A Viroinformatics Study: B-Cell Polytope Mapping of Envelope Protein to Develop Vaccine Candidate against Four DENV Serotype

Rahadian Zainul^1,2*, Viol Dhea Kharisma³, Santika Lusia Utami⁴, Nelson Chandra⁵,

Arif Nur Muhammad Ansori^6,7, Arli Aditya Parikesit⁵, Vikash Jakhmola⁷,

Daimon Syukri⁸, Edi Syafri⁹, Asri Peni Wulandari^10,11, Oski Illiandri¹²,

Khoirun Nisyak¹³, Bahrun¹⁴, Asmi Citra Malina A. R. Tasakka¹⁵

¹Department of Chemistry, Faculty of Mathematics and Natural Sciences,

Universitas Negeri Padang, Padang, Indonesia.

²Center for Advanced Material Processing, Artificial Intelligence, and Biophysic Informatics(CAMP-BIOTICS), Universitas Negeri Padang, Padang, Indonesia.

³Computational Virology Research Unit, Division of Molecular Biology and Genetics, Generasi Biologi Indonesia Foundation, Gresik, Indonesia.

⁴Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia.

⁵Bioinformatics Department, Indonesia International Institute of Life Sciences (i3L), Jakarta Timur, Indonesia.

⁶Postgraduate School, Universitas Airlangga, Surabaya, Indonesia.

⁷Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University, Dehradun, India.

⁸Department of Food and Agriculture Product Technology,

Faculty of Agriculture Technology, Universitas Andalas, Padang, Indonesia.

⁹Department of Agricultural Technology, Politeknik Pertanian Negeri Payakumbuh, Payakumbuh, Indonesia.

¹⁰Department of Biology, Faculty of Mathematics and Science, University of Padjadjaran, Bandung, Indonesia.

¹¹Center for Bioprospecting of Natural Fibre and Bioresources, University of Padjadjaran, Bandung, Indonesia.

¹²Department of Biomedicine, School of Medicine, Lambung Mangkurat University, Banjarmasin, Indonesia.

¹³Department of Pharmacy, Faculty of Public Health, Universitas Anwar Medika, Sidoarjo, Indonesia.

¹⁴Department of Chemistry, Faculty of Mathematics and Natural Sciences,

Hasanuddin University, Makassar, Indonesia.

¹⁵Faculty of Marine Science and Fisheries, Universitas Hasanuddin, Makassar, Indonesia.

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

ABSTRACT:

Nowadays, dengue virus (DENV) is still become a global problem, even though the virus infection issues have reached half of the population in some countries each year. DENV belongs to the enveloped virus with positive-sense single-stranded RNA (+ssRNA) genus Flavivirus and belongs to the Flaviviridae family. DENV has structural proteins which consist of the envelope protein (E), capsid (C), and membrane (M). There are four serotypes of this virus which are DENV-1, 2, 3, and 4. These four serotypes are transmitted to humans through Aedes sp. The development of this vaccine is still in progress and the challenge of this DENV vaccine candidate design is to overcome the heterotypic infection and the expansion of coverage protection to all virus serotypes. This research uses design simulation for vaccine candidates using B cell epitope in all DENV’s serotypes envelope to trigger the antibody formation through bioinformatics method that consists of protein modeling, immunogenicity, toxicity, and immune stimulation. DENV envelope protein was predicted to have polytope that can be recognized by B cells and act as an antigen, have low similarity with the composing sequence of cell surface receptors on the body, and non-toxic, and then can trigger the population increase of B cell and IgM antibody production with high avidity to neutralize four of the DENV serotypes. We recommend the B cell polytype which consists of A, C, E, and G peptides be examined by the wet-lab approach.

KEYWORDS: B-cell, Bioinformatics, DENV, Flavivirus, Polytope, Viroinformatics.

INTRODUCTION:

Nowadays, the dengue virus (DENV) is still a global problem, even though the virus infection issues have reached half of the population in some countries each year. DENV belongs to the enveloped virus with positive-sense single-stranded RNA (+ssRNA) genus Flavivirus and belongs to the Flaviviridae family. DENV has structural proteins which consist of the envelope protein (E), capsid (C), and membrane (M). There are four serotypes of this virus which is DENV-1, 2, 3, and 4 are transmitted to human through Aedes sp, DENV infection can cause several health problems such as abdominal pain, blushing, muscle pain, anorexia, dengue (DHF), and dengue shock syndrome (DSS). DENV infection can trigger the escalation of antibody titer production for neutralization and protection in immunity^1,2.

The body's immune system requires protection to fight DENV infection of the same serotype or homotypic, but referring to the presence of all four DENV serotypes can trigger heterotypic type infections, in these conditions antibodies with cross serotype protection are needed to fight heterotypic infections. The challenge for the development of DENV vaccine candidates is to be able to address heterotypic infections and expand the scope of protection across all viral serotypes. DENV has genetic material that is more dynamic, this can be shown in the mutation ability of each serotype when the virus experiences increased adaptation to changes in the host environment and vectors^1,3,4. The development of DENV vaccine candidates with increased protection against viral heterotypic is necessary to trigger a decrease in cases of DENV infection in humans.

Virion DENV has the E protein and is present on the surface, the E protein is located in the outermost domain and allows it to be recognized by Immune cells. The results of in vitro studies show the ability of protein E from DENV serotype 1 can trigger an increase in the production of antibodies naturally in patients^5,6. The immune response by B cells plays an important role in producing specific antibodies to anchor viral activity through a neutralization process⁷. The mechanism of B cell recognition in antigens consists of direct and indirect⁸.

This study used the simulated introduction of cell epitopes through a direct mechanism to trigger the formation of antibodies.

Multi-domain or polytope epitope constructions can trigger increased coverage of antibody neutralization and protection against certain viral infections^9,10. This study aims to design vaccine candidates with polytope protein E from DENV-1, 2, 3, and 4 through a bioinformatics approach.

METHOD:

Sample Retrieval:

DENV-1, DENV-2, DENV-3, and DENV-4’s E protein sequence with FASTA format were retrieved from NCBI (https://www.ncbi.nlm.nih.gov/) with “envelope protein dengue virus” keywords. This study used protein sequences of four DENV serotypes which include DENV-1 (AJW83537.1), DENV-2 (AJW83569.1), and DENV-3 (AFN85197.1), and DENV-4 (AFN85222.1).

3D Protein Modeling:

The 3D structure was obtained from the SWISS-MODEL modeling simulation (https://swissmodel.expasy.org/) [10]. The protein model was selected which meet the requirements, the protein must have scored above 20%, then downloaded as a protein databank format (PDB), Ramachandran Plot analysis via the PROCHECK server (https://servicesn.mbi.ucla.edu/PROCHECK/) was used to validate the structure of the model results¹¹.

B-cell Epitope Mapping Identification:

Prediction of B cell linear epitope on envelope proteins DENV-1, DENV-2, DENV-3, and DENV-4 was carried out through IEDB Analysis Resource (http://tools.iedb.org/bcell/) using BepiPred 1.0 and Emini Surfaces methods. Accessibility. Both predictions work by calculating the Hidden Markov Model (HMM) statistical method and propensity scale to indicate the presence of B cell epitopes in specific amino acid sequences¹².

Antigenicity, Similarity, and Toxicity Prediction:

The peptides that constructed the B cell epitope were then predicted for antigenicity through VaxiJen v.2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) to obtain antigenic peptides with viral organisms as targets. Then the similarity prediction was made through BLASTp (https://blast.ncbi.nlm.nih.gov/ Blast.cgi?PAGE=Proteins) the selection process for non-toxic peptides on non-allergenic peptides in this study was carried out through ToxIBTL (https://server.wei-group.net/ToxIBTL/Server.html)^13,14.

Physiochemical Analysis:

Prediction of physiochemical properties of peptides referring to various physicochemical properties consisting of atomic composition, theoretical pI, molecular weight, extinction coefficient, aliphatic index, grand average of hydropathicity (GRAVY), and others is done through ProtParam (https://web.expasy.org/protparam/)¹⁴.

Molecular Visualization:

Visualization of epitope positions and vaccine candidate peptides with 3D structures was performed using PyMol 2.5.2 version software (Schrödinger, Inc., USA) with an academic license. Coloring (by chain) and structural selection methods consisting of sticks, cartoons, and surfaces were used in this study¹⁵.

Immune Simulation:

Simulation of B cell immune response in producing antibodies by immunogenic peptide was identified through C-ImmSim (http://kraken.iac.rm.cnr.it/C-IMMSIM/). Simulation parameters such as simulation step of 1050, random seed 1234, volume 10, and adjuvant 100 were selected. Graphs of antibody production and decreased antigen levels are shown in the output of the simulation results¹⁶.

RESULT:

Identification of 3D Modeling Results and Validation of DENV Envelope Structure:

The 3D structure of the envelope modeling results from the four DENV serotypes are similar to the template because overall, the scores are >20%, all 3D protein target models have disallowed regions with a lower percentage than the allowed regions on the Ramachandran plot (Table 1), this shows that all DENV envelope models are valid and meet stereochemical rules refer to the results of the Ramachandran plot^17,18,19. Visualization of 3D modeling results with cartoons structure, transparent surfaces, and selection of staining on secondary proteins was performed on the four DENV envelope models^20,21,22 (Figure 1).

B-cell Polytope in the DENV Envelope Composing Protein Sequences:

B cells recognize epitopes through a direct mechanism by binding directly through the B-cell receptor (BCR), triggering the production of antibodies against pathogens²³. The virion envelope of the four serotypes consisting of DENV-1, DENV-2, DENV-3, and DENV-4 allows them to act as B cell epitopes. Peptides with A, C, E, and G labels are categorized as B cell epitopes, antigenic peptides, low similarity or low probability of triggering an allergic reaction, and are non-toxic (Table 2). B cell epitopes in the DENV-1, 2, 3, and 4 envelope domains indicated by the yellow region are positive predictions because they have a probability value above the threshold and negative predictions on the results of cell epitope predictions using the BepiPred 1 method (Figure 2).

Figure 1: Visualization of the viral envelope protein 3D model DENV (A) DENV-1 (B) DENV-2 (C) DENV-3 (D) DENV-4.

Table 1: The results of the analysis of homology modeling and model validation

GeneBank ID	Sequence Length (mer)	Ramachandran Plot		Homology Score
GeneBank ID	Sequence Length (mer)	Allowed Region	Disallowed Region	Homology Score
AJW83537.1	300	19,6%	0.6%	80,54%
AJW83569.1	325	12,9%	0.0%	97,23%
AFN85197.1	493	17,6%	0.1%	96,43%
AFN85222.1	495	16,5%	5.7%	63,84%

Tabel 2: Epitope mapping and antigenic peptide prediction results

Virus	BepiPred Prediction			VaxiJen v.2.0	BLASTp	ToxIBTL
Virus	Epitope Sequence	Residue Number	Peptide Label	VaxiJen v.2.0	BLASTp	ToxIBTL
DENV-1	SNTTTDSRCPTQGEATLVEE	66-85	A	Antigen	Non-similar	Non-toxic
DENV-1	TGDQHQVGNETTEHGTTAIITPQAPTSEIQL	145-175	B	Non-antigen	-	-
DENV-2	TNTTTASRCPTQGEPSLNEEQD	66-87	C	Antigen	Non-similar	Non-toxic
DENV-2	LPGADTQGSNWI	221-232	D	Non-antigen	-	-
DENV-3	TNVTTDSRCPTQGEAILPEEQDQN	66-89	E	Antigen	Non-similar	Non-toxic
DENV-3	YKGEDAPCKIPFSTEDGQGKAH	324-345	F	Non-antigen	-	-
DENV-4	TATRCPTQGEPYLKEEQDQQ	70-89	G	Antigen	Non-similar	Non-toxic
DENV-4	NGDTHAVGNDTSNHGVTATITPRSPSVEVKLP	145-176	H	Non-antigen	-	-

(A) (B)

Figure 2. Epitope mapping results on the viral envelope (A) DENV-1, (B) DENV-2 (C) DENV-3, and (D) DENV-4.

Peptide Vaccine Physiochemical Properties and Immune Simulation:

Peptides with antigenic, non-allergenic, and non-toxic properties consisting of peptides A, C, E, and G were identified in PortParam to determine the physiochemical properties of vaccine candidate peptides. The results showed that the physiochemical properties of the four peptides that make up the DENV epitope consisted of molecular weight, theoretical pI, instability index, aliphatic index, and GRAVY (Table 3). The peptides that make up the B cell epitope in the four DENV envelope serotypes were then predicted to stimulate the immune response and obtained positive predictions that could trigger IgM and IgG antibody isotype activity, population increase, and B cell activation (Figure 3).

Table 3: Physiochemical properties of peptide vaccine candidate

Peptide	Molecular Weight (kD)	Theoretical pI	Instability Index	Aliphatic Index	GRAVY
A	2139.23	4.00	287.00	39.00	-1.015
C	2379.45	4.00	59.35	22.27	-1.505
E	2646.78	3.83	70.50	48.75	-1.246
G	2322.49	4.41	62.17	24.50	-1.765

(A)

(B)

(C)

Figure 3: Predicted results of immune simulation (A) Type of antibody titer formed, (B) Total population of B cells, (C) Activity of B cells when recognizing antigens.

DISCUSSION:

The DENV-1, DENV-2, DENV-3, and DENV-4 envelopes are located outside the virion and allow for recognition by immune cells and can be used as key targets for vaccine candidate designs^{24,25,26,27,28,29,30}. The 3D envelope structure obtained from the four DENV serotypes has a high similarity to the template and is valid, the envelope domain that immune cell receptors can recognize is an epitope. Epitope prediction in this study is based on direct B-cell activation to trigger the production of specific antibodies. The release of antibodies by activated B cells aims to inhibit viral entry through a virus neutralization mechanism^{18,31,32,33,34,35,36}.

The results showed ten peptides with a length >10 mers that make up the epitope of B cells consisting of A, B, C, D, E, F, G, and H were found on the DENV-1, DENV-2, DENV-3, and DENV-3 envelopes. DENV-4. Epitope composing peptides must be able to trigger immune cell receptor activity or are referred to as antigens^24,25,26. Vaccine candidate peptides must have a low affinity with body cell surface receptors to avoid an autoimmune response and are non-toxic¹⁹. Peptides A, C, E, and G are antigenic, have non-autoimmune triggering sequences, and are non-toxic.

Direct B cell activation through epitope recognition on the B cell envelope can trigger the formation of antibodies with various isotypes such as IgM and IgG. IgM has advantages with high binding avidity for virus neutralization mechanisms^20,27,28. B cell epitopes from the envelope of the four DENV serotypes can trigger the formation of IgM antibody isotypes and are able to increase the B cell population that plays a role in the adaptive immune response. Polytope refers to the combination of peptides that make up the B cell epitope used in the design of vaccine candidates through the Bioinformatics approach^21,29,30. The study's results showed that the polytope composed of peptides A, C, E, and G was predicted to trigger an increase in vaccine coverage for the treatment of DENV infection from the four serotypes.

CONCLUSION:

In conclusion, the DENV envelope protein is predicted to have a polytope that allows it to be recognized by B cells and acts as an antigen, has low similarity with the body's cell surface receptor constituent sequences, and is non-toxic, which can then trigger an increase in the B cell population and the production of IgM antibodies with high avidity for neutralization of all four. DENV serotype. We recommend a B cell polytope composed of A, C, E, and G peptides for further testing through a wet-lab approach.

REFERENCES:

1. Triningrat AAMP, Agus Somia IK, Dwi Lingga Utama IM, Sri Yuliastini NPN, Wijayati MP, Handayani AT. Dengue hemorrhagic fever with severe ocular complication: case series. Bali Medical Journal. 2020; 9(3): 907–911. DOI: 10.15562/bmj.v9i3.1946

2. Deng SQ, Yang X, Wei Y, Chen JT, Wang XJ, Peng HJ. A Review on Dengue Vaccine Development. Vaccines (Basel). 2020; 8(1): 63.

3. Sudaryanto A, Ainnurriza US, Supratman S, Dewi SK. Mapping the prevalence of dengue fever in sragen regency Indonesia. Bali Medical Journal. 2021; 10(3): 1107–1110. DOI: 10.15562/bmj.v10i3.2821

4. Elmy Saniathi NK, Juffrie M, Rianto BUD, Soetjiningsih S. The dynamic of Soluble Vascular Cellular Adhesion Molecule -1 (sVCAM-1) level in Overnutritious children with Dengue Infection. Bali Medical Journal. 2021; 10(1): 261–265. DOI: 10.15562/bmj.v10i1.2304

5. Pinontoan OR, Sumampouw OJ, Ticoalu J, Nelwan JE, Musa EC, Sekeeon J. The variability of temperature, rainfall, humidity and prevalance of dengue fever in Manado City. Bali Medical Journal. 2022; 11(1): 81–86. DOI: 10.15562/bmj.v11i1.2722

6. Utama IMGDL, Agustini NMA. The role of macrophage Migration Inhibitory Factor (MIF) in pediatric dengue infection at Sanglah Hospital, Bali, Indonesia. Bali Medical Journal. 2020; 9(1): 224–228. DOI: 10.15562/bmj.v9i1.1740

7. Hoffman W, Lakkis FG, Chalasani G. B Cells, Antibodies, and More. Clin J Am Soc Nephrol. 2016; 11(1):137-54.

8. Rostaminia S, Aghaei SS, Farahmand B, Nazari R, Ghaemi A. Computational Design and Analysis of a Multi-epitope Against Influenza A virus. Int J Pept Res Ther. 2021; 12:1-14.

9. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T. SWISS-MODEL: homology modeling of protein structures and complexes. Nucleic Acids Res. 2018; 46(W1): W296-W303.

10. Maxwell PI, Popelier PLA. Unfavorable regions in the Ramachandran plot: Is it really a steric hindrance? The interacting quantum atoms perspective. J Comput Chem. 2017; 38(29):2459-2474.

11. Ansori ANM, Kharisma VD, Antonius Y, Tacharina MR, Rantam FA. Immunobioinformatics analysis and phylogenetic tree construction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Indonesia: spike glycoprotein gene. Jurnal Teknologi Laboratorium. 2020; 9(1):13-20.

12. Kharisma VD, Ansori ANM. Construction of epitope-based peptide vaccine against SARS-CoV-2: Immunoinformatics study. J Pure Appl Microbiol. 2020; 14 (suppl 1):999-1005.

13. Pan W, Chen DS, Lu YJ, Sun FF, Xu HW, Zhang YW, Yan C, Fu LL, Zheng KY, Tang RX. Bioinformatic prediction of the epitopes of Echinococcus granulosus antigen 5. Biomed Rep. 2017; 6(2): 181-187.

14. Susanto H, Kharisma VD, Listyorini D, Taufiq A. Effectivity of Black Tea Polyphenol in Adipogenesis Related IGF-1 and Its Receptor Pathway Through In Silico Based Study. J Phys Conf Ser. 2018; 1093(1): 012037.

15. Abraham Peele K, Srihansa T, Krupanidhi S, Ayyagari VS, Venkateswarulu TC. Design of multi-epitope vaccine candidate against SARS-CoV-2: an in-silico study. J Biomol Struct Dyn. 2021; 39(10): 3793-3801.

16. Swanstrom JA, Nivarthi UK, Patel B, Delacruz MJ, Yount B, Widman DG, Durbin AP, Whitehead SS, De Silva AM, Baric RS. Beyond Neutralizing Antibody Levels: The Epitope Specificity of Antibodies Induced by National Institutes of Health Monovalent Dengue Virus Vaccines. J Infect Dis. 2019; 220(2): 219-227.

17. He J, Huang F, Zhang J, Chen Q, Zheng Z, Zhou Q, Chen D, Li J, Chen J. Vaccine design based on 16 epitopes of SARS-CoV-2 spike protein. J Med Virol. 2021; 93(4): 2115-2131.

18. Sundling C, Lau AWY, Bourne K, Young C, Laurianto C, Hermes JR, Menzies RJ, Butt D, Kräutler NJ, Zahra D, Suan D, Brink R. Positive selection of IgG+ over IgM+ B cells in the germinal center reaction. Immunity. 2021; 11; 54(5): 988-1001.e5.

19. Han K, Zhao D, Liu Y, Huang X, Yang J, Liu Q, An F, Li Y. Design and evaluation of a polytope construct with multiple B and T epitopes against Tembusu virus infection in ducks. Res Vet Sci. 2016; 104: 174-80.

20. Tashiro Y, Murakami A, Hara Y, Shimizu T, Kubo M, Goitsuka R, Kishimoto H, Azuma T. High-affinity IgM+ memory B cells are defective in differentiation into IgM antibody-secreting cells by re-stimulation with a T cell-dependent antigen. Sci Rep. 2018; 8(1): 14559.

21. Himmah K, Fitriyah, Ardyati T, Afiyanti M, Rifa'i M, Widodo. Designing a polytope for use in a broad-spectrum dengue virus vaccine. J Taibah Univ Med Sci. 2017; 13(2): 156-161

22. Mannige RV, Kundu J, Whitelam S. The Ramachandran Number: An Order Parameter for Protein Geometry. PLoS One. 2016; 11(8): e0160023.

23. Yam-Puc JC, Zhang L, Zhang Y, Toellner KM. Role of B-cell receptors for B-cell development and antigen-induced differentiation. 2018; F1000Res. 7:429.

24. Gaurav A, Agrawal N, Al-Nema M, Gautam V. Computational Approaches in the Discovery and Development of Therapeutic and Prophylactic Agents for Viral Diseases. Curr Top Med Chem. 2022; 22(26): 2190-2206. DOI: 10.2174/1568026623666221019110334.

25. Sekaran SD, Liew ZM, Yam HC, Raju CS. The association between diabetes and obesity with Dengue infections. Diabetol Metab Syndr. 2022; 14(1): 101. DOI: 10.1186/s13098-022-00870-5.

26. Qusay KA, Rao AV, Husniza H, Najeeb ZT. Computational atom-based three-dimensional quantitative structure-activity relationship (3d qsar) model for predicting anti-dengue agents. Research Journal of Biotechnology. 2021; 16(10), pp. 50-58

27. Sekaran SD, Ismail AA, Thergarajan G, Chandramathi S, Rahman SKH, Mani RR, Jusof FF, Lim YAL, Manikam R. Host immune response against DENV and ZIKV infections. Front Cell Infect Microbiol. 2022; 12: 975222. DOI: 10.3389/fcimb.2022.975222.

28. Husen SA, Setyawan MF, Syadzha MF, Susilo RJK, Hayaza S, Ansori ANM, Alamsjah MA, Ilmi ZN, Wulandari PAC, Pudjiastuti P, Awang P, Winarni D. A Novel Therapeutic effects of Sargassum ilicifolium Alginate and Okra (Abelmoschus esculentus) Pods extracts on Open wound healing process in Diabetic Mice. Research J. Pharm. and Tech 2020; 13(6): 2764-2770. doi: 10.5958/0974-360X.2020.00491.6

29. Kharisma VD, Kharisma SD, Ansori ANM, Kurniawan HP, Witaningrum AM, Fadholly A, Tacharina MR. Antiretroviral Effect Simulation from Black Tea (Camellia sinensis) via Dual Inhibitors Mechanism in HIV-1 and its Social Perspective in Indonesia. Res J Pharm Technol. 2021; 14(1): 455-460. doi: 10.5958/0974-360X.2021.00083.4

30. Fadholly A, Ansori ANM, Kharisma VD, Rahmahani J, Tacharina MR. Immunobioinformatics of Rabies Virus in Various Countries of Asia: Glycoprotein Gene. Res J Pharm Technol. 2021; 14(2): 883-886. doi: 10.5958/0974-360X.2021.00157.8

31. Ansori ANM, Fadholly A, Proboningrat A, Hayaza S, Susilo RJK, Naw SW, Posa GAV, Yusrizal YF, Sibero MT, Sucipto TH, Soegijanto S. In vitro antiviral activity of Pinus merkusii (Pinaceae) stem bark and cone against dengue virus type-2 (DENV-2). Res J Pharm Technol. 2021; 14(7):3705-8. doi: 10.52711/0974-360X.2021.00641

32. Ansori ANM, Kharisma VD, Fadholly A, Tacharina MR, Antonius Y, Parikesit AA. Severe Acute Respiratory Syndrome Coronavirus-2 Emergence and Its Treatment with Alternative Medicines: A Review. Research Journal of Pharmacy and Technology 2021; 14(10):5551-7. doi: 10.52711/0974-360X.2021.00967

33. Husen SA, Ansori ANM, Hayaza S, Susilo RJK, Zuraidah AA, Winarni D, Punnapayak H, Darmanto W. Therapeutic Effect of Okra (Abelmoschus esculentus Moench) Pods Extract on Streptozotocin-Induced Type-2 Diabetic Mice. Res J Pharm Technol. 2019; 12(8): 3703-3708. doi: 10.5958/0974-360X.2019.00633.4

34. Ansori ANM, Kharisma VD, Solikhah TI. Medicinal properties of Muntingia calabura L.: A Review. Res J Pharm Technol. 2021; 14(8): 4509-2. doi: 10.52711/0974-360X.2021.00784

35. Proboningrat A, Kharisma VD, Ansori ANM, Rahmawati R, Fadholly A, Posa GAV, Sudjarwo SA, Rantam FA, Achmad AB. In silico Study of Natural inhibitors for Human papillomavirus-18 E6 protein. Res J Pharm Technol. 2022; 15(3): 1251-6. doi: 10.52711/0974-360X.2022.00209

36. Kharisma VD, Ansori ANM, Jakhmola V, Rizky WC, Widyananda MH, Probojati RT, Murtadlo AAA, Rebezov M, Scherbakov P, Burkov P, Matrosova Y, Romanov A, Sihombing MAEM, Antonius Y, Zainul R. Multi-strain human papillomavirus (HPV) vaccine innovation via computational study: A mini review. Res J Pharm Technol. 2022; 15(8).

Received on 05.09.2022 Modified on 17.04.2023

Research J. Pharm. and Tech 2024; 17(3):973-978.

DOI: 10.52711/0974-360X.2024.00150