Advances in Dental Research

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The Specific Pathogen-Free Human: a New Frontier in Oral Infectious Disease Research M.A. Taubman, R.J. Genco and J.D. Hillman ADR 1989 3: 58 DOI: 10.1177/08959374890030010501 The online version of this article can be found at: http://adr.sagepub.com/content/3/1/58

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THE SPECIFIC PATHOGEN-FREE HUMAN: A NEW FRONTIER IN ORAL INFECTIOUS DISEASE RESEARCH M.A. TAUBMAN 1 , RJ. GENCO 2 , AND J.D. HILLMAN 3 department of Immunology and department of Molecular Genetics, Forsyth Dental Center, 140 The Fenway, Boston, Massachusetts 02115; and department of Oral Biology, State University of New York at Buffalo, Buffalo, New York 14214 Adv Dent Res 3(l):58-68, May, 1989

ABSTRACT he indigenous flora acts as a deterrent to the establishment of some pathogenic species. We propose that advances in oral health research will lead to control of oral infections by altering the indigenous microflora to create a specific pathogen-free human. Investigations of important endogenous and exogenous factors which affect the oral flora and the interactions among these parameters, in health and disease, will have to be undertaken for this goal to be achieved. Several approaches to produce a specific pathogen-free human include: (1) introduction of individual or collective moieties which inhibit detrimental interactions on a genetic and molecular level; (2) genetic modification of salivary flow and protein composition by use of transgenic techniques; (3) therapeutic replacement with altered bacterial strains; (4) alteration of host immune responses to produce specific isotype immunity at the most appropriate time in the ontogeny of the oral environment; (5) production of isotype and/or antigen-specific regulatory molecules at the most appropriate time in development; (6) use of synthetic vaccines; (7) genetic alteration or replacement of cells with defective protective capabilities; and (8) use of anti-idiotype vaccines.

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INTRODUCTION Humans. Bacteria, and Health Humans are exposed to bacteria for the first time during their passage through the birth canal. Within a very short time thereafter, each individual acquires a resident or indigenous flora consisting of hundreds of different types of bacteria that persistently colonize various tissues and organs in large numbers. In addition, each person is exposed daily to other bacteria that are present in the environment, and which may be found in association with the host for various periods of time, and which constitute the transient flora. The indigenous and transient flora offer an enormous challenge to the health of the host. Human beings are healthy and pathogen-free most of the time (Brock, 1966). By examining factors responsible for this state of affairs, we may gain insight into the prevention of and cures for those relatively few bacterial and preA report of the AADR ad hoc Committee on New Frontiers in Oral Health Research

disposing host conditions that are responsible for overt microbial disease. The common state of health in humans could be explained if indigenous bacteria were almost always non-pathogenic. This is likely since host-parasite relationships are inherently unstable (Person, 1968). Evolutionary processes select for mutations that confer increased resistance of the host and decreased virulence by the parasite. Given enough time, this will yield a climax state in which the parasite has become adapted to its niche in the host to such an extent that it can no longer cause overt disease, a condition known as amphibiosis. In many cases, one (commensalism) or both (mutualism) of the partners may actually benefit from the relationship. Most indigenous microorganisms, by virtue of their long association with man, have achieved one or another of these climax states. Indigenous bacteria that possess some pathogenic potential are near, but have not yet reached, a climax state. They usually require a predisposing environmental alteration to exert that potential {e.g., colonization of heart valves by Streptococcus sanguis requires previous cardiac damage or unusually high

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numbers of bacteria). Such bacteria are frequently termed "opportunistic pathogens". This class of infections could be largely eliminated either by prevention of the predisposing conditions or by alteration of the bacteria for further reduction of their alreadyminimal pathogenic potential, thereby bringing them to a climax state. Opportunistic infection may also be prevented by reconstituting host resistance or increasing host resistance in an otherwise-normal individual. Exogenous infections are caused by those transient bacteria which are virulent. The exogenous pathogenic bacteria are less-well-adapted to the human host than are indigenous bacteria. Exogenous pathogens typically do not require predisposing host conditions or unusual environmental alterations to exert pathogenic potential. Several factors normally limit transient bacterial infections and thereby preserve an ongoing state of health. One such factor is the ability of indigenous bacteria to prevent either initial colonization by transients, or their emergence to the level necessary for pathogenic effect. Host resistance is also an important factor in prevention or limitation of infections caused by transient bacteria. Infections caused by exogenous pathogens could be prevented in several ways: (a) by eliminating exposure to the infectious agent by destroying or avoiding its primary environmental reservoir; (b) by finding or adapting indigenous organisms to occupy the niche in susceptible host tissues which would be occupied by the pathogen; (c) by adapting indigenous organisms which produce factors antagonistic to the exogenous pathogens; or (d) by increasing resistance of the host to the pathogen. Almost any change in an ecosystem may affect either the host's susceptibility or a pathogen's virulence. In the modern era, many of the environmental alterations of consequence are the result of man's intervention. Improved sanitation, better diets, and vaccines are examples of measures that have decreased the incidence and severity of many microbial diseases. Environmental alterations may also upset the balance of organisms in the climax state, resulting in a return to a host-parasite relationship. This effect has been observed a number of times, notably in relationships between modern sanitation and poliomyelitis and between high-sugar diets and dental caries. The Concept of a "Specific Pathogen-free Human" Louis Pasteur suggested (1885) that host and microorganism develop an association which is compatible with co-existence. He believed that this was essential for the normal life of the host. Germ-free animals have many host defense abnormalities, including underdeveloped lymphoid organs, low immunoglob-

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ulin levels, high levels of digestive hydrolases, and enormously enlarged caeca (Luckey, 1963). Such animals are often more susceptible to infections than those raised conventionally. Studies with gnotobiotic animals have clearly demonstrated both beneficial (probiotic) and antagonistic (antibiotic) relationships between the host and its microflora. Pioneering work by Dubos and Schaedler (1960) showed that germ-free animals housed in a clean environment and allowed to be colonized by various combinations of autochthonous flora grow and develop far better than do their conventional counterparts. Such SPF (specific pathogen-free) mice produced larger letters of more uniform size, which develop into larger adults. The optimal intestinal flora of SPF mice can be altered detrimentally by antibiotics. In the early studies, the selection of the SPF flora was arbitrary; however, Schaedler and his colleagues (1965) were the first to colonize germ-free mice with specific members of the autochthonous microflora. Syed and co-workers (1970) showed that association of the host with some 130 strains could reverse abnormalities associated with the germ-free state. Abnormal cecal enlargement was eliminated, and numbers of E. coli in the small bowel and thinning of its lamina propria were reduced. Later (1972), Freter and Abrams were able to reduce this SPF flora to 59 strains (45 obligate anaerobes and 14 facultative anaerobes). Similar studies reinforcing the importance of the intestinal microflora for resistance to enteric pathogens were performed by Bohnoff and colleagues (1954). They showed that elimination of the intestinal flora by antibiotics resulted in a 100,000-fold increase in susceptibility to infection by Salmonella. This was not due to a lack of immune competence of germ-free animals, even though these animals have reduced amounts of lymphoid tissue. Germ-free animals can respond immunologically as well as conventional animals when challenged (Taubman and Smith, 1974). The obligately anaerobic flora established by the second week of life inhibited subsequent infections by coliforms, enterococci, and other facultatively anaerobic bacteria. This phenomenon was called " colonization resistance" by Van der Waaij et al. (1971). The work of several others indicated that a single type of fusiform bacteria alone was able to inhibit colonization of pathogenic and non-pathogenic bacteria. This information can be used to establish a "defensive" bacterial flora which can repel invading pathogens. The indigenous flora can deter the establishment of some pathogenic species. Even when some of these pathogenic organisms infect the host —for example, pneumococci and meningococci — their numbers seem to be held to levels below the threshold for pathogenesis (Hardie and Bowden, 1974; Bowden et al., 1979). This phenomenon has also been observed in the oral cavity (Krasse et al., 1967; Mikx et al., 1976; Svanberg and Loesche, 1978). Manipulating the indigenous flora to provide an environment for creation of specific pathogen-free


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humans, who are also resistant to pathogens, has only recently been considered. The microbial flora of humans can, to some extent, be manipulated to eliminate potentially pathogenic micro-organisms. For example, plasmid-free E. coli were used in infants to reduce subsequent infection with antibiotic-resistant E. coli strains (Duvall-Iflah et al., 1982). Significant information in this regard was also provided by experience with David the "Bubble-Boy", who suffered from severe immunodeficiency. Although David was born by Caesarean section, and was maintained behind sterile plastic barriers for virtually his entire life, he did have a microflora (Bealmear etal., 1985; Guerra and Shearer, 1986). By the time he was 4 years old, approximately 35 species of micro-organisms, mostly transient contaminants, had been isolated. He was colonized by species including: Bacteroides oralis, Staphylococcus epidermidis, Enterobacter agglomerans,

Candida tropicalis, and several potentially pathogenic species of Pseudomonas (Guerra and Shearer, 1986). However, despite lacking all lymphocyte function, he was not ill nor did he develop viral infections (Guerra and Shearer, 1986). This natural human experiment underscores the possibility of combating infection by provision of an appropriate microflora. A complementary approach to creation of a pathogen-free human would be to alter the host by bolstering protective responses which engender an active resistance to pathogens. In order for a pathogen-free human to be established, it will be necessary to identify the pathogens, the mechanisms by which they exert pathogenic effects, and either alter the pathogenic potential or the host resistance. The remainder of this report will survey the existing information and will present future prospects for achieving the goal of a specific pathogen-free human. Ecosystems: The Basis for the Establishment of a "Defensive" Flora Ecosystems are dynamic. Constant interactions between the components and their environment lead to the eventual establishment of the "climax community", in which there is a fairly stable balance. Environmental conditions such as food supply contribute to the establishment of the climax community. Drastic changes in the amount or nature of the food, or other environmental changes, can alter the balance of the community and result in a new ecosystem. In the oral cavity, attachment of micro-organisms to various surfaces is essential for the formation of plaque on these surfaces. Oral micro-organisms have specific and non-specific mechanisms to attach to teeth, other oral surfaces, or to each other (Gibbons and van Houte, 1971). HOST-BACTERIAL INTERACTIONS Mechanisms of Interaction in Dental Plaque Within minutes, a newly erupted or experimentally clean tooth acquires an organic pellicle from salivary

proteins to which bacteria promptly attach. Potentially odontopathic and periodontopathic organisms are present in the indigenous microflora of most individuals. However, it is conceivable that these micro-organisms can be prevented from colonizing by utilization of various "new frontier" methods. We will emphasize the use of a competing flora as well as the host immune systems to curtail colonization of oral pathogenic bacteria. Most initial colonization of the human comes from the mother, with lesser contributions from other humans. These contacts are of considerable importance, because most environmental micro-organisms are saprophytic and, therefore, are not permanent colonizers of the human flora. In humans, the major source of the dental pathogenic mutans streptococci (S. mutans and S. sobrinus) is maternal (Berkowitz and Jordan, 1975; Kohler and Bratthall, 1977). Initial colonization events provide the most propitious time for modulation of the establishment of bacteria (Smith and Taubman, 1987; Taubman and Smith, 1987,1988a). The oral cavity contains numerous unique host factors and exogenous factors influencing the flora. The potential for health or disease resides within this environment. We will identify some unique factors in the oral cavity, or unique aspects of more common features of the oral cavity, which determine the nature of the oral environment. Potential areas for regulation of these factors to the benefit of the host will be emphasized. Factors Influencing the Oral Environment There are endogenous and exogenous factors which influence the general environment and micro-environments in the oral cavity. The endogenous factors are derived from the host, and they include: salivary proteins, glycoproteins, enzymes, teeth, pellicle, shedding mucous membrane, barrier functions of oral mucous membranes, humoral immune factors (salivary and serum antibody), cellular immune factors (e.g., lymphocytes, neutrophils, and cytokines), and protective factors. They also may include lysozyme (Iacono et al., 1982), lactoferrin (Arnold et al., 1982), and oral peroxidase systems (Pruitt and Reiter, 1985). The second group of factors is exogenous. Examples include: bacteria, viruses, diet, and nutrition. While bacteria can be considered exogenous factors, there appear to be at least two types of potential pathogens among the indigenous flora, i.e, enduring indigenous and transient indigenous micro-organisms; the latter are also called exogenous pathogens (Genco et al, 1988). Microbial Succession in the Oral Cavity — Pathogens Reconsidered The enduring indigenous microbiota includes micro-organisms that are ubiquitous to the host species for the duration of life or for extended periods during life. The delayed type (transient) would be indige-


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nous in some individuals for a less extended duration. Colonization with these transient indigenous organisms may occur later in life and could be temporary. Examples of organisms that are likely transient indigenous are Bacteroides gingivalis and Actinobacillus actinomycetemcomitans. Examples of en-

during indigenous organisms are S. sanguis and mutans streptococci (Carlsson et al., 1975). Although mutans streptococci appear to inhabit virtually everyone's oral cavity (Littleton et al., 1970; van Houte and Duchin, 1975), the work of Kohler et al. (1983) showed that children may not necessarily become infected even when the mother is carrying high numbers of mutans streptococci. While S. sanguis colonizes earlier than mutans streptococci, and there are possibly other differences in ease and potential permanence of their elimination, for purposes of this discussion both can be considered as enduring indigenous organisms. Mutans streptococci are present at higher levels in those who have caries (Bowden et al., 1976), indicating the pathogenic capacity of this organism. Thus, mutans streptococci appear to be pathogenic if present at high levels. There are other examples of indigenous organisms which can express pathogenic roles, e.g., Candida can overgrow to cause disease (Arendorf and Walker, 1980). Another example of an organism which is enduring indigenous, but unique, is the herpes simplex virus, which resides in the nervous tissue and, upon activation, goes from a latent to an active state. Further delineation of pathogens on the basis of enduring or transient status may provide an approach to interference with the establishment of these micro-organisms. This approach may be particularly fruitful in periodontal diseases. Perhaps some periodontopathogens are indigenous and enduring, and simply overgrow to cause inflammatory responses. An example might be gingivitis associated with an overgrowth of Actinomyces. However, there also appear to be specific transient indigenous organisms, such as B. gingivalis and Actinobacillus, which may not be residents of the normal oral flora. They are present only at low levels, if at all, in normal individuals (Zambon et al., 1986). A clear identification of which species are enduring and which are transient pathogens, and elucidation of their relationship to the development of a specific pathogen-free oral cavity, is necessary in the development of a specific pathogenfree human. It might be more difficult to obtain a specific pathogen-free oral cavity if the pathogens in question are indigenous enduring, since they may be hard to eliminate. On the other hand, transient pathogens such as B. gingivalis and Actinobacillus may be amenable to complete elimination from the oral cavity, a result which could be consistent with health. There is evidence to support long-term elimination of such organisms (Nord and Heimdahl, 1986). Furthermore, once transient indigenous pathogens have been eliminated, re-infection may be difficult. Hence, provision of a specific pathogen-free mouth with re-

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gard to pathogens which are transient indigenous may be feasible. It may even be possible to eliminate an enduring indigenous pathogen such as S. mutans. Reduction of the level of S. mutans infection in the mother can reduce the risk that the infant is infected by the bacteria (Kohler et al., 1983). This results in reduced caries incidence in the infants (Kohler et al., 1984; Alaluusua and Renkonen, 1983). Topical application of fluorides reduces the ability of bacteria to attach to the teeth, and a low sucrose intake does not support the establishment of mutans streptococci. In infected persons, the number of mutans streptococci can be reduced to undetectable levels for long periods of time. Re-emergence could be due to regrowth of micro-organisms which have not been eliminated (Emilson et al., 1987) or to recolonization which can be delayed by the use of fluorides (Zickert et al., 1987). Antiseptics, such as a chlorhexidine varnish, might eliminate the mutans streptococci completely (Sandham et al, 1988). Diet and nutrition are important exogenous factors from two perspectives: First, diet establishes the portion of the ecologic niche for bacteria in the oral cavity which is provided by exogenous nutrients. Second, the diet will have a direct bearing, ultimately, on the host's nutritional status, which, in turn, can directly affect the ability to resist bacterial challenges and disease. Any physiological or pathological state can increase nutrient requirements and exacerbate the potential for nutrient deficit. Recognition of these complexities is important to understanding exogenous factors and their roles in prevention and therapy of oral disease. METHODS TO MODIFY BACTERIA Important Oral Interactions For the goal of establishing a specific pathogen-free human to be accomplished, approaches involving identification and investigation of new frontiers in oral ecology will have to be undertaken. This will entail elucidation and investigation of important endogenous and exogenous factors and their interactions in health and disease. The question arises as to how the potential interactions of importance will be identified. Bacterialimmune factor interactions might be expected to be different for enduring as compared with transient colonizers. Normally, there can be ineffectively low levels of specific immunity to transient indigenous bacteria, which are probably not often encountered by the host. On the other hand, there may be a low level of immunity to enduring indigenous bacteria. Hence, further induction of immunity to enduring bacteria may not be as effective in total elimination, since there is already a low level of immunity in health. On the other hand, this low level of immunity may keep the enduring indigenous non-pathogenic bacteria at relatively low levels, and it is conceivable that only when these bacteria overcome the host immunity do they become


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pathogens. It could then be inferred that transient bacteria would be more easily interfered with by specifically induced immunity. In order for bacteria to be significantly modified as a strategy in provision of a pathogen-free human, advances in our understanding of bacterial-host interactions must be made. These interactions include the salivary factor-tooth interaction, pellicle-bacteria interaction, bacteria-bacteria interaction (bacterial coaggregation), bacteria-immune factor interaction, and bacteria-nutrient interaction.

Advances in Microbial Coaggregation Expected to Aid in Our Understanding of Bacterial-Bacterial Interactions Interactions of microbial coaggregation have been studied extensively (Cisar et al, 1979, 1984; Weerkamp et al, 1984; Kolenbrander et at., 1983). For example, Cisar and his colleagues (1979) studied coaggregation of S. sanguis and A. viscosus. A high percentage of S. sanguis and Actinomyces isolates from dental plaque coaggregated by a mechanism involving lectin-carbohydrate interactions. Coaggregations mediated by lectins on Actinomyces and the complementary polysaccharide receptors on specific strains of S. sanguis (or S. mitis) were similar in that all were inhibited by lactose. In contrast, coaggregations that were not inhibited by this sugar involved lectin-like adhesins on streptococcal strains and carbohydrate-containing receptors on the Actinomyces. With certain coaggregating pairs, lectins on each cell type contributed to the interaction. The specific cellcell interactions of these and various other oral bacteria are of particular interest, since they may contribute to the formation of the characteristic microbial communities found on different oral tissue surfaces.

Structural Studies Leading to Future Molecular Inhibitors The types of investigations described above illustrate the importance of structural studies on the interactive moieties. The reactive carbohydrate receptor from S. sanguis strain 34 is composed primarily of anhydro sugars. If it were desirable to inhibit interactions involving this receptor, specific inhibitors bearing anhydro sugars could be synthesized. Such syntheses would be designed to produce individual inhibitors or collective (group) inhibitors based on methodology for peptide or carbohydrate syntheses or by genetic manipulation procedures. Further studies would be concerned with regulation of these interactive molecular domains on a genetic and molecular level. An example might involve modulation of expression of important bacterial surface components which would enhance the oral colonization potential of a useful defensive indigenous organism.

Genetic Alteration of Micro-organisms Genetic alterations of bacteria may also aid in the design of a useful defensive oral organism (Freedman et al., 1981). This can be approached with mutant micro-organisms or by use of recombinant DNA technology. Several approaches to implementation of this strategy can be considered. These include: (1) Mutational alteration of bacteria to eliminate harmful characteristics while maintaining features necessary for continued presence in the oral cavity. Such organisms can be used in "replacement therapy" to colonize teeth purposely with a non-virulent mutant of a pathogenic species such as S. mutans (Hillman, 1978; Johnson et al, 1980; Johnson and Hillman, 1982). By occupying the niche on the teeth normally inhabited by decay-causing bacteria, the mutant or altered strain could prevent colonization by wildtype pathogens. Such protection might last a lifetime and might even be transmitted from mother to child. (2) Antagonistic micro-organisms could be used to establish a flora resistant to colonization by pathogens, or alter the flora during disease, leading to elimination of pathogens. (3) Natural or artificial production of bacterial surface moieties might be used to favor healthy bacterial interactions or to compete with detrimental bacterial interactions. Replacement Therapy The natural selective pressures to decrease the virulence of mutans streptococci and to increase the host resistance to caries may require several millennia to bring the mutans streptococci into a climax state with the human bacteria in which there is no caries. One aim of replacement therapy is to achieve the climax state much sooner by introduction of mutans streptococci of lower virulence. The approach depends on finding an effector strain of bacteria which does not cause disease, and which persistently colonizes the host tissues that are prone to infection by this particular pathogenic bacterium. It is also important that this effector strain prevent colonization of the host by wild-type pathogens. In the case of dental caries, mutants of cariogenic streptococci which produce little acid and an atypical strain of S. salivarius have shown promise as effector strains (Hillman and Socransky, 1987; Tanzer et al, 1985). Replacement therapy may also be an approach in the prevention and cure of certain periodontal diseases (Hillman and Socransky, 1987). Plaque taken from healthy subjects invariably contains indigenous bacteria (notably S. sanguis and S. uberis) that make hydrogen peroxide in amounts sufficient to kill a variety of presumed periodontal pathogens such as Actinobacillus actinomycetemcomitans. Patients lacking such

inhibitors may be treated by replacement therapy to add these defensive hydrogen peroxide-producing streptococci, which would antagonize colonization by periodontal pathogens.


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METHODS TO MODIFY THE HOST Introduction of cloned DNA into the mouse genome by micro-injection of fertilized eggs was achieved early in this decade (Constantini and Lacy, 1981; Gordon et al., 1980). Insertion of DNA into the germline by this method can dramatically change the physiology of the animal. For example, mice that receive the mouse metallothionein promotor and regulatory region fused to the rat growth hormone structural gene can grow 2-3 times as fast as controls and reach a size twice that of normal mice (Palmiter et al., 1982). Genetic engineering of salivary flow characteristics and protein composition might be considered for directing the formation of pellicles. Permanent changes in genetic composition are important so that salivary changes can be made stable to provide a lifetime of protection against colonization by pathogens. Another approach involves purification and synthesis of active peptides from salivary proteins (or glycoproteins). It would be important to select components which were significant in maintenance of healthy pellicle(s) and interactions between salivary components and useful (host beneficial) micro-organisms. These components could be selectively added to growing pellicles to produce a "healthy" plaque. Host Immune Response The introduction of changes in host immune response to produce specific isotype immunity at the most appropriate time in ontogeny of the oral environment would involve alteration of regulatory mechanisms. T-lymphocytes exert regulatory effects on both T- and B-cell classes (Katz and Benacerraf, 1972; Gershon, 1974). This regulation of the immune system can range from enhancement to suppression and involves complex communication processes. The T-cells participating in different regulatory functions or exerting different effector functions in cell-mediated immunity belong to distinct subclasses identifiable by phenotypic expression of cell markers (Cantor and Weissman, 1976). Physiological interactions of T-cells and B-cells involve genetic restriction associated with major histocompatibility complex genes. These regulatory interactions are often complex multicellular events in which the members may be organized into circuits (Germain and Benacerraf, 1980). The induction of suppressor pathways depends upon the action of a so-called "inducer population". This inducer population may also be one of the sites of action of the amplification steps. Such circuits are illustrated by immunologic suppression in which the first suppressor cell is induced to act to cause the production of a second class of suppressor (Benacerraf et al., 1982). Amplification is achieved by proliferation of precursors of effectors after antigen exposure and from helper signals generated by cell products. Suppression is achieved by specific subpopulations of cells which are themselves induced by

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direct or indirect consequences of antigenic stimulation. These cells are effective either by reducing amplification signals provided by the helper cells or by acting on the effector cell precursors. Production of Isotype and Antigen-specific Regulatory Molecules by Genetic or Protein Synthetic Means Cytokines are products of various cells of the immune system which appear to be involved in regulation of a variety of cellular functions involving the host immune capability. Several of these human factors have been produced by recombinant technology and are available for use. However, the use of any of these individual cytokines in isolated fashion has usually not resulted only in beneficial effects, because cytokines have multiple biologic activities, some of which may be deleterious to the host. A more complete understanding of the structure-function relationship of portions of the molecules of cytokines will facilitate selection of those portions with beneficial effects for selective synthesis and use in amplifying desired host responses. Host Cell Replacement Therapies Defective effector or regulatory cells could conceivably be corrected by genetic alterations or by replacement. Major knowledge and advances in histocompatibility would be needed before this approach could be implemented. Development of New Types of Vaccines The best way to create a pathogen-free human may be by vaccination. Vaccination is the simplest, safest, and most effective form of prevention of disease, and has achieved remarkable success by eliminating pathogens from the human flora. Vaccination has not been a top priority of dental researchers for caries and periodontal diseases, because the causative organisms were not clearly known, and because past generations of vaccines have had adverse side-effects. Some of the side-effects of older vaccines are serious enough to constitute a risk not worth taking for non-life-threatening diseases such as caries and periodontal diseases. This has changed since we now have a knowledge of the major bacteria causing caries and periodontal disease. Furthermore, present strategies promise to provide safe, more effective vaccines. Recent advances have resulted in new types of vaccines (see Taubman and Smith, 1988b). These include: subunit vaccines, recombinant vaccines, recombinant infection vectors, internal image anti-idiotypes, and synthetic peptides. Small Synthetic Peptides to Elicit Antibodies Reactive with Native Proteins The potential antigenic determinants of many proteins coincide with regions exposed to solvent (Sutcliffe et al., 1983), and these segments often represent areas of considerable molecular flexibility (Williams


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and Moore, 1985). Structurally, these regions may represent a continuous part of the polypeptide chain ("sequential or primary determinant") or areas that approximate each other resulting from folding of the polypeptide chain ("topographical") (Atassi, 1984). For example, antibody to the carboxy terminus of the transforming protein of Rous sarcoma virus was produced by immunization of rabbits with a synthetic peptide which corresponded to the six carboxy terminal amino acids of this protein (Sefton and Walter, 1982). Many laboratories have utilized synthetic peptides as vaccines. Systems which have received attention include: diphtheria toxin, the M protein of Streptococcus pyogenes, and viral systems such as foot and mouth disease and influenza (Arnon et al., 1983). For example, a 35-residue synthetic polypeptide fragment of type 24 streptococcal M protein has been demonstrated to elicit protective anti-streptococcal antibodies in rabbits (Beachey et al., 1981) and mice (Jolivet et al., 1983). Antibodies to synthetic peptides of influenza hemagglutinin (HA) 1 neutralize the cytopathic effect of the virus on canine kidney cells (Bittle et al., 1982). Four peptides of different lengths corresponding to different regions of the H3 influenza hemagglutinin were synthesized. Two peptides gave rise to antibodies that bound to the intact virus, and one of these led to a protective effect upon challenge in vivo (Em-

A.

B. ~

C.

D.

Fig. — Idiotypes and anti-idiotypes as potential vaccine moieties. Antigen was injected into animal 1, and the antibody produced (AB 1 = idiotype) is injected into animal 2, which forms AB 2 ( = anti-idiotype). The AB 2 recognizes an idiotype (Id) on AB 1, and also some AB 2 molecules may look like and behave like epitopes (X, Y) on the antigen. A variety of "internal image" anti-idiotypes can be generated. [Some may recognize idiotypes, while others will mimic the epitope (determinant) against which the idiotype was directed.] A major reason why idiotypes behave like the origional epitope is that these can share the identical amino acid sequence with the immunogenic epitope (A). An additional possibility would suggest that the epitope is mimicked on a stereochemical level (B). Additional antibody (AB 2a) may recognize the interaction of AB 1 and AB 2. Further injection of AB 2 into animal 3 might produce antibody molecules quite similar to those of AB 1 (see C). These antibody molecules (AB 3) could react with the original antigen (D).

tage et al., 1980). The length of the synthetic peptide was critical in determination of antigenicity, presumably by ensuring the proper folding required to mimic the native structure of the hemagglutinin. Recombinant DNA methods have also been used to create anti-influenza peptides. The cloning of the HA gene in E. coli (Davis et al., 1981; Gething and Sambrook, 1981) and in SV40 (Paoletti et al, 1984) leads to the possibility of large-scale production of the HA. The preparation of safe and effective vaccines from synthetic peptides used in combination with adjuvants or carriers to make them more immunogenic is a feasible goal. There are several advantages to such an approach. First, linking various relevant peptides from different organisms would allow for the creation of multivalent vaccines. Second, this approach offers the opportunity to mass-produce relevant antigens, thereby making a vaccine more economical and hence practical. Third, and perhaps most significant, this approach would eliminate the induction of host antibody to irrelevant or potentially harmful cross-reactive antigens, as is known to occur when complex immunogens, such as intact bacteria or live attenuated viral agents, are employed. Anti-idiotype Vaccines The antigen-binding region on the antibody molecule is based on six hypervariable loops (3 on heavy chain, 3 on light chain) that contain highly diverse amino acid sequences. X-ray crystallographic studies of the antibody-combining site, the part that actually binds to the antigenic determinant or epitope, show that it is made up of amino acids from other hypervariable regions. These stretches of amino acids form a flattened area of about 700 A2, which is unique for each antibody. Since this region is unique, the antibody-combining site itself is an antigen, or idiotype, and potentially an immunogen capable of inducing immunity. If immunity is induced to the idiotypic determinants, anti-idiotypic antibodies result. Since the antigen receptors on T-cells also display idiotypic determinants, anti-idiotypic T-cells may result. Thus, idiotypes (the hypervariable region determinants) are unique on each antibody molecule. Jerne (1974), in his Idiotype Network Hypothesis, suggested that lymphocytes should be able to recognize these hypervariable shapes on the receptors of other lymphocytes or other antibodies. Thus, idiotypes on one lymphocyte would react with complementary anti-idiotypes on another lymphocyte. A vast network spreading through the lymphocyte array of each individual was envisioned. These theoretical considerations generated experiments in which the antigen was injected into animal 1, and the antibody produced (Ab2 = idiotype) is injected into animal 2, and this animal forms antibody (Ab2 = anti-idiotype) (Fig.). The Ab2 recognizes an idiotype (Id) on Ab2; also, some Ab2 molecules may look like and behave like epitopes on the antigen. The activity of the im-


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mune network is also postulated to generate a variety of "internal image" anti-idiotypes (some may recognize idiotypes, while others will mimic the epitope against which the idiotype was directed). The immune system cannot distinguish between an idiotype or an anti-idiotype vaccine. Anti-idiotypes behave like the original epitope, since they share the identical amino acid sequence with the immunogenic epitope (Fig., A). Thus, Greene and colleagues showed that Ab2 possesses the identical amino acid sequence as the original antigen (virus) epitope (Gaulton and Greene, 1986). An additional possibility would suggest that the epitope can be recognized by the antiidiotype antibody at the stereochemical level (Fig., B). This is particularly significant for non-protein antigens such as carbohydrates. Thus, certain Ab2's can be used as substitute antigens which can induce specific immunity to viruses (Kennedy and Dreesman, 1984; Sharpe et al., 1984), bacteria (McNamara et al., 1984; Stein and Soderstrom, 1984), or parasites (Grzych et al., 1985; Sacks and Sher, 1983). The potential of anti-idiotype vaccines resides in their use to replace antigens that are unsafe or toxic. Another use is induction of anti-carbohydrate immunity. For example, infants are not immunologically competent to respond to certain polysaccharide antigens. Idiotype vaccines which are proteins could be used to induce a protective immune response to carbohydrates. This could be of major significance in altering the developing flora of the oral cavity, where many of the important molecules in adherence and colonization are carbohydrates. This system combines the exquisite specificity of the primary immunogen with the flexibility of immunoglobulin manipulation.

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TABLE APPROACHES TO ERADICATION OF ORAL PATHOGENS AND APPROXIMATE TIME FOR IMPLEMENTATION Short-range Goals (to 5 years)

Cataloguing microbial surface structures (Cariogenic organisms and periodontal pathogens) Cataloguing interactions between: Salivary factors and the teeth Pellicle and bacteria Bacteria and bacteria (coaggregation) Bacteria and immune factors Bacteria and nutrients Investigation of the ontogeny of the oral environment. Investigation of the molecular pathogenesis of oral diseases. Bolstering of host immune system to resist pathogens. Oral immunization with conventional antigens, viral vaccines. Mutant bacteria with desirable properties. Intermediate-range Goals (5-15 years)

Understanding of host and bacterial molecules which are of primary importance in establishment and maintenance of the oral environment. Artificial production of vaccine epitopes to interfere with oral infection. Synthetic peptide and/or carbohydrate moieties as competitive inhibitors of detrimental interactions (e.g., plaque reduction).

SUMMARY AND TIMETABLE

Utilization of immune system at most opportune time in development (childhood immunocompetence).

The major emphasis of the approach to investigations of the ecology of the oral cavity will involve study of the important interactions between host and environment to distinguish beneficial from detrimental factors. Knowledge of these interactions will be used to modify the molecular composition of host and environmental factors to favor beneficial interactions and to exclude harmful relationships. The genetic and molecular mechanisms to accomplish some of these goals are available now. The Table summarizes an overall approach to the attainment of such goals. These are divided into anticipated short-range advances (approximately five years), anticipated intermediate-range advances (approximately 10 years), and anticipated long-range advances (approximately 15-25 years). Most of the goals to be undertaken and accomplished in the short range represent advances in obtaining new information. Thus, the understanding of important host-bacterial interactions in the oral cavity will probably proceed for more than five years, although important inroads in elucidating the most sig-

Introduction of antagonistic microflora. Introduction and synthetic production of bacterial surface moieties. Long-range Goals (more than 15 years) Use of immunocompetent cells as therapeutic agents. Genetic alteration and/or replacement of defective host cells to enhance protective capabilities. Anti-idiotype vaccines. Genetic engineering of salivary flow characteristics and/or protein composition of saliva. Genetically altered strains which are infective but not pathogenic and/or strains which antagonize potential pathogens. Production of custom-made immune molecules, salivary proteins, or bacterial products as constant sources in the oral cavity.


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nificant interactions and their molecular basis should occur sooner. Such studies in the past have resulted in a better understanding of the molecular pathogenesis of human dental infections and have led to approaches to enhance host protective capabilities (Smith and Taubman, 1987). Information about the development of the host salivary factors (Smith et al., 1987), the important immune responses (Smith et al., 1986), and bacterial colonization and succession are of critical importance to eventual manipulation of the oral flora in creating a pathogen-free human. The primary emphasis in the intermediate range will be the implementation of existing methodology and information to manipulate the host response and the oral microbiota to begin achieving a pathogenfree state. Such measures as competitive synthetic salivary peptides and bacterial inhibitors will likely be tested. The ultimate goal anticipated by the longrange advances will be to control the host micro- and macro-environments which control the flora in such a way as to eliminate bacterial-mediated disease. Some of these goals will have been achieved by engineering the host to enhance protective responses. Anti-idiotype vaccines will eliminate potentially hazardous microbes. These molecular biological techniques hold promise for an oral environment that is free of disease. We have attempted to focus on factors which may lead to the creation of a specific bacterial pathogenfree human, who would be free of dental caries and periodontal disease for a lifetime by virtue of resisting infection with cariogenic and periodontopathic bacteria. It is clear that several pathogen-free states may be necessary, reflecting difficult environments and challenges. For example, there may be one effective pathogen-free state for children and another for adults. There may be one required for persons living in an environment with low levels of cariogenic organisms or periodontal organisms, and another for persons living in environments with high levels of these organisms. These variations represent challenges to the ingenuity of future researchers. We purposely did not discuss the specific "viral" or "fungal" pathogen-free human, but clearly many of the principles involved in the development of bacterial pathogen-free humans will apply to one free of viruses or fungi. However, there are many unique features of viral infection, including latency, which offer challenges substantially different from those of bacteria. In considering all of the difficulties bearing on the creation of a pathogen-free human, one could easily become discouraged. At one time, the elimination of smallpox and the reduction in incidence of whooping cough, tetanus, and pneumonia must have appeared to be hopeless. Yet, through vaccination and infection control, these diseases have largely been subdued. A pathogen-free human free from infectious disease is a high aspiration. It clearly represents a new

frontier in oral health research which may be achieved by progress through the anticipated advances in the short, intermediate, and long range. ACKNOWLEDGMENTS Some of the research work and effort presented in this manuscript was supported by USPHS Grants DE03420, DE-04733, DE-06153, DE-04529, and DE-07009. The author(s) thank Professor Bo Krasse for review of this manuscript, and Donna Freedman and Judy Balsamo for excellent secretarial assistance. REFERENCES ALALUUSUA, S. and RENKONEN, O.V. (1983): Streptococcus mutans Establishment and Dental Caries Experience in Children from 2 to 4 Years Old, Scand ] Dent Res 91:453-457. ARENDORF, T.M. and WALKER, D.M. (1980): The Prevalence and

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