T-Cell Epitope Vaccine Prediction Analysis Targeting Phosphoprotein (P) Rabies Virus Based on the Presence of HLA-I Alleles A, B, and C Loci Throughout Southeast Asia: An Immunoinformatics Study

Achmad Makin Amin¹, Arif Nur Muhammad Ansori², Viol Dhea Kharisma³,

Days Chelsealani Kaaro⁴, Muhammad Hermawan Widyananda^3,5, Arli Aditya Parikesit⁶,

Joko Pebrianto Trinugroho⁷, Dora Dayu Rahma Turista⁸, Imam Rosadi⁹,

Sergazy Duyssembaev¹⁰, Maksim Rebezov^11,12, Pavel Burkov¹³, Pavel Scherbakov¹⁴,

Vikash Jakhmola¹⁵, Rahadian Zainul¹⁶*

¹Master Program of Biotechnology, Graduate School, Universitas Gadjah Mada, Yogyakarta, Indonesia.

²Postgraduate School, Universitas Airlangga, Surabaya, Indonesia.

³Division of Molecular Biology and Genetics, Generasi Biologi Indonesia Foundation, Gresik, Indonesia.

⁴Bachelor Program of Biotechnology, Technical University of Berlin, Berlin, Germany.

⁵Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang, Indonesia

⁶Department of Bioinformatics, School of Life Sciences, Indonesia International Institute for Life Sciences, Jakarta, Indonesia.

⁷Bioinformatics and Data Science Research Center, Bina Nusantara University, Jakarta, Indonesia.

⁸Department of Educational Biology, Faculty of Teacher Training and Education, Mulawarman University, Samarinda, Indonesia.

⁹Department of Biology, Faculty of Mathematics and Natural Sciences, Mulawarman University,

Samarinda, Indonesia.

¹⁰Department of Veterinary, Sharakim University of Semey, Semey, Kazakhstan.

¹¹Faculty of Biotechnology and Food Engineering, Ural State Agrarian University,

Yekaterinburg, Russian Federation.

¹²Department of Scientific Research, K.G. Razumovsky Moscow State University of Technologies, and Management (The First Cossack University), Moscow, Russian Federation.

¹³Center for Biotechnology of Animal Reproduction, South Ural State Agrarian University,

Troitsk, Russian Federation.

¹⁴Department of Infectious Disease and Veterinary, South Ural State Agrarian University,

Troitsk, Russian Federation.

¹⁵Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun, India.

¹⁶Department of Chemistry, Faculty of Mathematics and Natural Sciences,

Universitas Negeri Padang, Padang, Indonesia.

*Corresponding Author E-mail: rahadianzmsiphd@fmipa.unp.ac.id

ABSTRACT:

T cell immunity, like responses of CD4⁺and CD8⁺ T-cell, plays an important role to fight against viral infections and pathological harm. Several previous studies have shown the results that rabies virus (RABV) protein can act as an ideal receptor for rabies neuroseptic vaccine by inducing a response of T-cell. In this research, we evaluated possible vaccine epitopes based on the Rabies virus sequence and human lymphocyte antigen (HLA) distribution. First, this study used the rabies virus protein P sequence obtained from the NCBI database. Next, we predicted rabies CTL protein epitopes based on the frequency of HLA-I distribution allele locus A, B, and C in Southeast Asia region (> 1%) using Immune Epitope Database and Analysis Resource (iedb.org). Our results predict the presence of 12 epitopes of the protein P RABV. A cluster analysis of epitopes shows that seven P-protein clusters cover 97.47% of the Southeast Asian population. After a conservative epitope analysis, 8 epitopes of protein P showed protection in 11 different types of isolated Rabies virus. Finally, 4 epitopes (SQTVEEIISY_119-128; RSSGIFLYNF_218-227, ASGPPALEW_178-186, IISYVTVNF_125-133) were used to vaccinate 97.47% of people in Southeast Asia. Our results suggest that both single and combined CTL epitopes which were predicted in this study can be used as a more effective alternative vaccine against rabies virus’ infections and development

KEYWORDS: Immunoinformatics, Rabies Virus, T-Cell Epitope, Human Leukocyte Antigen (HLA).

INTRODUCTION:

Rabies is an animal-to-human infection (zoonotic) caused by infection with a virus from the genus Lyssavirus and the Rhabdovirus family, known as the Rabies virus (RABV), which invades the brain and nervous system. Rabies is a dangerous disease because it carries a risk of death in humans since this infection causes moderate or even lethal irritation of the cerebrum and spinal rope. Clinically, it has two structures: (1) Enraged rabies described by hyperactivity and mental trips; (2) Immobile rabies described by loss of motion and extreme lethargies. The virus that causes Rabies is transmitted to human or animal by bites, saliva, or cratching of a RABV-infected animal^1,2. The main animal host of the RABV isdog. In addition,many other animals, such as monkeys, cats, civets, and rabbits, are highly potential to transmit to humans¹.

Rabies is assessed to cause 59000 human deaths yearly in more than 150 nations and territories, with 95% of cases happening in Asia and Africa. Due to underreporting and questionable evaluations, the actual number may bemuch higher. The weight of illness is disproportionally borne by rustic unfortunate populaces, with roughly 50% of cases owing to youngsters under 15 years old^2,3. WHO, FAO and OIE have focused on rabies under a One Health approach and have sent off the 'United Against Rabies Forum' (UAR), a multi-partner stage. UAR advocates and focuses on rabies control and facilitates the worldwide rabies-disposal endeavors to accomplish zero human passings from rabies by 2030³. Cases of rabies infection in Indonesia are currently increasing, with initial status as a low to moderate rabies endemic country.

The RABV has five main proteins that act as constituents and aid in the virus replication. The 5 proteins are RNA polymerase (L), phosphoprotein (P), matrix protein (M), nucleoprotein (N) and glycoprotein (G). Proteins P and L will interact at the stage of transcription and viral replication. The positive driver RNA strand and all five mRNAs are synthesized during the transcription stage.

At the stage of viral replication, a nucleocapsid containing a full-length antisense RNA genome is formed,which is used as anarrangement for the process of synthesizing the viral genome RNA⁴.

The protein P (RABV) is a cofactor that has non-catalytic properties, and this protein acts as a regulatory protein that is active when interacts with L protein in the polymerase complex, which then binds to the N protein. Protein P has two binding sites to protein N, one site binds to the protein P to the binding region of the 177-terminal amino residue that binds to N° (the part that is unconnected and non-interacting with viral RNA) and the second site, closely related to the domain of terminal carboxylic that binds to N-RNA⁵. RABV phosphoprotein (P) is an important protein that has many roles apart from a key role in viral transcriptional stage and the viral replication process.The protein P is competent of interacting with many other proteins to take over the function in the signalling transduction pathway that directs the signals to the replication of viral process^6,7,8, and can cause mitochondrial malfunction in neurons resulting in severe degenerative damage^9,10. Therefore, protein P can be used as the main candidate protein target for the development of inhibitors^8,11. Furthermore, protein P has immunogenic potential that can be used as the candidate of prime target protein for the RABV vaccine.

Nowadays, a medical treatment for rabies exposure such as postexposure (PEP) RABV vaccine is the mainstream treatment for the people infected RABV. A rabies vaccine can be designed using an epitope mapping approach from antibodies to specific antigens T cells for molecular recognition of the adaptive immune response. Epitope immunizations will be perceived more rapidly by T lymphocytes, so it is to be relied upon to speed up the arrangement of antibodies. Early rabies vaccines mostly evolved from inactivated viral vaccinesregulated either in human diploid cell culture (HDC) or in filtered chick embyonic cell culture (CEC)¹². This inactivated rabiesvaccines requires some investment to be perceived by B cells to create antibodies.

Recent studies in bioimmunochemistry showed the peptides from viruses that can be used as important parts of epitopes to trigger immune responses^13,14,15. In this study, bioinformatics analysis was used to reveal T-cell epitope prediction data for the rabies virus protein P as a target. Protein P samples were collected from Southeast Asia Isolate via the GeneBank database. The results of this study can be used as reference data for vaccine design studies to eliminate the rabies virus.

MATERIALS AND METHODS:

Materials:

Bioinformaticanalysis in thisstudy were performed using aHP computer hardware, the specifications are processor Intel^RCore^TM i5-1035G1 CPU 1.19 GHz RAM 8.00 GB HDD 500 GB OS Windows 11 Home Single Language Version 21H2. The web serverwhichwasused in this studyincludesthe Immune Database and Analysis Resource(IEDB) (https://www.iedb.org/) for specific T-cell protein epitope prediction and analysis andonline prediction(https://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py#forms::PEP-FOLD3) website for 3D modelling protein (PEP-FOLD).

Sequence Retrieval of Rabies Virus Protein P:

Protein P (Phosphoprotein) isolated rom Rabies Virus with GenBank ID: KC148266.1 wasretrieved fromNCBI (https://www.ncbi.nlm.nih.gov/nuccore/ KX148266.1/).

Epitope for T-celland HLA-I Allele Analysis:

Cytotoxic TLymphocyte (CTL)-explicit epitope for white blood cellacknowledgment of the Protein P RABV were analyzed by utilizingcombined expectation ofexpectation of proteasomal debasementhandling, TAP, and restricting of MHC I to obtain a worth of all out score that can be interpreted as an intrinsic potential value of various peptide fragments from the protein P as an Epitope of T-cells. The truncated peptide is a significant material for identifying peptides that will be specifically recognized by T cells. CTL is able to recognize specific epitopes with specific peptide fragments that are foreign to the hand part of MHC class I. Allele frequencies of 1% or higher were used as the percentage threshold value to determine the HLA allele used in this study. The HLA allele analysis was compiled from the Immune Epitope Database and Analysis Resource (IEDB) (http://tools.iedb.org/processing/). The out of score was utilized to anticipate an amount proportional to the number of peptides that the MHC molecules were able to present to the cell surface. The total score is equivalent to the epitope efficiency value that can be shown by the MHC molecule. Therefore, if the total score is higher, it can be interpreted, that the possibility of epitope to be recognized by MHC is also higher. In this case, the worth of the out of score is utilized as a restricting incentive for epitope choice in this examination.

Peptide Clustering Analysis:

Epitopes are clustered using an analysing tool for epitope clustering (http://tools.iedb.org/cluster/). This grouping system refers to the sequence of peptides that have been processed so that they can be recognized by MHC. Clustering performed on peptides can be defined as clustering to create groups based on the peptide sequence that is greater than the minimum identity limit of the specified peptide sequence. In this research, a bunch is a cluster analysis is set by default.

Coverage Population Analysis:

The population coverage analysis method is carried out by counting individual groups that are able to recognize certain specific epitopes based on the known specific MHC recognition (http://tools.iedb.org/population/). This analysis has the principle that the frequency of the HLA genotype is assumed to have an imbalance in the segregation of HLA loci in each individual¹⁶. The final result of this study, we chose the limit value of the population coverage limit with a percentage value > 95%.

Conservancy Epitope Analysis:

The analysis of the preservation of the immune system T cell epitope was performed to determine the nature of the conservation of the epitope in the protein P sequences of 11 RABV isolates analyzed for each sequence (http://tools.iedb.org/conservancy/). The level of conservation was characterized collectively from peptide arrangements that contained epitope at a specific personality level, so the particular epitope completely relates to somewhere around two rabies isolates that have been broke down in this study. This analytical method was used as a tool to analyze the percentage similarity of identity protein sequences P between KX148266.1 (Rabies Isolate 03003INDO Indonesia) and 10 other rabies viruses, namely JN786877.1 (Rabies Isolate QS-05 Thailand); KX148248.1 (Rabies Isolate 99015BIR Myanmar); KX148254.1 (Rabies Isolate 01016VNM Viet Nam); LC018626.1 (Rabies Isolate RV/R3.PHL/2013/Tra-457 Philippines); MN075931.1 (Rabies Isolate TH13-42-07240 Thailand); KX148258.1 (Rabies Isolate 02003LAO Laos); KX148255.1 (Rabies Isolate 99010LAO Laos); KX148253.1 (Rabies Isolate 99012CBG Cambodia); KX148250.1 (Rabies Isolate 99016CBG Cambodia); MG201923.1 (Rabies Isolate GX074 China).

Epitope Immunogenicity and Allergenicity Prediction:

Immunogenicity analysis was carried out to decide the capacity of the chosen peptide as an epitope to set off an insusceptible immune response (http://tools.iedb.org/immunogenicity/). This analytical method has the principle that the amino acid properties and their position in a peptide sequence can be used to foresee the conceivable immunogenicity characteristics of the peptide-MHC complex (pMHC)¹⁷. Then, the epitope with a score > 0 or higher was subsequently subjected for allergenicity analysis (https://www.ddg-pharmfac.net/AllerTOP/index.html).

Protein Modeling:

Epitopes with immunogenicity scores > 0 or higher and non-allergenic properties will be modeled using webPEP-FOLD3 (https://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py #forms::PEP-FOLD3).

RESULTS AND DISCUSSION:

Currently, many methodologies are being developed to design a vaccine. Immunoinformatics is one of the new strategic methods for designing and synthesizing vaccines based on peptide epitope specific antigens against viral infections. In this research, we predicted HLA-associated CTL epitopes by recognition of the protein P (phosphoprotein) sequence of RABV using an immunoinformatics method. Our results found that there were four T-cell-specific antigenic peptides that were non-allergenic probable with immunogenicity values of 0.0206 - 0.390(Table 5). In addition, the four peptide epitopes also had a match percentage of 90.91%-100% among 11 different RABV isolates (Table 4).The population data on HLA alleles were selected from Southern populations East Asian^{18,19,20,21,22,23,24}, with the average percentage of HLA allele frequenciesgreater than 1% of the entire population samples. In total, 18 HLA Class I alleles were selected in this study, consisting of 4 HLA-IA alleles, 9 HLA-IB alleles and 5 HLA-IC alleles for analysis (Table 1).

Table 1: Alleles Data of HLA Class 1 Loci A, B, and C frequently found in Southeast Asian population samples.

HLA-I Loccus	Allele	Total
HLA-IA	A11:01, A24:02, A33:03, A31:01	4
HLA-IB	B13:01, B15:02, B15:25, B18:01, B35:01, B40:01, B46:01, B57:01, B*58:01	9
HLA-IC	C03:02, C04:01, C06:02, C07:02, C*08:01	5

A total of 36,162 rabies virus protein P epitopes were predicted against 18 alleles of the HLA-IA, -IB, and -IC. Table 2 shows that there were 12 epitopes from the epitope prediction analysis. The results of the epitope cluster analysis and the population coverage obtained in this study were used as the basis for determining the epitope so that from these data we chose a limit value for epitope selection, which is a minimum value of 0.70 (12 epitopes). From these values, we could identify several epitopes that cover a population coverage of greater than 95%. The results showed that the population coverage in Southeast Asia was 97.47%, this indicates that this epitope peptide can serve as an epitope candidate for rabies vaccine for most of the Southeast Asian population. The protein P RABV epitope showed a mean score of the complex combination of specific epitope and HLA that the population could recognize was 2.55, with the minimum score that could be recognized by 90% of the population was 1.51 (Table 2).These data indicate that the expected epitope peptide can be an epitope candidate for a rabies vaccine that is capable of being recognized by at least 90% of the targeted population.

Table 2: Number of epitopes, number of clusters, and population coverage predicted by Rabies Virus protein P.

ID	Peptide	Peptide Length	Amino Acid Position	Total Score	Population Coverage			Clustering
ID	Peptide	Peptide Length	Amino Acid Position	Total Score	Coverage^a	hit^b	PC90^c	Clustering
P8	MATAGESKY	9	69-77	1.64	97.47	2.55	1.51	3.1
P4	RSSGIFLYNF	10	218-227	1.61	97.47	2.55	1.51	1.1
P3	FPSRSSGIF	9	215-223	1.46	97.47	2.55	1.51	1.1
P2	FPSRSSGIFLY	11	215-225	1.13	97.47	2.55	1.51	1.1
P9	SQTVEEIISY	10	119-128	1.08	97.47	2.55	1.51	4.1
P10	IISYVTVNF	9	125-133	1.03	97.47	2.55	1.51	5.1
P5	RSSGIFLY	8	218-225	0.94	97.47	2.55	1.51	1.1
P11	RQMRSGERF	9	106-114	0.89	97.47	2.55	1,51	6.1
P7	ASGPPALEW	9	178-186	0.82	97.47	2.55	1.51	2.1
P6	MVAQTASGPPALEW	14	173-186	0.81	97.47	2.55	1.51	2.1
P12	IMQDDLNRY	9	286-294	0.74	97.47	2.55	1.51	7.1
P1	KFPSRSSGIFLYNF	14	214-227	0.73	97.47	2.55	1.52	1.1

^aresult%: population coverage

^baverage: epitope hits / HLA combinations recognized by the population

^cminimum: epitope hits / HLA combinations recognized by 90% of the population

Peptide epitope vaccine was designed from the protein P RABV sequence. Phosphoprotein (P) RABV is one of the proteins that has a main function in the initiation of damage to nerve cells with early invasiveness in the peripheral nerve infection and functions as an interferon antagonist that enhances immunity. Inflammatory immune reactions are necessary to protect the host from the spread, development, and infection of RABV²⁵. In rhabdovirus RABV, protein P (phosphoprotein) is known as a cofactor that associates with L protein. P and L bridge with viral nucleocapsid, antisense RNA genome completely encapsulated by viral nucleoprotein (N)⁵. In addition, the protein P of RABV has many important functions and contains many specific proteins that function to bind viruses. Protein P can bind to protein N and then binds to nonspecific host RNA, and it can also bind to protein L. Protein P also functions as a co-factor for RdRp during the RABV RNA synthesis process. On the other hand, Protein P acts as a potential regulatory protein in the RABV replication process and acts a significant function in avoidance of the innate immune system by binding to the transcription factor STAT.

Protein P is a substantial protein in the evasion process of the innate immune response. This makes protein P a possibleobjective protein for the medication of post-exposure prophylactic RABV contamination (PEP) or RABV antiviral drugs. At the time of RABV invasion into infection, protein P is able to interact with L9, resulting in translocation of the P-L9 complex to the cytoplasm from the nucleus, which then inhibits the initiation recognition of the transcriptional process of RABV. Furthermore, the protein P can bind to the LC8 dynein light chain in the cytoplasm, which acts to accelerate the RNA transcription process from RABV but is also able to promote the viral axonal transport process in neurons. Then, the amino acid PCTD_106-131 interacts with focal adhesion kinases to process the initiation and progression of RABV infection^{6,26, 27}.

Yin, et al. (2021)²⁸ stated that the P protein (297aa) of RABV has several functions, including interacts with L protein to shape an active RNA polymerase for the regulation and the viral RNA packaging process; interacts with LC8 to trigger the initiation of RNA transcription and provide viral intermediaries in the axonal transport of neurons; is able to combine with BECN1 to trigger the failure of the autophagy stage so that it will disrupt the response of the immune system and trigger viral genome replication.Protein P is also capable of linking with FAK to induce the stage of RABV infection and is able to attenuate IRF3 phosphorylation to block the formation of IFN and block the JAK/STAT signal transduction pathway that mediates IFN^29,30,31, where this signal has an important function in interferon antagonist (IFN) type I which acts in helping the viral proliferation in host cells. The inhibition of such signal transduction acts a primary role in the viral escapefrom the innate response of the host.

In this study, it is hoped that the administration of an epitope-based T-cell vaccine can increase the immune system response to RABV exposure because T-cell is an adaptive immunity. The immune system is expected to be able to quickly respond to an infection and eliminate pathogens from the body. The mutated epitope may be prevented the CTL response of the host cell, this needs to be solvedto obtain the immune system response from natural vaccine or a therapeutic vaccine induction based on the peptide epitope of the proteinP RABV. In this study, we used protein sequences from RABV (KC148266.1) obtained from NCBI for epitope analysis and found that there were 12 specific epitope peptides from one isolate and showed a specific identity percentage against 11 different RABV isolates (Table 2).

The epitope cluster analysis used peptides that have been selected based on the total score (> 0,70). The predicted epitopes formed a group into 7 clusters. Cluster 1 consists of 5 epitopes represented by HLA-IA*24:02, two recognitions HLA-IB*35:01, HLA-IB*57:01, HLA-IB*58:01, cluster 2 consists of 2 epitopes which is represented by two recognitions HLA-IB*58:01, cluster 3 consists of singleton represented by HLA-IB*35:01 and clusters 4-7 consists of singletons represented by HLA-IB*15:25 (Table 3). The analysis process of the epitope classification was then continued with an analysis of the conservation of epitope identity between the rabies virus isolates.

Table 3: Epitope sequence, amino acid position and HLA allele presented from Rabies Virus protein P by cluster analysis.

Cluster Number	Epitope Number	Alignment	Epitope	Amino Acid Position	HLA-Alleles
1.1	Consensus	KFPSRSSGIFLYNF	-	-
	1	KFPSRSSGIFLYNF	KFPSRSSGIFLYNF	214-227	HLA-IA*24:02
	2	-FPSRSSGIFLY--	FPSRSSGIFLY	215-225	HLA-IB*35:01
	3	-FPSRSSGIF----	FPSRSSGIF	215-223	HLA-IB*35:01
	4	----RSSGIFLYNF	RSSGIFLYNF	218-227	HLA-IB*57:01
	5	----RSSGIFLY--	RSSGIFLY	218-225	HLA-IB*58:01
2.1	Consensus	MVAQTASGPPALEW	-	-	-
	1	MVAQTASGPPALEW	MVAQTASGPPALEW	173-186	HLA-IB*58:01
	2	-----ASGPPALEW	ASGPPALEW	178-186	HLA-IB*58:01
3.1	Singleton	MATAGESKY	MATAGESKY	69-77	HLA-IB*35:01
4.1	Singleton	SQTVEEIISY	SQTVEEIISY	119-128	HLA-IB*15:25
5.1	Singleton	IISYVTVNF	IISYVTVNF	125-133	HLA-IB*15:25
6.1	Singleton	RQMRSGERF	RQMRSGERF	106-114	HLA-IB*15:25
7.1	Singleton	IMQDDLNRY	IMQDDLNRY	286-294	HLA-IB*15:25

Tabel 4: Epitope similarity analysis results for protein P RABV from 11 Rabies Viruses

ID^a	Epitope Sequence	Sequence Matches^c	Identity Percentage (%)d
ID^a	Epitope Sequence	Sequence Matches^c	KX148266.1	JN786877.1	KX148248.1	KX148254.1	LC018626.1	MN075931.1
P1	KFPSRSSGIFLYNF	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%
P2	FPSRSSGIFLY	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%
P3	FPSRSSGIF	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%
P4	RSSGIFLYNF	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%
P5	RSSGIFLY	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%
P6	MVAQTASGPPALEW	18,18% (2/11)	100,00%	-	-	-	-	-
P7	ASGPPALEW	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%
P8	MATAGESKY	9,09% (1/11)	100,00%	-	-	-	-	-
P9	SQTVEEIISY	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%
P10	IISYVTVNF	90,91% (10/11)	100,00%	100,00%	100,00%	100,00%	-	100,00%
P11	RQMRSGERF	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%
P12	IMQDDLNRY	90,91% (10/11)	100,00%	100,00%	100,00%	100,00%	100,00%	100,00%

Continew Table 4

ID^a	Epitope Sequence	Sequence Matches^c	Identity Percentage (%)d
ID^a	Epitope Sequence	Sequence Matches^c	KX148258.1	KX148255.1	KX148253.1	KX148250.1	MG201923.1
P1	KFPSRSSGIFLYNF	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P2	FPSRSSGIFLY	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P3	FPSRSSGIF	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P4	RSSGIFLYNF	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P5	RSSGIFLY	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P6	MVAQTASGPPALEW	18,18% (2/11)	-	-	-	-	100,00%
P7	ASGPPALEW	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P8	MATAGESKY	9,09% (1/11)	-	-	-	-	-
P9	SQTVEEIISY	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P10	IISYVTVNF	90,91% (10/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P11	RQMRSGERF	100,00% (11/11)	100,00%	100,00%	100,00%	100,00%	100,00%
P12	IMQDDLNRY	90,91% (10/11)	100,00%	100,00%	100,00%	100,00%	-

^aEpitope Name

^bEpitope Length

^cPercent of protein sequence matches at identity 100%

^dthe identity percentage results of each 12 epitopes with 11 protein P sequences

The analysis was then continued by measuring the probability value of the immunogenicity of the chosen epitope. Epitope immunogenicity analysis uses the basis of amino acid properties and their position in peptides to predict the immunogenicity of the MHC peptide complex (pMHC), where it is indicated by the higher score showing a greater possibility of the peptide to cause an immune response. In this analysis, the 12 tested epitopes showed that there were only 6 epitopes with positive values (> 0). Epitope analysis results with ID P9 have the highest score: 0.39008 (Table 5).

Table 5: Class I Immunogenicity

ID^a	Peptide	Length^c	Score^d
P9	SQTVEEIISY_119-128^b	10	0,39008
P5	RSSGIFLY_218-225	8	0,21084
P4	RSSGIFLYNF_218-227	10	0,20666
P7	ASGPPALEW_178-186	9	0,07501
P10	IISYVTVNF_125-133	9	0,05038
P12	IMQDDLNRY_286-294	9	0,02066

^aEpitope ID

^bAmino-acid Position

^cLength of Peptide

^dA higher score : a greater eliciting an immune response.

After obtaining the probability of immunogenicity, the chosen epitope was analyzed for allergenicity response (Table 6). This prediction is used to determine the possibility of whether the peptide is able to trigger the occurrence of allergies or not. The six epitopes that showed positive values in the class I immunogenicity analysis were then continued with allergenicity prediction analysis with the results: ID P9, P4, P7, P10 probable non-allergen epitope and ID P5 and P12 probable allergen epitope.

Tabel 6: Allergenicity Prediction

ID^a	Peptide	Result
P9	SQTVEEIISY_119-128^b	Probable Non-Allergen
P5	RSSGIFLY_218-225	Probable Allergen
P4	RSSGIFLYNF_218-227	Probable Non-Allergen
P7	ASGPPALEW_178-186	Probable Non-Allergen
P10	IISYVTVNF_125-133	Probable Non-Allergen
P12	IMQDDLNRY_286-294	Probable Allergen

In addition, further tests showed that 6 of the 12 peptide epitopes that we selected in this study had high immunogenicity scores. This indicates that the higher the score, the greater the chance of the peptide epitope to trigger an immune response.Furthermore, of the 6 epitopes, there are 4 epitopes that are probable non-allergen, and we chose these epitopes in this study which can function as candidate epitopes for the RABV vaccine because the protein sequences are identical (90.91%-100%) against 11 RABV protein P isolates(Table 4). The four epitopes are SQTVEEIISY_119-128, RSSGIFLYNF_218-227, ASGPPALEW_178-186, IISYVTVNF_125-133; In addition, these four epitopes could cover 97.47% of the individual population of Southeast Asia (Table 2; Table 6).

Tabel 7: Protein Modelling

ID	Peptide	Best Model
P9	SQTVEEIISY
P4	RSSGIFLYNF
P7	ASGPPALEW
P10	IISYVTVNF

Epitope with ID: P9 (SQTVEEIISY_119-128) presented by HLA-IB*15:25 showed the highest score in immunogenicity analysis compared to the other three epitopes selected in this study. However,P4 (RSSGIFLYNF_218-227) presented by HLA-IB*57:01, P7 (ASGPPALEW_178-186) presented by HLA-IB*58:01, and P10 (IISYVTVNF_125-133) presented by HLA-IB*15:25 are able to cover 97.47% of individuals in the Southeast Asia population. Thus, the combination of these four epitopes can be used as vaccine candidates with a T cell-specific epitope peptide analysis approach to treat RABV infection based on protein P (Table 2; Table 3).

Our results indicate that there is a potential to use the predicted results of CTL-specific epitope as a reference for vaccine production to strictly control RABV infection and progression of RABV via post-exposure prophylaxis. Based on this study, we obtained four CTL-specific epitope combinations on the distribution of HLA-IA, -IB, and -IC alleles which were able to have a coverage of 97.47% of individuals in the region located in Southeast Asia (Table 2).Nevertheless, the CTL-specific epitope that we chose in this study was predicted by incorporating the epitope-binding ability with MHC-specificmolecules that determined the absolute score. The absolut score is used as the basis for the threshold value for epitope choosing.Therefore, the analysis we used led to the neglect of several different peptide epitopes of the protein P RABV because the total score was below the limit value set in this study (protein P with a total score greater than 0.70).

In past investigations, it has been realized that proteins G, L, N and P fill in as the primary focuses for the RABV vaccine^32,33,34,35. However, this past research focuses on general immune induction which will be responded by the humoral immune system which tends to take longer to neutralize antigens than the cellular immune response. In thisstudy, the protein P is very potential as an antigen candidate because of its crucial protein capacity. In addition, the development of peptide epitope for T Lymphocytes, able to recognize fasteron the outer membrane of antigen-presenting cells (APC).The presented antigen on APC surface will bind to MHC which will cause animmune response^15,36,37. Thus, immune response is relied upon to have the option to rapidly answer the presence of antigens and have the option to rapidly wipe out antigens from the body.

CONCLUSION:

The results of the T-cell epitope prediction showed that there were 4 types of epitopes, which had a match percentage of more than 90% against 11 RABV isolates,and were able to cover 97.47% of Southeast Asian individuals. The four epitopes are SQTVEEIISY119-128, RSSGIFLYNF_218-227, ASGPPALEW_178-186, IISYVTVNF_125-133; In addition, these 4 epitopes also have a high immunogenicity score (0.050-0.390) and are non-allergenic probable. These studies have predicted epitope using T cell-specific epitope prediction analysis. However, this study should be confirmed using peripheral blood mononuclear cells that are sensitive to specific epitope peptides or CD8⁺ CTL response analysis isolated in vivo or in vitro from experimental animals. The results of this study can contribute to the exploration and prediction of the possible use of epitopes for vaccines against RABV infection.

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Received on 17.01.2023 Modified on 08.06.2023

Research J. Pharm. and Tech 2024; 17(5):2001-2008.

DOI: 10.52711/0974-360X.2024.00317