Department of Medical Microbiology, Turkey
*Corresponding author:Gülnur Tarhan, Department of Medical Microbiology, Turkey
Submission: September 27, 2019; Published: October 16, 2019
ISSN 2578-0190 Volume3 issues2
Synthetic peptide antigens; molecular biology, genetics, biochemistry, microbiology, etc. in the field of antibiotic drug development, antibody production and vaccine development has been the subject of numerous research studies. These antigens are widely used in the industry for the diagnosis, treatment and prevention of genetic and metabolic diseases, especially infectious diseases. This technology is mainly based on the selection of epitopes found in natural structures and causing strong immune response, synthesis of amino acid sequences by various methods, coupling with the carrier molecule and delivery to the organism with the appropriate adjuvant. The most important advantage is that these molecules can be synthesized in the desired amount and in pure form. In various studies, vaccines prepared in this way received various levels of immune response. However, due to the absence of secondary and tertiary structures of the synthesized molecules, the immune response was low.
Keywords:Synthetic peptide antigens; Usage area; Vaccine
Recent advances in molecular biology, biochemistry and immunology; It has made it possible to identify the antigenic structures of bacteria, viruses and parasites that cause infectious diseases and to develop vaccines that can be effective against them [1,2]. Today, antigen synthesis using chemically peptide synthesis and recombinant DNA technology has become an important technological tool in the development of new diagnostic kits . Infectious agents have numerous antigenic determinants or epitopes. These are the basic structures that give the agent antigenic specificity. However, a very limited number of these structures is important for immunospecificity. In particular, it is sufficient to have one or more of these for protective immunity [4-6]. In order to synthesize protein sequences of such specific regions by DNA cloning and sequencing techniques, the selection of the regions (oligopeptides) to be used as the antigen must be made correctly [7-10]. The main methods used for this purpose:
In this method, proteins are separated into specific regions using enzymes or chemical agents such as cyanogen bromide. Immunologically active fragments which are capable of binding to specific antibodies (monoclonal antibodies) and capable of antigen-antibody interaction are selected after separation.
Synthetic fragments of proteins can be identified using neutralizing antibodies. This application has been used in particular to identify determinants related to phenomena such as virus neutralization.
Antigenic determinants can be determined by this method. However, the lack of crystal structure of many epitopes limited the use of this system.
Identification of amphipathic helical region, flexibility β return and water attractiveness. Specifically, the amino acid sequences of the molecule with their attractiveness to the liquid (hydrophobicity) or flexibility (flexibility) are found by calculation. If the protein structure is considered, the hydrophilic amino acids present on the surface and play a role in immunization should be included in the synthetic peptide structure. Systematically synthesizing hexapeptide units in the sequence and measuring antibodies to natural proteins and their antigenic activity [11,12]. After selecting the functional region on the protein, another issue that needs to be decided is to determine the minimum and maximum amino acid number in the structure of oligopeptides capable of applying the immune response. In 1981, Hopp and Woods developed a computational method that determines the ideal peptide sequences that can be used as antigens, starting from the protein structure. They evaluated the environment hydrophilicity parameters for segments with various amino acid sequences. According to the results, the most suitable size for synthetic peptides was found to be 6-10 amino acids. However, in subsequent studies where the epitope used as the antigen plays a role in the carrier molecule, suitable antigens have been obtained in smaller and larger peptides . In the first studies with synthetic antigens, the basic antigenic structures that cause the antibody response of the microbial agents causing metabolic diseases such as diabetes and common infectious diseases have been identified, some have been synthetically produced and antibody response formation has been provided against them in various experimental animal models [10,14-16].
Made obtained 50 amino acids by chemical technique, which is called solid phase peptide synthesis . In this method; active groups were formed on which amino acid can bind; synthetic resins were used. The first amino acid was bound to the resin by ester bonds to release the amino group. After protecting the amino group of the second amino acid with t-butoxycarbonyl chloride, the peptide bond was formed by interacting with dicyclo hexyl carbodimide. The first amino acid was bound to the resin by ester bonds to release the amino group. A polypeptide containing the desired number of amino acid residues was synthesized in the same manner. Peptides were separated from the root at the final stage with hydrogen fluoride addition. Many of the synthetic peptides are weak immunogens. They result in a very low immune response even when administered alone with a strong adjuvant. They are therefore attached to various carrier molecules for administration. The choice of suitable carrier is very important, especially if synthetic peptides are to be used for vaccine preparation. For this, a carrier must be an enhancing immune response to the peptide, injectable to humans, minimal side effects and an inexpensive material. The most commonly used carriers are; Keyhole limpet hemosiyanin (KLH), Lumiulus polyphemus hemocyanin(LPH), Bovine serum albumin (BSA), Tetanoz toxoid(TT), Silika beads thyroglobulin and IgG, β galaktosidaz (β gal), ovalbumin, swine IgG, Diphtheria toxoid, Flagellin, Pneumoccal polysaccharide, Meningococcal protein, synthetic polymers of α amino acids such as poly (L-lysine), A.L (multichain poly [DL-alanine), ficoll, E. coli heat labile toxin, Liposomes, PPD, human erythrocytes, cholera toxin, influenza hemagglutinin and the peptides of P. falciparum obtained from circulating sporozoite proteins and various nanoparticles [18-22]. Although these carriers increase the immunogenicity of the synthetic peptides, some have been observed to cause some hypersensitivity reactions, particularly during repetitive injections. Therefore, the studies have focused on the development of synthetic carriers that will not produce reactions. In order for the peptides bound to the carrier to produce a good immune response, they were administered to the organism together with adjuvants [23-25].
The most commonly used adjuvants for this purpose are:
a. CFA (Complete Freund’s adjuvant)- Decrease in metabolism of mineral oils, local reactions due to mycobacteria, granulomas, inflammation and fever were observed. It is therefore not suitable for human use.
b. Aluminum salts: Al (OH)3, Alum, Aluminum phosphate.
c. MDP (N-acetyl-muramyl-L alanyl-D isoglutamine) is a substance of peptidoglycan structure from the cell wall of mycobacteria.
e. Squalene emulsions.
f. ISCOM (Immuno stimulating complex): Biologically compatible detergent and complex adjuvant consists of Quil A.
g. Protein cohleate-protein + calcium + phospholipid: It was used for the presentation of multiple antigens.
h. Calcium phosphate.
i. Cord factor-trehalose dimycolate.
Apart from the use of synthetic peptides as antigens and being very pure, there are other benefits. For example; Proteins whose amino acid sequence has been conserved throughout evolution (tubulin, actin, calmodulin, etc.) are not good immunogens. When they were used for immunization by known methods, it was found that they did not stimulate the immune system, but when synthetic fragments were used, the ELISA or RIA antibody titer was shown as one hundred times higher [1,5,10,26,27]. Furthermore, since it is also possible to use radioactive amino acids during the preparation of synthetic peptides, the peptides can be labeled, thereby facilitating tracking and quantitation of the peptide [28- 35]. In addition, it is also possible to add an amino acid which is not normally present in the protein structure. For example; tyrosine addition to the carboxyl terminus allows the peptide to bind to the carrier protein easily. The most important point to consider when applying this technique is the selection of the part of the proteins that will interact with the antibody. If this selection is made correctly, the polyclonal antibodies to be obtained from the immunization will in fact have monoclonal antibody specificity. Because the antibody prepared is specific to a single region of the protein. Synthetic peptides may be used for the production of certain antibodies or may be used to obtain different molecular antibodies for purification of other antibodies by affinity chromatography. The functional sequence found on a protein may not be immunogenic when present in the native protein [5,8-10]. However, when the same sequence is synthetically prepared and added to a suitable carrier, it can strongly stimulate the immune response. Thus, it is possible to increase the immunogenicity of certain epitopes using synthetic peptide and different carrier combinations [19,20]. The use of synthetic peptides has been extended with the introduction of peptide antibodies for the production of therapeutic proteins and for the diagnosis of infectious diseases. These products are used in the following areas [36-41].
Detection of gene products: The product protein of any gene can be investigated with antibodies prepared against short sequences of several amino acids from the gene sequence. It is also possible to carry out studies in this way to test whether a cloned gene has been identified correctly. Antibodies obtained by this method have been widely used in the field of virology, especially for oncogenic viruses. Once peptide antibodies have been prepared, the immune peptide can be used for asion or western blotting.
Protein purification: The use of peptide antibodies for the detection of the protein region or whole protein is particularly important for trace amounts of proteins present in the cell. For example; human erythropoietin is a protein with very low rates and it is not possible to purify and use it in a standard immunoassay. However, it is possible to prepare antibodies against a known portion of this protein and develop a sensitive immunological test. Peptide antibodies can be used in the separation and purification of proteins from cellular extracts. They cloned human interferon and produced it in E. coli cells in 1981.Then, they used affinity columns containing peptide antibodies in the purification of interferon from their extracts and were able to purify the interferon bound to the column by a simple washing technique to remove it from the column. Determination of functional regions of proteins: A peptide antibody that binds only to the functional region of a protein can be used as a “probe”. This method is important in determining both functional area of protein and whole protein. These types of studies are mostly used for oncogene proteins and useful results are obtained. Schaffhausen et al. in their studies investigating viral and cellular components causing polyoma virus transformation; They prepared antibodies against fragments of 9 amino acids in the T antigen and showed that in vitro kinase activity was destroyed when complexed with the T antigen. Later, they showed that tyrosine specific protein kinases are involved in viral transformation and homology in their active centers by cross-reacting for different viruses. In 1984, they prepared peptide antibodies against G protein from Vesicular stomatitis virus envelope proteins, marked with a fluorochrome dye and injected into the cell following viral infection and followed the path of G protein in the cell. They inhibited transformation proteins using peptide antibodies in cell culture and returned the transformed phenotype. So far, the most important problem with peptide antibodies is the possibility of unwanted cross-reactions. For example; When the antibody against the C-terminal fragments of the transforming protein of RSV (Raus sarcoma virus) was prepared and tested, it was observed that it gave non-specific reaction with cellular proteins at low concentrations of antibodies. However, the activity of this type of reaction is low. Such low affinity and nonspecific binding are present in monoclonal antibodies, which can eliminate unwanted side activities by adjusting antigen and antibody concentrations during in vivo use.
Vaccine preparation: It has been a step forward in the development of synthetic peptide vaccines of the chemical structure of antigenic regions or bacterial toxins of bacteria and viruses. In their first study, the hexapeptides corresponding to the C-terminal end of the tobacco mosaic virus were purified by enzymatic digestion. They were then conjugated to bovine serum albumin and given to rabbits, and it was reported that antibodies formed in these animals precipitated and neutralized the tobacco mosaic virus. Peptides corresponding to the P2 capsid protein region of the MS-2 bacteriophage were synthesized and showed that neutralizing antibodies in the rabbits were generated. Today, synthetic peptide vaccines are grouped into three groups: synthetized bacterial, viral and parasitic vaccines. In the immunization studies carried out with synthetic antigens which are important in immunity and pathogenesis of various infectious agents, successful results have been obtained in various experimental animals. A few examples of these studies are given below for each group of organisms.
Studies on synthetic bacterial peptide vaccines: When a 14-amino acid synthetic hexapeptide of the 186-201 amino acids of the diphtheria toxin A chain is administered to the guinea pigs together with the synthetic muarmildipeptide adjuvant and the synthetic multichain poly DL alanine carrier, it has been described that these animals provide protective antitoxic immunity [42,43]. Peptide vaccines against lipopolysaccharides and cholera toxins of V. chloreae were prepared and tested. Many 11-18 amino acid peptides belonging to V. chloreae B subunit were prepared and used together with tetanus toxoid or combined with multi-chain poly DL-alanine and tested in rabbits. Neutralizing antibodies have been observed in these animals . Synthetic peptides corresponding to heat-resistant and non-resistant epitopes of enterotoxigenic E. coli were prepared and disclosed to reduce the severity of infection in experimental animals. As another approach, it has been observed that antibodies which cross-react with each other against the common sequences between the labile toxin and cholera toxin of E. coli, which show very high homology . Have indicated that antibodies against CTP1 and CTP3 peptides, which inhibit the biological activity of cholera toxin, also neutralize the biological activity of labile toxin of E. coli. All data suggest that a vaccine based on synthetic peptides may provide general protection against the coli-cholera family . Another important study with synthetic bacterial peptides was done with S. pyogenes M proteins. Have showed a synthetic peptide consisting of 35 amino acid residues corresponding to a segment of the M protein of S. pyogenes . It has been observed that both cellular and humoral immune responses occur in rabbits against this protein. In addition, mice with antibodies obtained from these rabbits have been Shown to be protected against a challenge infection with Type 24 streptococci when mice are passively immunized. Furthermore, it has recently been disclosed that this synthetic peptide can be used as a carrier for several types of peptides which are distinguished from other infectious agents (for obtaining a multivalent synthetic vaccine).
Studies on synthetic viral peptide vaccines: Synthetic peptide vaccines with viral antigen synthetic hepta and endoka peptides corresponding to the N and C-terminal regions of the large T antigen of the Simian virus 40 virus were prepared and it was found that the antibodies formed by these rabbits reacted with the original T40 antigen of Simian virus 40. Pentapeptide corresponding to the nucleotide sequences at the 3 ends of the Moloney leukemia virus RNA was made and antibodies were detected in rabbits [47,48]. Synthetic peptides corresponding to nucleotide sequences 138-146 of the hepatitis B virus were prepared and it was disclosed that the antibodies formed by them reacted with the virus. In addition, 117-123 and 124-137 nucleotides were synthesized in the opposite amino acids and given to the mice and said to form antibodies. The amino acids (55 amino acids) corresponding to the nucleotides of the pre-S region of HBV have also been shown to form a high proportion of group-specific antibodies (when administered with liposomes). However, it is stated that the evaluation of their protective effects will require further studies.
Synthetic peptide (p120-145), which belongs to a significant portion of Pre-S, is reported to be more immunogenic in animals. Synthetic peptide (p120-145), which belongs to a significant portion of Pre-S, have been reported to be more immunogenic in animals. It was found that the N-terminal end of this peptide recognizes T cells and B cells in the C-terminal sequences. It was determined that the antibodies produced by the peptide cross-reacted with native pre-S HBsAg [2,5,9,11,49]. The antigenic specificity of influenza virus depends on the main determinants in hemagglutinin. These are constantly changing due to differences in the amino acid chain. In contrast, there are several kinds of fixed amino acid extensions within the hemagglutinin chain. Considering the folded structure of hemagglutinin, peptide of 98-110 nucleotides of Type A H3N2 virus was prepared and given to mice by combining with tetanus toxoid and Freund’s adjuvant. It has been reported that this combination produces both neutralizing and hemagglutination inhibition antibodies in mice and provides partial protection in mice. It has been also reported that 18 of the 20 synthetic peptides corresponding to 75% of the HA1 region of the HA molecule form antibodies. Herpes simplex Type 1 (HSV-1) glycoprotein D was found to react with anti gpD monoclonal antibodies that neutralize HSV-1 and 2, which are opposed to the 8-23 region. It was emphasized that it reacts with native glycoprotein D consisting of synthetic 16 amino acids and also neutralizes HSV-1 and 2 [9,11,50,51]. The 20 amino acid peptides corresponding to the surface protein 8vp1) 141-160 regions of foot and mouth virus (FMDV) type 1 has been reported to stimulate neutralizing antibody synthesis and protect guinea pigs in cattle and guinea pigs when administered with a carrier protein. It has been reported that antibodies produced by synthetic peptide belonging to amino acid regions 55-179 belonging to surface protein of Type A have protective effects in pigs and cattle. In addition, the synthesis of VP1’s 201-213 regions were performed. Many small peptides corresponding to the hydrophilic domain of the VP1 capsid protein of the poliovirus were synthesized and bovine was administered with serum albumin and complete Freund’s adjuvant. Furthermore, their reaction with polyovirus neutralizing antibodies is also indicated [52,53].
Studies on Synthetic Parasitic Peptide Vaccines: The sequence in which the antibody response to sporozoites of all P. falciparum strains can be increased is made by repeating a guadropeptide consisting of asn-ala-asn-pro (NANP) 3 3-fold. (NANP)3Al(OH)3 and tetanus toxoid have been shown to produce antibody response to sporozoite when administered to humans. The major circum sporozoite protein (CS) of the sporozoites of the malaria parasite have immuno- dominant repetitive epitopes. Antibodies against these epitopes have been reported to stop infection. Merozoite surface Ag MSA2 studies continue [54,55].
One of the features expected from an ideal vaccine is effective immunization, no unwanted side effects, easy delivery and low cost. In the case of antiviral vaccines, viruses need to be produced in living systems and the possibility of contamination of vaccines with other substances increases. Simian virus 40 contamination, which has occurred many years ago in salk polio virus vaccines, is a good and unforgettable example of this. The use of recombinant DNA technology for this purpose has brought great innovations. In this technique, the gene that encodes the immune-generating protein is isolated, generated in the bacterium, and in a sense the protein to be used for immunization is synthesized to the bacterium. The next step is the purification of the protein to be used in immunization and the possibility of contamination of the vaccines with other substances increases. Because the immunogenic protein needs to be purified from bacterial proteins It is currently contemplated that vaccines prepared with synthetic peptides can overcome these problems. By adding more than one active peptide to the same carrier, it is possible to prepare uniform vaccines against different infections with synthetic peptides [9,11]. Synthetic peptides do not have secondary and tertiary structures such as proteins. Since most of them are primary structures, their immunogenicity is low. Therefore, it is used in combination with adjuvant and carrier molecules.
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