, as delivery vehicles for the development of new mucosal vaccines. These non-pathogenic Gram-positive bacteria have been safely consumed by humans for centuries in fermented foods. They thus constitute an attractive alternative to the attenuated pathogens (most popular live vectors actually studied) which could recover their pathogenic potential and are thus not totally safe for use in humans. This chapter reviews the current research and advances in the use of LAB as live delivery vectors of proteins of interest for the development of new safe mucosal vaccines. The use of LAB as DNA vaccine vehicles to deliver DNA directly to antigen-presenting cells of the immune system is also discussed.
Schistosomiasis is a worldwide parasitic disease. Currently, chemotherapy is the main effective method to treat schistosomiasis; however, it does not prevent reinfection. No effective vaccine is currently available to prevent schistosomiasis. Sj-F1 (GenBank accession number AY261995) is a novel gene that was discovered through screening adult Schistosoma japonicum worm cDNA library with female S. japonicum antigen-immunized sera. Streptococcus gordonii, a normal inhabitant of the human oral cavity, has been a prime candidate in recent investigations toward developing a live oral vaccine vector. One of the approaches for the surface expression of heterologous antigens in S. gordonii is to surface-localize them with the M6 protein from Streptococcus pyogenes. Here, we develop a recombinant S. gordonii strain that expresses the M6-Sj-F1 fusion protein on the bacterial surface. Intranasal immunization in mice with such M6-Sj-F1-expressing S. gordonii bacteria induced strong serum IgG, serum IgA, and saliva IgA against Sj-F1. The results of protective immunity against a challenge with cercariae of S. japonicum showed statistically significant protection following this treatment, with a worm reduction rate of 21.45 % and an egg reduction rate of 34.77 %. Our data indicate that the described M6-Sj-F1-expressing S. gordonii is highly immunogenic and can partially protect mice from challenge infection with S. japonicum. Intranasal immunization with recombinant S. gordonii may be an alternative to developing a novel S. japonicum vaccine in a safe, effective, and feasible way.
Introduction LAB: The Context Behind Their Use as Live Vectors New and Future Uses of LAB Lactococcus lactis: The Model LAB Genetic Tools for the Production of Heterologous Proteins in L. lactis Expression and Targeting Systems of Heterologous Proteins Cellular Targeting Systems of Heterologous Proteins Genetically Modified Strains LAB as Live Vaccines Conclusions References
Lactic acid bacteria (LAB) have proved to be effective mucosal delivery vehicles that overcome the problem of delivering functional proteins to the mucosal tissues. By the intranasal route, both live and killed LAB vaccine strains have been shown to elicit mucosal and systemic immune responses that afford protection against infectious challenges. To be effective via oral administration, frequent dosing over several weeks is required but new targeting and adjuvant strategies have clearly demonstrated the potential to increase the immunogenicity and protective immunity of LAB vaccines. Oral administration of Lactococcus lactis has been shown to induce antigen-specific oral tolerance (OT) to secreted recombinant antigens. LAB delivery is more efficient at inducing OT than the purified antigen, thus avoiding the need for purification of large quantities of antigen. This approach holds promise for new therapeutic interventions in allergies and antigen-induced autoimmune diseases. Several clinical and research reports demonstrate considerable progress in the application of genetically modified L. lactis for the treatment of inflammatory bowel disease (IBD). New medical targets are on the horizon, and the approval by several health authorities and biosafety committees of a containment system for a genetically modified L. lactis that secretes Il-10 should pave the way for new LAB delivery applications in the future.
The charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of
heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP of
Streptococcus mutans and a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacterium Streptococcus gordonii. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, the dlt mutant strain, which has a mutation in the dlt operon preventing d-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface
proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both the dlt mutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged
cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca2+, Mg2+, and K+, presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition
of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the
level of PrsA, a peptidyl-prolyl cis/trans isomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system
LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous
SpaP-S1 protein expression in S. gordonii and that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory
system.
Abstract: Rotavirus is the major cause of gastroenteritis in infants, young children and animals worldwide. Variable efficacies in rotavirus vaccine trials were attributed to maternal antibody (MatAb) interference. We demonstrated first that a vaccine consisting of oral (PO) attenuated human rotavirus (AttHRV) and oral 2/6-virus-like-particles (VLP) with immunostimulating complexes (ISCOM) (AttHRV/PO-VLP) induced high protection rates against virus shedding and diarrhea, and high serum IgA, IgG and intestinal IgA antibody titers in seronegative gnotobiotic pigs. We next investigated effects of different titers of MatAb on protection and immune responses to the above vaccine but with VLP given intranasally (IN) [(AttHRV/IN-VLP) or IN VLP/ISCOM (VLP3x) vaccines]. High titer circulating MatAb contributed to partial protection against HRV challenge; however, MatAb interference led to no or low intestinal IgM, IgA and IgG antibody titers and significantly reduced intestinal IgA and IgG effector and memory B cell responses in the AttHRV/INVLP pigs pre- and post-challenge. Suppression was not alleviated by extending the vaccination/challenge interval from 28 to 42 days. Suppression by high titer MatAb was also occurred in VLP3x vaccinated pigs, indicated by reduced intestinal IgG antibody secreting cells (ASC), serum and intestinal IgA antibodies and pre-challenge memory B cell responses. Low titer (Lo) MatAb had both enhancing and suppressing effects on B cell responses, depending on tissue, antibody isotype and vaccine. Differential effects of LoMatAb on IgA responses suggest that LoMatAb did not suppress induction of IgA ASC and memory B cells in the induction site (ileum) but impaired homing of activated B cells to secondary lymphoid or effector tissues, reducing IgA ASC and antibodies at these sites. Finally we investigated cytokines transferred from sow colostrum/milk to their suckling and weaned pigs. The peak mean cytokine concentrations (IL-4>TGF-[beta]1>IL-6>IL-12>IFN-[gamma]>IL-10) in pig sera were detected at 1-2 days post-partum. High IL-4 and TGF-[beta]1 concentrations in serum of suckling pigs may explain the Th2-biased neonatal immune responses. Increased serum IL-6 and IL-12 concentrations in weaned pigs suggest acclimation to post-suckling microbial flora. This knowledge provides a basis for future studies of influences of maternally-derived cytokines, in addition to MatAb, on development of the neonatal immune system. Title from first page of PDF file. Document formatted into pages; contains xxv, 449 p.; also includes graphics (some col.). Thesis (Ph. D.)--Ohio State University, 2005. Includes bibliographical references (p. 376-449). System requirements: World Wide Web browser and PDF viewer.
The oral commensal bacterium Streptococcus gordonii has been gathering interest as a candidate live mucosal vaccine delivery vector. S. gordonii has been shown to be capable of activating antigen presenting immune cells in a manner which leads to their activation and maturation, yet the mechanism used by S. gordonii to do so is poorly understood. The aim of this work was to investigate the immunostimulatory components of S. gordonii in inducing murine dendritic cell (DC) activation and maturation. Lipoteichoic acid (LTA), lipoprotein (LP), peptidoglycan (PGN), and DNA were isolated from S. gordonii, and used to stimulate murine DC. Cytokine production and DC surface marker upregulation in response to the bacterial components was quantified by enzyme-linked immunosorbent assay and flow cytometry respectively. The results were contrasted against data obtained from DC derived from MyD88, TRIF [TIR(Toll/Interleukin-1 Receptor)-domain-containing adapter-inducing interferon-beta] or toll-like receptor-2 (TLR-2) knockout mice. The four S. gordonii bacterial components were found to differentially induce cytokine production and surface marker upregulation by murine DC. Activation of DC by both whole S. gordonii cells and the four bacterial components was abrogated in the absence of MyD88, but not in the absence of TRIF. LTA, LP and PGN, but not DNA and whole S. gordonii, required TLR-2 to induce a DC response. The results collectively indicate that S. gordonii activates DC predominantly through a MyD88-dependent and TRIF-independent pathway. This activation can be attributed to multiple immunostimulatory components present within S. gordonii bacterial cells.
Bacterial vectors offer a biological route to gene and protein delivery with this article featuring delivery to antigen-presenting cells (APCs). Primarily in the context of immune stimulation against infectious disease or cancer, the goal of bacterially mediated delivery is to overcome the hurdles to effective macromolecule delivery. This review will present several bacterial vectors as macromolecule (protein or gene) delivery devices with both innate and acquirable (or engineered) biological features to facilitate delivery to APCs. The review will also present topics related to large-scale manufacture, storage, and distribution that must be considered if the bacterial delivery devices are ever to be used in a global market.
In recent years, there has been considerable interest in using the oral commensal gram-positive bacterium Streptococcus gordonii as a live vaccine vector. The present study investigated the role of d-alanylation of lipoteichoic acid (LTA) in the interaction of S. gordonii with the host innate and adaptive immune responses. A mutant strain defective in d-alanylation was generated by inactivation of the dltA gene in a recombinant strain of S. gordonii (PM14) expressing a fragment of the S1 subunit of pertussis toxin. The mutant strain was found to be more susceptible to
killing by polymyxin B, nisin, magainin II, and human β defensins than the parent strain. When it was examined for binding
to murine bone marrow-derived dendritic cells (DCs), the dltA mutant exhibited 200- to 400-fold less binding than the parent but similar levels of binding were shown for Toll-like receptor
2 (TLR2) knockout DCs and HEp-2 cells. In a mouse oral colonization study, the mutant showed a colonization ability similar
to that of the parent and was not able to induce a significant immune response. The mutant induced significantly less interleukin
12p70 (IL-12p70) and IL-10 than the parent from DCs. LTA purified from the bacteria induced tumor necrosis factor-alpha and
IL-6 production from wild-type DCs but not from TLR2 knockout DCs, and the mutant LTA induced a significantly smaller amount
of these two cytokines. These results show that d-alanylation of LTA in S. gordonii plays a role in the interaction with the host immune system by contributing to the relative resistance to host defense peptides
and by modulating cytokine production by DCs.
A single-chain variable fragment (scFv) antibody library against Bordetella pertussis was constructed using M13 phage display. The library was enriched for phages surface displaying functional scFv by biopanning against B. pertussis immobilized on polystyrene plates. Two hundred eighty-eight individual clones from the enriched library were screened for binding to B. pertussis cells, filamentous hemagglutinin (FHA), and pertactin (PRN) in enzyme-linked immunosorbent assays (ELISAs). Based on the binding ability, the clones were put into eight groups. The scFv DNA inserts from the 288 clones were digested with BstOI, and 18 unique restriction patterns, named types 1 to 18, were found. Eight clones (types 1 to 7 and 18) were selected for further testing against FHA, PRN, and B. pertussis by ELISA. The results showed that types 1, 5, 7, and 18 bound strongly to B. pertussis cells as well as FHA and PRN. Type 3 bound strongly to the cells and FHA but weakly to PRN. Types 4 and 6 bound FHA only, and type 2 did not bind to the cells or antigens. The ability of the eight clones to inhibit B. pertussis from binding to HEp-2 cells was assayed. Types 1, 5, and 7, but not the remaining clones, inhibited the adherence of B. pertussis to HEp-2 cells. The scFvs were sequenced, and the deduced amino acid sequence showed that the scFvs were different antibodies. Maltose-binding protein (MBP) fusion proteins composed of three different regions of FHA (heparin-binding domain, carbohydrate recognition domain, and the RGD triplet motif) were constructed. The three fusion proteins and Mal85 (MBP-FHA type I domain) were used to map the binding sites for scFvs of types 1, 5, and 7 by ELISA. The results showed that all three scFvs bound to the heparin-binding domain fusion protein but not the other fusion proteins. BALB/c mice who received recombinant phage-treated B. pertussis had reduced bacterial counts in the nasal cavity, trachea, and lungs compared to the control groups.
The mucosa represents the primary target site and thus the first barrier for most microbial pathogens. Nevertheless, nearly all present-day vaccines are applied by an invasive route, target the systemic immune system, and do not confer efficient mucosal protection. Currently, mucosal immunity can only be achieved by the delivery of antigens via the mucosal route. Therefore, multiple efforts are under way to develop mucosal vaccines and particularly live oral vaccines as these would confer considerable advantages. We have engineered the AIDA autotransporter system for the surface presentation and/or release of heterologous polypeptides. This study is focused on the development and evaluation of a tripartite live bacterial vector system for oral vaccination based on the AIDA autotransporter, heterologous virulence factor-derived (poly-)peptides, and the apathogenic Escherichia coli Nissle 1917 strain as a live carrier. Potentially with this system also attenuated Salmonella or Shigella strains might be employed as carriers. Model antigens included e.g. the p60 antigen of Listeria monocytogenes, the OspA/OspG antigens of B. burgdorferi, the LT-B subunit of E. coli, and Stx-B subunits of enterohemorrhaghic E. coli (EHEC), all representing crucial virulence factors of important bacterial pathogens. Exemplary oral immunization studies were conducted using different regimes in BALB/c mice with candidate vaccines expressing Stx B-subunits and OspA and OspG proteins. To monitor the induction of immune responses, specific antibody titers in serum as well as secreted mucosal antibodies of local and distal mucosal surfaces were determined. Antigen-specific mucosal as well as systemic antibodies could be induced; however, thus far the response turned out to be heterogeneous and appeared not to be sufficient to mediate protection.
Studies of lactic acid bacteria (LAB) as delivery vehicles have focused mainly on the development of mucosal vaccines, with much effort being devoted to the generation of genetic tools for antigen expression in different bacterial locations. Subsequently, interleukins have been co-expressed with antigens in LAB to enhance the immune response that is raised against the antigen. LAB have also been used as a delivery system for a range of molecules that have different applications, including anti-infectives, therapies for allergic diseases and therapies for gastrointestinal diseases. Now that the first human trial with a Lactococcus strain that expresses recombinant interleukin-10 has been completed, we discuss what we have learnt, what we do not yet understand and what the future holds for therapy and prophylaxis with LAB.
Here, we show that bacteria induce de novo synthesis of both major histocompatability complex (MHC) class I and II molecules in a mouse dendritic cell culture system.
The neo-biosynthesis of MHC class I molecules is delayed as compared with that of MHC class II. Furthermore, bacteria stabilize
MHC class I molecules by a 3-fold increase of their half-life. This has important consequences for the capacity of dendritic
cells to present bacterial antigens in the draining lymph nodes. In addition, a model antigen, ovalbumin, expressed on the
surface of recombinant Streptococcus gordonii is processed and presented on MHC class I molecules. This presentation is 106 times more efficient than that of soluble OVA protein. This exogenous pathway of MHC class I presentation is transporter
associated with antigen processing (TAP)-dependent, indicating that there is a transport from phagolysosome to cytosol in
dendritic cells. Thus, bacteria are shown to be a potentially useful mean for the correct delivery of exogenous antigens to
be presented efficiently on MHC class I molecules.
The B monomer of the Escherichia coli heat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer
expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers
of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the
enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces
and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1
when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery,
suggesting, in this case, the recruitment of a Th1 type of immune response.
Human colostrum, parotid saliva, and serum were assayed for the presence of naturally occurring antibodies to five serotypes of Streptococcus mutans. Appreciable levels of agglutinins to strains AHT, BHT, 10449, 6715, and LM-7 (groups a leads to e, respectively) were detected in normal colostrum and saliva, whereas relatively low levels were found in serum. No agglutinins could be detected in the colostrum or saliva of immunodeficient patients. Molecular sieve chromatography of the colostrum on Sephadex G-200 revealed agglutinin activity in the secretory immunoglobulin A (s-IgA)-rich fraction only. Titration of purified colostral s-IgA confirmed the IgA nature of this agglutinating activity. Indirect immunofluorescence tests with anti-s-IgA, -IgG, and -IgM revealed S. mutans specificity only in the s-IgA class. The presence of s-IgA antibodies to indigenous oral microorganisms in colostrum, as well as in saliva, suggests that antigenic stimulation occurs at a site remote from the oral mucosa.
We have developed a system in which a foreign antigen replaces nearly all of the surface-exposed region of the fibrillar M protein from Streptococcus pyogenes and is fused to the C-terminal attachment motif of the M molecule. The fusion protein is thus expressed on the surface of Streptococcus gordonii, a commensal organism of the oral cavity. The antigen chosen to be expressed within the context of the M6 molecule was the E7 protein (98 amino acids) of human papillomavirus type 16. Stable recombinant streptococci were obtained by integrating genetic constructs into the chromosome, exploiting in vivo homologous recombination. The M6-E7 fusion protein expressed on the S. gordonii surface was shown to be immunogenic in mice. This is the first step in the construction of recombinant live vaccines in which nonpathogenic streptococci as well as other gram-positive bacteria may be used as vectors to deliver heterologous antigens to the immune system.
An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because of variable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells.
This article reviews the ontogeny of immune systems in the human oral cavity that may influence the colonization, accumulation, or pathogenesis of oral microbiota. The prenatal development of cellular components associated with the secretory immune system reveals that the initial organization of tissue into Peyer's patches can first be detected immunohistologically at 11 weeks gestation. Epithelial cells positive for secretory component and immunocytes positive for IgM can be detected in salivary gland tissue by 19 to 20 weeks and continue to predominate during gestation. After birth, immunocytes containing IgA begin to dominate. Essentially, no IgA can be detected in saliva at birth. However, salivary IgA and IgM often appear soon thereafter, presumably in response to environmental antigenic and mitogenic challenges. Salivary IgA in young infants has molecular characteristics of secretory IgA and becomes the quantitatively predominate Ig in saliva. Both IgA subclasses are present in proportions characteristic of adult pure glandular salivas in many 1- to 2-month-old infants, although the appearance of IgA2 is delayed in some subjects. Many innate, antibody, and cellular immune components are found in maternal colostrum and breast milk. The antibacterial properties of these maternal factors are diverse and can exert multifaceted protective effects on the infant's alimentary tract. The infant apparently can mount mucosal immune responses quite early in life. For example, salivary antibody activity to organisms that originally colonize the gut (e.g., E. coli) or the oral cavity (e.g., S. mitis, S. salivarius) can be detected by 1 to 2 months of age. Most of this antibody activity has characteristics of secretory IgA, although some IgM antibody can also be initially detected. Salivary IgA1 and IgA2 antibody specificities to S. mitis and S. salivarius components increase qualitatively and quantitatively during the first few years of life. Salivary IgA antibody to components of streptococci that require hard surfaces for colonization (e.g., S. sanguis and mutans streptococci) generally appear after tooth eruption. The loss of placentally derived maternal IgG antibody specificities to these microbiota in the circulation is replaced by de novo synthesis, presumably as a result of the teething process. These IgG antibodies can enter the oral cavity in the gingival crevicular fluid and by the process of teething.(ABSTRACT TRUNCATED AT 400 WORDS)
Antibodies to S. salivarius, S. sanguis, and S. mutans cells and to glucosyltransferases (GTF) prepared from these micro-organisms were measured in the sera of 133 infants and children by enzyme-linked immunosorbent assay (ELISA). IgG antibody activity to each cell type and GTP was present at birth (presumably derived from maternal transfer) and declined significantly thereafter. IgG antibody levels to S. salivarius and S. sanguis were next detected in young children (2 to less than 3 yr group). However, an increase in IgG antibody to S. mutans cells was not seen until children were older (4 to less than 8 yr group), possibly reflecting the later colonization of this organism. In contrast, IgG antibody to GTF of all three streptococcal species remained at low levels throughout the first four years of life. IgG antibody to S. mutans GTF was then the first to appear (4 to less than 8 yr group). Serum IgA antibodies to all GTFs were not detected until after this time. Fifteen sera were used to develop IgG immunoblots with the GTF antigens. Some positive sera (7/12) demonstrated reaction(s) with GTF from each of the three streptococcal species. Individual sera showed IgG antibody bonds to GTF from several serotypes of the mutans streptococci. No immunoblot reaction was observed with GTF and sera (3) from the four-to-seven-year and younger age groups. These results indicate the presence of serum antibody to bacteria and bacterial products associated with plaque formation very early in life and during and after the pre-adolescent years. The potential exists for this serum antibody to modulate bacterial colonization or accumulation in the oral cavity.
Immunoglobulin A (IgA) and IgG antibodies against Streptococcus mutans K1R and 10449 were measured in serum and in stimulated whole saliva from two groups of naval recruits, representing high or low caries susceptibility. The antibody assays were performed by using the enzyme-linked immunosorbent assay, and the results were expressed by a method able to estimate the amount of high-avidity and total specific antibodies. As a control, concentrations of salivary total immunoglobulins were related to the amounts of specific antibodies. Further, antibodies were assayed against three antigens, unrelated to the streptococci. No clear differences were observed in serum antibodies between the subjects with high or low caries susceptibility. However, in saliva, low caries susceptibility was associated with a high amount of total antigen-specific IgA, and possibly IgG, against S. mutans. This difference between the groups still existed when the amounts of specific antibodies were related to the amounts of salivary immunoglobulins. There were no differences in the amounts of total specific antibodies against the unrelated antigens. No differences were observed in the estimates of high-avidity anti-S. mutans antibodies between the groups, either in serum or saliva. Thus, within the limitations of the assays and crude antigen, lack of high-avidity antibodies is not responsible for caries susceptibility. Instead, the amount of anti-S. mutans antibodies seems to be linked with caries protection. The results of the present study indicate that salivary antibodies are linked with the control of human dental caries.
To circumvent the need to engineer pathogenic microorganisms as live vaccine-delivery vehicles, a system was developed which allowed for the stable expression of a wide range of protein antigens on the surface of Gram-positive commensal bacteria. The human oral commensal Streptococcus gordonii was engineered to surface express a 204-amino acid allergen from hornet venom (Ag5.2) as a fusion with the anchor region of the M6 protein of Streptococcus pyogenes. The immunogenicity of the M6-Ag5.2 fusion protein was assessed in mice inoculated orally and intranasally with a single dose of recombinant bacteria, resulting in the colonization of the oral/pharyngeal mucosa for 10-11 weeks. A significant increase of Ag5.2-specific IgA with relation to the total IgA was detected in saliva and lung lavages when compared with mice colonized with wild-type S. gordonii. A systemic IgG response to Ag5.2 was also induced after oral colonization. Thus, recombinant Gram-positive commensal bacteria may be a safe and effective way of inducing a local and systemic immune response.
Genetically engineering bacteria to express surface proteins which can antagonize the colonization of other microorganisms is a promising strategy for altering bacterial environments. The fimbriae of Porphyromonas gingivalis play an important role in the pathogenesis of periodontal diseases. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to be an important virulence factor, which likely promotes adherence of the bacterium to saliva-coated oral surfaces and induces host responses. Immunization of gnotobiotic rats with synthetic peptides based on the predicted amino acid sequence of fimbrillin has also been shown to elicit a specific immune response and protection against P. gingivalis-associated periodontal destruction. In this study we engineered the human oral commensal organism Streptococcus gordonii to surface express subdomains of the fimbrillin polypeptide fused to the anchor region of streptococcal M6 protein. The resulting recombinant S. gordonii strains expressing P. gingivalis fimbrillin bound saliva-coated hydroxyapatite in a concentration-dependent manner and inhibited binding of P. gingivalis to saliva-coated hydroxyapatite. Moreover, the recombinant S. gordonii strains were capable of eliciting a P. gingivalis fimbrillin-specific immune response in rabbits. These results show that functional and immunologically reactive P. gingivalis fimbrillin polypeptides can be expressed on the surface of S. gordonii. The recombinant fimbrillin-expressing S. gordonii strains may provide an effective vaccine or a vehicle for replacement therapy against P. gingivalis. These experiments demonstrated the feasibility of expressing biologically active agents (antigens or adhesin molecules) by genetically engineered streptococci. Such genetically engineered organisms can be utilized to modulate the microenvironment of the oral cavity.
In this study, the expression of the Bordetella pertussis S1 subunit was tested in Streptococcus gordonii, a commensal oral bacterium which has the potential to be a live oral vaccine vehicle. The DNA fragment encoding the N-terminal 179 amino acids of the S1 subunit was ligated into the middle part of spaP, the surface protein antigen P1 gene originating from Streptococcus mutans. The resulting construct, carried on the Escherichia coli-Streptococcus shuttle vector pDL276, was introduced into S. gordonii DL-1 by natural transformation. One of the transformants (RJMIII) produced a 187-kDa protein (the predicted size of the SpaP-S1 fusion protein) which was recognized by both the anti-pertussis toxin (anti-PT) and anti-SpaP antibodies, suggesting that an in-frame fusion had been made. Results from immunogold-electron microscopic studies and cellular fractionation studies showed that the fusion protein was surface localized and was mainly associated with the cell wall of RJMIII, indicating that SpaP was able to direct the fusion protein to the cell surface. A rabbit antiserum raised against heat-killed S. gordonii RJMIII recognized the native S1 subunit of PT in Western blotting and showed a weak neutralization titer to PT by the Chinese hamster ovary cell-clustering assay. BALB/c mice immunized with the heat-killed S. gordonii RJMIII were protected from the toxic effect of PT in the leukocytosis-promoting and histamine sensitization assays. In conclusion, a fragment of the S1 subunit of PT was successfully surface expressed in S. gordonii; the recombinant S1 fragment was found to be immunogenic and could induce protection against the toxic effect of PT in mice.
Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.
Bacteria are being actively investigated as vaccine carriers for inducing or boosting protective immune responses. In this
study, human monocyte-derived dendritic cells (DCs) and normal B cells were compared for their capacity to present the C fragment
of tetanus toxin (TTFC), expressed on the surface of recombinant Streptococcus gordonii, to specific CD4+ T lymphocytes. DCs were more efficient than B cells at presenting soluble TTFC and remarkably more capable of presenting
bacterium-associated TTFC both in terms of the amount of antigen required to obtain a given T-cell response and on a per-cell
basis. This difference was associated with a much lower capacity of B cells to endocytose soluble TTFC and phagocytose recombinant
S. gordonii. In addition, S. gordonii induced the phenotypic maturation of DCs but not of B cells. The results thus indicate that DCs but not B cells play a crucial
role in the amplification of class II-restricted immune responses induced by immunization with recombinant gram-positive bacteria.
The srtA gene product, SrtA, has been shown to be required for cell wall anchoring of protein A as well as virulence in the pathogenic
bacterium Staphylococcus aureus. There are five major mechanisms for displaying proteins at the surface of gram-positive bacteria (P. Cossart and R. Jonquieres,
Proc. Natl. Acad. Sci. USA 97:5013–5015, 2000). However, since many of the known surface proteins of gram-positive bacteria
are believed to be exported and anchored via the sortase pathway, it was of interest to determine ifsrtA plays a similar role in other gram-positive bacteria. To that end, the srtA gene in the human oral commensal organism Streptococcus gordonii was insertionally inactivated. The srtA mutant S. gordoniiexhibited a marked reduction in quantity of a specific anchored surface protein. Furthermore, the srtA mutant had reduced binding to immobilized human fibronectin and had a decreased ability to colonize the oral mucosa of mice.
Taken together, these results suggest that the activity of SrtA plays an important role in the biology of nonpathogenic as
well as pathogenic gram-positive cocci.
The use of live microorganisms as an antigen delivery system is an effective means to elicit local immune responses and thus represents a promising strategy for mucosal vaccination. In this respect, lactic acid bacteria represent an original and attractive approach, as they are safe organisms that are used as food starters and probiotics. To determine whether an immune response could be elicited by intranasal delivery of recombinant lactobacilli, a Lactobacillus plantarum strain of human origin (NCIMB8826) was selected as the expression host. Cytoplasmic production of the 47-kDa fragment C of tetanus toxin (TTFC) was achieved at different levels depending on the plasmid construct. All recombinant strains proved to be immunogenic by the intranasal route in mice and able to elicit very high systemic immunoglobulin G (IgG1, IgG2b, and IgG2a) responses which correlated to the antigen dose. No significant differences in enzyme-linked immunosorbent assay IgG titers were observed when mice were immunized with live or mitomycin C-treated recombinant lactobacilli. Nevertheless, protection against the lethal effect of tetanus toxin was obtained only with the strains producing the highest dose of antigen and was greater following immunization with live bacteria. Significant TTFC-specific mucosal IgA responses were measured in bronchoalveolar lavage fluids, and antigen-specific T-cell responses were detected in cervical lymph nodes, both responses being higher in mice receiving a double dose of bacteria (at a 24-h interval) at each administration. These results demonstrate that recombinant lactobacilli can induce specific humoral (protective) and mucosal antibodies and cellular immune response against protective antigens upon nasal administration.
Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis. P. gingivalis fimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation. P. gingivalis fimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development against
P. gingivalis-associated periodontitis. We have previously shown that biologically active domains of P. gingivalis fimbrillin can be expressed on the surface of the human commensal bacterium Streptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with two S. gordonii recombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes of P. gingivalis FimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss
following P. gingivalis infection was also evaluated. The results of this study show that the oral delivery of P. gingivalis FimA epitopes via S. gordonii vectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses
and that the immune responses were protective against subsequent P. gingivalis-induced alveolar bone loss. These results support the potential usefulness of the S. gordonii vectors expressingP. gingivalis fimbrillin as a mucosal vaccine against adult periodontitis.
Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.
Sortase is a newly discovered transpeptidase that covalently links LPXTGX-containing surface proteins to the gram-positive
bacterial cell wall. In this study, the sortase gene (srtA) was isolated from Streptococcus mutans NG8 by PCR. The gene encoded a 246-amino-acid protein, including a 40-amino-acid signal peptide. The srtA gene was insertionally inactivated by a tetracycline resistance cassette. P1, a major surface protein adhesin previously
shown to anchor to the peptidoglycan by the LPXTGX motif, was secreted into the culture medium by the srtA mutant. In contrast, the wild-type P1 remained cell wall associated. Complementation of the mutant with srtA restored the P1 surface expression phenotype. P1 produced by the mutant, but not that produced by the wild type and the srtA-complemented mutant, was recognized by an antibody raised against the hydrophobic domain and charged tail C terminal to the
LPXTGX motif. These results suggest that the failure to anchor P1 to the cell wall is due to the lack of cleavage of P1 at
the LPXTGX motif. The srtA mutant was markedly less hydrophobic than the wild type and the complemented mutant. The srtA mutant failed to aggregate in the presence of saliva or salivary agglutinin and adhered poorly to saliva- or salivary agglutinin-coated
hydroxylapatite. In rats, the srtA mutant colonized the teeth poorly when sucrose was absent. When sucrose was present, the srtA mutant colonized the teeth but less effectively and induced significantly less caries (P < 0.05) than the wild-type strain. In conclusion, the sortase enzyme in S. mutans is responsible for anchoring P1 to the cell surface and plays a role in modulating the surface properties and cariogenicity
of S. mutans.
The ability of a recombinant Streptococcus gordonii RJM4 expressing the N-terminal 179-amino acid fragment of S1 subunit of pertussis toxin (PT) as a SpaP/S1 fusion protein was tested for oral colonization and immunogenicity in BALB/c mice. Following two consecutive oral inoculations, 100% of the mice were colonized by the recombinant strains for up to 9 weeks and >60% of the animals still retained the strains after 19 weeks. RJM4 recovered from the animals continued to express the SpaP/S1 fusion protein indicating the stability of the fusion gene in vivo. Secretory (S) IgA antibody against PT could be detected in saliva from RJM4-colonized mice but not from the control groups of mice. SIgA against SpaP was also detected in saliva from the RJM4-inoculated and the S. gordonii SL3 (control)-inoculated mice. Serum antibodies against PT and SpaP were not detected in these animals. In conclusion, long-term oral colonization of BALB/c mice with our recombinant S. gordonii was established and the colonization elicited mucosal antibodies against PT and SpaP.
Most available vaccines for enteric diseases are cinactivated whole cell preparations which provide incomplete cprotection against infection and have undesirable side effects. cAs alternative vaccines for enteric infection, several live oral cvaccines have been recently developed. These vaccines are cderived from virulent pathogens by introduction of stable cattenuating mutations in metabolic pathway genes. Ideally, cthese mutations are non-reverting and genetically well defined. cThe Salmonella
typhi live oral vaccine Ty21a has been tested in cseveral field trials, first in Egypt and more recently in four cseparate trials in Chilean schoolchildren (Ferreccio et al, 1989; Levine et al 1987a; Wahdan et al, 1982). The results of cthose trials indicate that Ty21a is safe, well tolerated, and cprovides 66% protection in a typhoid fever endemic area. cProtection against typhoid fever was at least that achieved with cparenterally administered whole cell vaccine preparations and cwas retained for at least three years.
An auxotrophic mutant which requires a metabolite not available in vertebrate tissues should be unable to grow in such tissue and thus be non-virulent. Most mutations to auxotrophy do not affect virulence1-3, presumably because the required metabolites are available at sufficient concentration. However, Bacon et al.1,2 noted that a Salmonella typhi mutant requiring p-amino-benzoic acid (p AB) was less virulent (for mice) than its parent. Salmonella, unlike vertebrates, cannot assimilate exogenous folate and must synthesize it from p AB; the virtual absence of p AB from vertebrate tissues is shown by the efficacy of sulphonamide chemotherapy. Yancey et al.4 reported reduced virulence for a S. typhimurium mutant with a requirement for 2,3-dihydroxybenzoate (DHB), the precursor of the bacterial iron-acquisition compound, enterochelin. As DHB is not a known vertebrate metabolite, it would be expected to be absent from mouse tissues. Salmonella synthesize p AB and DHB from chorismate, the final product of the aromatic biosynthetic (aro) pathway. A complete block at any step of this pathway should make S. typhimurium auxotrophic for two compounds not available in vertebrate tissues, and thus non-virulent. We describe here the use of a tetracycline-resistance transposon, Tn10 (refs 5, 6), inserted in gene aroA to produce non-reverting, aromatic-requiring derivatives of virulent S. typhimurium strains. These derivatives were virtually non-virulent; their use as live vaccines conferred excellent protection against challenge with a virulent strain.
The agglutinin titers for three Streptococcus mutans serotypes (AHT, BHT, and 10449, representing serotypes a, b, and c, respectively) were measured in the saliva, tears, and serum of 19 human subjects. Naturally occurring S. mutans agglutinins were routinely present in all fluids tested in the absence of overt local stimulation by antigen. The immunoglobulin A nature of this secretory agglutinin activity was suggested by blocking with alpha heavy-chain-specific antiserum and by the demonstration of S. mutans-reactive immunoglobulin A in the saliva and tears by indirect immunofluorescence. This finding is consistent with stimulation and antigen commitment of immunoglobulin A precursor lymphocytes at remote sites and subsequent homing to the lacrimal system. The relationship of anti-AHT agglutinins to anti-10449 agglutinins differed among the body fluids tested. The tears had more agglutinins for strain AHT than for strain 10449, whereas the reverse was true for saliva and serum. A possible explanation is local antigen-driven expansion of AHT-reactive committed lymphocytes in the lacrimal tissues.
The fimbriae of Porphyromonas gingivalis plays an important role in the pathogenesis of periodontal disease. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to promote adherence of the bacteria to host surfaces and also induce an immune response. Biologically active domains of fimbrillin responsible for adherence or eliciting immune responses have been determined. In a previous study, we engineered the human oral commensal organism Streptococcus gordonii to express such biologically active domains on the surface of the bacteria as a vaccine delivery system. In this study we report an alternative approach of secreting fimbrillin polypeptide domains into the medium by modification of the surface-expression system described earlier. Such recombinant S. gordonii, in addition to being a source for antigen presentation to trigger a protective immune response, may have the added advantage of directly blocking the fimbriae-mediated adherence of P. gingivalis to the oral cavity following implantation. This approach can also be utilized for secreting other biologically important therapeutic molecules on mucosal surfaces for modulating local microenvironments.
A genetic system for surface display of heterologous proteins has been developed in Streptococcus gordonii, a gram-positive human oral commensal that is naturally competent for genetic transformation. Our approach is based on chromosomal integration downstream from a resident promoter and translational fusion to an M6 protein. Using this strategy a variety of proteins, of different origin and size, were displayed on the cell surface and were shown to be stably expressed both in vitro and in vivo. Animal models of mucosal colonization (oral and vaginal) and intragastric immunization with recombinant S. gordonii were developed and the local and systemic immune responses were studied. Here we report the techniques for the construction of recombinant bacteria, use of animal models, and analysis of the immune response.
Tetanus toxin fragment C (TTFC) was expressed on the surface of the vaccine vector Streptococcus gordonii, a Gram-positive commensal bacterium of the human oral cavity. The immunogenicity of recombinant S. gordonii expressing TTFC was assayed in mice immunized by the parenteral and mucosal routes. High serum TTFC-specific IgG responses were induced in both BALB/c and C57BL/6 mice immunized subcutaneously. A total of 82% of vaccinated BALB/c mice were protected from the lethal challenge with 50 LD(50) of tetanus toxin (TT) and a direct correlation between the serum TTFC-specific IgG concentration and survival time of unprotected animals was observed. Intranasal immunization of BALB/c mice was also effective in inducing TTFC-specific serum IgG and local IgA in lung washes. Furthermore, 38% of animals immunized intranasally were protected from the lethal challenge with 10 LD(50) of TT while all control animals died within 24 h. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after parenteral immunization in BALB/c mice (IgG1/IgG2a ratio congruent with6) while following mucosal immunization a mixed IgG1 and IgG2a pattern (IgG1/IgG2a ratio congruent with1) was observed. These data show that TTFC expressed on the surface of S. gordonii is immunogenic by the subcutaneous and mucosal routes and the immune response induced is capable of conferring protection from the lethal challenge with TT.
The ability of a recombinant Streptococcus gordonii RJM4 expressing the N-terminal 179-amino acid fragment of S1 subunit of pertussis toxin (PT) as a SpaP/S1 fusion protein was tested for oral colonization and immunogenicity in BALB/c mice. Following two consecutive oral inoculations, 100% of the mice were colonized by the recombinant strains for up to 9 weeks and >60% of the animals still retained the strains after 19 weeks. RJM4 recovered from the animals continued to express the SpaP/S1 fusion protein indicating the stability of the fusion gene in vivo. Secretory (S) IgA antibody against PT could be detected in saliva from RJM4-colonized mice but not from the control groups of mice. SIgA against SpaP was also detected in saliva from the RJM4-inoculated and the S. gordonii SL3 (control)-inoculated mice. Serum antibodies against PT and SpaP were not detected in these animals. In conclusion, long-term oral colonization of BALB/c mice with our recombinant S. gordonii was established and the colonization elicited mucosal antibodies against PT and SpaP.
To test the effect of co-expression of immunomodulatory molecules, together with target antigen, two recombinant Streptococcus gordonii strains were constructed which secreted either murine interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) in addition to a surface anchored test antigen (the conserved C-repeat region (CRR) of the M6 protein of Streptococcus pyogenes). The secretion of functional cytokines by S. gordonii was achieved by in-frame fusion of sequences encoding mature IL-2 or IFN-gamma to the sequences encoding the leader signal of the M6 protein. Expression of the M protein CRR region from a separate chromosomal site produced double recombinants expressing a secreted cytokine and the M protein CRR region anchored to the surface. Protein expression was verified by streak blot, immunoblot, and ELISA on both the single and double recombinants. A cytokine bioassay using HT-2 cells verified biological activity of recombinant IL-2 secreted from S. gordonii. When mice were immunized subcutaneously with the different S. gordonii expression strains, cytokine co-expression apparently modulated the systemic immune response. These results show that streptococci can deliver biologically active molecules such as cytokines along with antigens to the immune system. These results demonstrate that a cytokine-secreting, noninvasive, bacterial vaccine vector can be used to modulate immune responses to a co-expressed antigen.
The Gram-positive bacterium Streptococcus gordonii has been genetically engineered to allow the simultaneous expression of two heterologous proteins at the cell surface. A family of recombinant streptococci displaying two different antigens was constructed. All the strains were genetically stable and expressed both proteins at the surface of the same bacterial cell. S. gordonii co-expressing the immunomodulating molecule LTB (B monomer of Escherichia coli heat-labile toxin) and the V3 domain of HIV-1 gp120 were inoculated subcutaneously to BALB/c mice. Animals were capable of responding to both antigens, producing LTB- and V3-specific serum IgG. The V3-specific IgG titer was four-fold higher in mice immunised with the double protein-expressing bacteria, as compared to control animals inoculated either with S. gordonii expressing the V3 domain alone or with a mixture of the two strains expressing LTB and V3, separately. Therefore, LTB was able to potentiate the antibody response towards the V3 domain, and this effect was observed only when LTB was co-expressed on the same bacterial cell.
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