PERIODONTAL VACCINE-A SHOT IN THE ARM !

Dr. Apeksha Gajghate, Dr. Salman Ansari, Dr. Rajvir Malik, Dr. Namrata Khetal and Dr. Rashmi Bele P.G Student, Department of Periodontology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Wanadongri Road, Hingna, District Nagpur441110 ................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 15 August 2020 Final Accepted: 18 September 2020 Published: October 2020


3.Tannerela forsythia (T.forsynthia)
These bacteria produce an array of antigens that stimulate pro-inflammatory cells and leads to the production of a wide variety of cytokines. [3] These antigens may stimulate T-helper cells-1 (Th1) or T-helper cells-2 (Th2) cells. Antigens are taken up by dendritic cells and presented to CD-8 or CD-4 cells along with MHC antigens. [4] The host produces anti-bacterial substances such as defensins, cathelicidins and saposins, which protect the host tissues from bacterial products and forms the first line of defense. However, sometimes these are inactivated by the bacterial virulence factors. Once bacteria break this barrier, cytokines are produced, which can be both proinflammatory and anti-inflammatory. Production of inappropriate cytokines results in periodontitis. [5] Host response in periodontal disease: The presence of periodontal pathogens triggers the host immune response. The host defense against periodontal pathogens includes of Innate and Acquired immunity. Saliva, Gingival crevicular fluid (GCF) and epithelial cells play a key role in innate immune response. [6] Neutrophils, Macrophages and Dendritic cells are also important innate immune cells which express pattern recognition receptors (PRRs) that interact with the specific molecular structures on microorganisms called microbe associated molecular patterns (MAMPs) to signal immune responses. Innate immune response is nonspecific and results in excessive host tissue damage without effective antigenic clearance. [7] Adaptive immunity is slower and reliant on complex interactions between antigen-presenting cells and T and B lymphocytes, cytotoxic T cells and antibodies. [8] The immune response to pathogenic microorganisms involves the combination at the molecular, cellular, and organ level of elements often categorized as being part of the innate immune system or the adaptive immune system. However, Periodontal pathogens have developed mechanisms to inhibit and evade cell-mediated and humoral immune responses. [9] To stop the progression of periodontal disease, multiple approaches including host immune modulation and pathogen-specific approaches could be used.

Vaccines:
Vaccination is a process that induces specific immune resistance to a bacterial or viral infection. It is the development of immunity or resistance to infection, after a secondary response (booster) that is adequate to consider the individual immune to a subsequent infection. [10] More than 200 years ago, Edward Jenner showed a new outlook in preventive medicine with the introduction of Small Pox vaccine and its successful use to eradicate the epidemic. Advances in Microbiological and Biochemical methodologies led to revolutionary era that ultimately resulted in the formulations of vaccines against serious epidemic and endemic diseases affecting humans, including Tuberculosis, Tetanus, Typhoid, Cholera and Plague. [11] The prime step in vaccine development is identification of an antigenic component from various organisms that can provide immune protection.
Vaccines may be prepared by: [12] 1.Killing the organism using formalin-called inactivated or killed vaccine.
2.Using only antigenic part of the disease causing organism, like the capsule, the flagella, the part of the protein cell wall-acellular vaccines.
3.By weakening a live microorganism by aging it or altering growth condition-attenuated vaccines. 4.Toxoids are vaccines from toxins, which are adsorbed onto aluminum salts to decrease their harmful effects and is administered with an ''adjuvant'' which can have effects on antigen delivery, immune modulatory cytokines, and antigen-presenting cells. [13] Types of Vaccination: Active Immunization: Here an individual immune system is stimulated by administrating killed or live attenuated products derived from micro-organisms.

Passive immunization:
Here, the antibodies formed in one individual are transferred to another.

DNA vaccination:
Here, DNA plasmids encoding genes required for antigen production are transferred to an individual. [14] Characteristics of an effective vaccines are-Safety, Protectivity, Sustained protection, production of neutralizing antibodies, Stimulation of protective t-cells. Practical considerations like cost-effectiveness, biological stability, access, minimum contraindications and side effects are also significant. [15] Periodontal vaccines: What is the prerequisite for periodontal vaccines? Periodontal pathogens associated with periodontitis predominantly are gram-negative, anaerobic bacteria-P. gingivalis, A. actinomycetemcomitans, T. denticola and T. forsythia etc. Various immunization approaches both as active and passive immunization, against periodontal pathogens have been explored either using the whole organism or specific virulence factors. [16] Periodontal vaccines can be helpful in decreasing the incidence of Periodontal diseases. Periodontal diseases result in higher systemic levels of inflammatory markers which causes systemic changes leading to various systemic conditions. The subgingival biofilm associated bacteria are found to exacerbate diabetes and cardiovascular disease and it has been suggested that immunotherapy for periodontal disease may be considered as a second indication for controlling atherosclerosis. [17] Another group of patients that would benefit from the use of such a vaccine are immunocompromised patients affected by the disease through various acquired or congenital causes. [18] Periodontal treatment lays a monetary load on the individuals suffering from it. Availability of vaccine for preventing periodontal disease would be of great help for those individuals. So, the development of periodontal vaccines is of supreme importance in the management of periodontitis.

History:
In the early 20th century, 3 periodontal vaccines were employed. Pure cultures of streptococcus and other organisms, Autogenous vaccines and Stock vaccines-Vancott's vaccine, Goldenberg's vaccine and Inava endocarp vaccine. [19] Strategies employed for development of Periodontal Vaccines: Several immunization approaches against periodontitis have been tested, both as active and passive immunization. Besides, the target antigens have progressed from the whole organism in the past to current approaches wherein the specific virulence factors aim to confer immunity against colonization or the virulent activity of putative periodontal pathogens. Socranskyet al, analyzed 13,261 plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization and proposed five color-coded 'complexes' based on the extent of its presence in periodontitis-affected sites. They suggested that the organisms of the Red complex, namely P. gingivalis T.denticola and T. forsythus, are the predominant disease-associated organisms. [20] The current arrival of advanced molecular diagnostic techniques, such as Polymerase Chain Reaction (PCR), viruses like herpesvirus and their interaction with the periodontal pathogenic bacteria have also been an emphasis of studies. [21][22][23][24] The new additions to this outwardly ever-growing list of implicated bacteria are Desulfovibrio fairfieldensis [25] and Methanogenic archaea. [26] Thus, the development of a vaccine has become more exciting, taking into consideration, the recent developments in the etiology and pathogenic mechanisms involved.

Mechanism of action: Active immunization:
Various target organisms for vaccine preparation have been tried, of which P. gingivalis and A. actinomycetemcomitans are of major importance owing to their omnipresent role in the pathogenesis of periodontal disease. P. gingivalis is a gramnegative, non-spore/forming, nonmotile, assacharolytic, obligate anaerobic coccobacillus. The virulence factors of P. gingivalis which have been used as subunits for the development of active immunization. [27] It is known to survive in a hostile environment by successfully evading host antimicrobial defenses by utilizing a variety of virulence factors, such as cysteine proteases, gingipains, fimbriae, lectin-like adhesins, capsular polysaccharide, lipopolysaccharide, outer membrane , heat shock protein and the release of toxic metabolic products. [28] Apart from the whole cell, all these factors havebeen tested as target antigen for immunization that can produce functional protection against periodontal tissue destruction induced by the organism.

Outer membrane protein:
It was seen that transcutaneous injection of 40 kDa of outer membrane protein (OMP) inhibits coaggregation of outer membrane protein (OMP) inhibits co-aggregation of P. gingivalis with Streptococcus gondonii. This also can be used 384 for vaccine development for passive immunization. Polyclonal anti-40 kDa. OMP antibody exhibited potentially protective, complement-mediated bactericidal effect. [29] 'Whole cell' as a target antigen-It was one of the first approaches tried in various animal models. Perssonet al. reported that active immunization of non-human primate, Macaca fascicularis, with killed P. gingivalis whole cell conjugated with syntex adjuvant formulation-M, inhibits the progression of periodontal tissue destruction. [30] Page also confirmed the similar results employing the same animal model. [31] These studies showed that only humoral immune response was elicited that lasted for a short period and no cell-mediated immune response was triggered that could provide immune memory and thus provide long-term protection. Hence, there have been minimal advances, with respect to vaccine development, by means of this approach.
Gingipains-Gingipains is the specific term used to describe a host of cysteine proteases that impart major pathogenic capability to P. gingivalis. It was Coined by Travis and Colleague. These are cysteine proteinases which cleave synthetic and natural substrates after arginine or lysine residues and are referred to as arginine gingipain (RgpA and RgpB) and lysine gingipain (Kgp) respectively. [32] Kgp is most potent fibrinogen degrading enzyme of 3 gingipains in human plasma and involved in bleeding tendency at diseased gingival. They are expressed on the outer membrane of P. gingivalis. Rgp and Kgp are key determinants in the growth and virulence of P. gingivalis. Gingipains vaccines are mainly DNA vaccines. DNA vaccines induce both humoral and cellular immunity. They possess a hemagglutinin domain that plays an essential role in the adherence of microorganism to erythrocytes, while the catalytic domain present in RgpA, RgpBand Kgp plays an important role in the evasion of the host defense system by extensively degrading immunoglobulins, complement proteins and by disturbing the functions of neutrophils [33][34] . This activity also degrades C3-derived opsonin, thus rendering P. gingivalis resistant to phagocytosis [35] . Thus, a vaccine targeting this virulence factor may provide protection against both invasive and noninvasive strains of P. gingivalis. Other reports also suggested that immunization with the RgpA-Kgp proteinase-adhesin complexes of P. gingivalis protected against periodontal bone loss by inducing a high titer of serum IgG 2a response in the rat periodontitis model, similar to that seen in animals immunized with formalin-killed P. gingivalis whole cells [36] . So, this approach seems to be an essential way forward to achieving the goal of 'successful' vaccination.
Fimbriae as target antigens-Fimbriae from P.gingivalis play an important role in adhesion to oral tissue and are highly immunogenic. Electron microscopy studies of P. gingivalis have shown that it possesses at least three types of fimbriae [37] . It consists of major fimbriae (product of fimA gene) and other antigenically distinct fimbriae, designated as minor fimbriae, composed of a protein of 67 kDa [38] . Another distinct fimbrial structure, Pg-II (72-kDa protein) has also been detected by immunoelectron microscopy. [39] Although both major and minor fimbriae partake in the pathogenic process, major studies have been taken on in averting thepathogenic effects of major fimbriae. In a study on rats, it was observed that when rats were parenterally immunized with highly purified 43-kDa fimbrial(Fim) protein, the induced FimA-specific antibodies in serum and saliva gave 100% protection against P. gingivalis induced alveolar bone loss. [40] On the contrary, a study demonstrated that rabbits immunized with 43-kDa fimbrillin polymer of P. gingivalis did not show evidence of any protection against all the strains of P. gingivalis, suggesting that opsonic target sites are not shared across serotypes or five types of P. gingivalis fimbriae [41] These results suggest that the fimbrial protein of P. gingivalis could be used as a target antigen to yield an effective periodontal vaccine.
Hemagglutinins-Non-fimbrial adhesion hemagglutinin B (HagB) is a potential vaccine candidate. Rats immunized subcutaneously with recombinant HagB were protected against periodontal bone loss induced by P.gingivalis strain ATCC 33277. Human antibody against hemagglutinin should be ideal for practical use in immunotherapy. [42] GroEL heat shock protein -Heat shock proteins have significant role in inflammatory mechanism and autoimmune diseases. Rats immunized with P. gingivalis HSP60 showed decrease in bone loss induced by infection with multiple periodontal bacteria. Significant association between HSP90 concentration and microbial colonization has been observed. [43] Synthetic peptides-These require synthesis of linear and branched polymers of 3-10 amino acids based on the known sequences of microbial antigens. Such peptides are weakly immunogenic by themselves and need to be coupled to large proteins to induce antibody response. Genco 1992 found that synthetic peptides based on the protein structure of fibrillin inhibit the adhesion of P. gingivalis to saliva-coated hydroxyapatite crystals in vitro.

Passive immunization:
Passive immunization is short lived because host does not respond to immunization and protection lasts only as long as injected antibody persists. Antigens are injected into vector that produce antibodies. These antibodies when inoculated into host bring about passive immunization. Passive immunization against Porphyromonas gingivalis-Developing monoclonal anti-bodies against the colonization factor of P. gingivalis could also be a potential target for immunotherapy. Two major colonization factors of P. gingivalis are coaggregation factor (outer membrane proteins [OMPs]) and hemagglutinins, both are involved in the adsorption, colonization and penetration of bacteria into host cells. [44 -45] Antibodies against coaggregation factor-There are several mechanisms by which P.gingivalis can cause disease, but perhaps the most important step is coaggregation between P. gingivalis and Actinomyces viscosus. P. gingivalis can also adhere to Streptococcus gordonii, mediated by the specific OMP on the cell surface and in extracellular vesicles. [46] This coaggregation contributes to the formation and maturation of biofilm, which is mainly implicated in causation periodontal disease. [47] Actinomyces naeslundii has also been implicated in coaggregation with P. gingivalis. Based on this fact, a panel of monoclonal antibody (mAb) was prepared by immunizing mice with purified r40-kDa OMP, which revealed that several mAbs specifically inhibited coaggregation of A. naeslundii with several strains of P. gingivalis. [48] A genomic analysis of P. gingivalis was conducted and it was reported that recombinant OMP antigens PG32 and PG33 (both known to play an important role in bacterial growth, coaggregation with other bacteria and transcription) are potential vaccine candidates. [49] Hence, OMPs have been reported to be highly immunogenic due to the easy access to host cells, it can be crucial in the development of a highly effective vaccine against P. gingivalis infection.
Immunization against Aggregatibacter actinomycetemcomitans-After P. gingivalis, A. actinomycetemcomitansis considered significant pathogen in human periodontal disease, especially in aggressive forms. In a study, it was demonstrated that sub-cutaneous and intranasal immunization of mice with capsular serotype b-specific polysaccharide antigen (SPA) produced a specific antibody, which efficiently opsonized A. actinomycetemcomitans serotype b, suggesting that antibodies to the SPA of the organism might have a protective role. [50] Furthermore, when mice were immunized with antisurface associated material from A. actinomycetemcomitans (anti-SAM-Aa), it was found to result in rapid healing of the primary lesions and a rise in protective antibody levels by acting as an opsonin against challenge with live A. actinomycetemcomitans. [51] However, astonishingly only a few studies have been conducted on developing vaccines targeting A. actinomycetemcomitans. Even though preliminary studies are promising, further research has to be carried out to depict a potential antigen for developing vaccine against A. actinomycetemcomitans.
Plantibodies-Apart from various microorganisms, plants are being increasingly used for the production of recombinant immune-therapeutic agents owing their high efficiency and low cost and the fact that they do not initiate an immunological reaction when administered orally. Molecular biological techniques to express bacterial or viral antigens in plants which could be used as orally administrated vaccines. Ma (2000) characterized a secretory IgG antibody against streptococcus mutans, produced in transgenic plants. [52] These studies are promising in that the plantibodies can be used to prevent specific microbial colonization in the oral cavity. Additional studies must be carried out to test the efficacy of plantibodies in eliminating periodontopathic bacteria.
Immunization targeting antecedent plaque microbes-In human plaque, Fusobacterium nucleatum colonizes prior to P. gingivalis and high levels of F. nucleatum have been demonstrated in association with P. gingivalis, as well as other bacteria associated with periodontal disease. [53] In a recent report, it was demonstrated that when mice were immunized with F. nucleatum prior to P. gingivalis, there was a significantly increased IgG 2a (Th1) response to P. gingivalis. [54] Besides, the inhibition of neutrophil phagocytosis of immune serum opsonized P. gingivalis was modulated by the presence of anti-F.nucleatum antibody.

Genetic immunization:
By the early 1990's, Scientists had initiated to study new approaches for the production of vaccines. This involves genetic engineering or recombinant DNA technology.
Plasmid vaccines-DNA does not have the ability to grow, whereas plasmids have the ability to grow. With this ability of the plasmids, they are fused with the DNA of a particular pathogen of interest and inoculated in an animal for the production of antibodies. This is then transferred to the host for immunization. Disadvantage-It may lead to oncogenesis.

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Live, viral vector vaccines-A variety of infectious but non-disease causing DNA or RNA viruses or bacteria have been engineered to express the proteins of a disease-producing organism. The vector enters the body cells where the proteins are generated and then induce humoral or cellular immune responses. [55] Methods of DNA vaccine administration-Intranasal, Intramuscular and Gene gun. Breivik and Rook conducted a series of immunization trails in wistr rats employing various routes of administration. They reported that the subcutaneous injection of killed Mycobacterium vaccae, SRL 172, prophylactically [56] , and therapeutically [57] , and also oral administration of killed M. vaccae, SRP299 [58] , diminished the Th2 response in wistar rats, thus reducing periodontitis induced bone loss.

Host modulation:
Pathogen-triggered host response is the foremost culprit in tissue destruction. Therefore, the focus of the immunization approach has shifted from targeting specific microbes to modulating the host innate immune response by altering specific immune cell functions. Excess inflammatory mediators as a result of host immune response can be blocked to prevent inflammation induced bone loss. It was demonstrated that immunization with formalin killed P. gingivalis blocked bone loss by depressing prostaglandin E2 levels, a major component involved in bone resorption. [59] Vaccines consisting of antigen alone are often not very effective in inducing the anticipated immune responses. So, adjuvants are commonly used to enhance the host response to the vaccine antigen. Yang et al. demonstrated that intranasal immunization of rHagB and monophosphoryl lipid (MPL) A, a nontoxic derivative of lipid A region of lipopolysaccharide, acts as mucosal adjuvant and potentates the response to rHagB. [60] There was an increase in the serum IgG1 antibody activity and amplified Th2 response. So, Host modulation could render satisfactory protection against pathogenic agents and formulating an immunization approach in this way could be possible in future.

Barriers in periodontal vaccine development:
As Periodontal disease is a multifactorial disease, eradication of certain bacteria will not stop the onset and progression of disease. Several Problems like maintaining adequate levels of antibodies for long time, generating Tcell mediated response needs to be overcome. The few comparations between the conventional animal models and human beings, and incidence of toxic reactions to inactivated whole cell vaccines add to our worries. Also, the functional differences between the antibodies produced by the infected host and that produced after immunization is to be borne in mind, as the former is usually present but is unsuccessful in resolving the disease. Hence, a vaccine that can create functionally viable antibodies will be the most desirable.

Future immunization approaches:
An effective periodontal vaccine for humans will probably require multiple bacterial species responsible for disease pathogenesis to be targeted. [61] The immunization approach could include introduction of nonpathogenic engineered microorganisms that could competitively and permanently adsorb on to the epitopes in the biofilm and eventually prevent coaggregation and maturation of biofilm. However, studies to validate such a possibility are yet to be carried out. Recent dawn of Nanotechnology opens an entire range of nanospheresand liposomes for controlled release of protein or nucleic acid for the particular delivery of vaccine in adequate amount. Also, delivery routes, likeoral drops, nasal spray, dermal patch and subcutaneous or intramuscular injections, are to be effectively studied in order to determine the most effective route of administration. Local drug delivery of the active ingredient is also a feasible choice.

Conclusion:-
The most critical step in developing a successful immunization approach against periodontal disease would be the identification of the individuals prior to the initiation of disease process. Several immunization approaches have been tested targeting virulence factors as antigens or by various forms of host modulation to modify the response against the pathogens. Many research groups focusing on vaccine development in the past and at present have developed effective immunization for animals and the same may be developed for human use soon.