Viroinformatics study: polytope mapping of envelope glycoprotein to tackle HIV-2 infection and develop vaccine candidate

Human Immunodeficiency Virus type 2 (HIV-2) has been identified to exhibit an ability to resist antiretroviral administration and many scientists has predicted increases in the pathogenicity of HIV-2. The development of a vaccine against the type 1 virus (HIV-1) infection has reached the phase 3 clinical trial stage, but currently there is no information on the development of a vaccine against HIV-2. Vaccine development to trigger an increase in the coverage of the expansion of protection can be done through B cell polytope. This study aims to provide an important preliminary for the construction of vaccine candidates by identifying the peptides that make up the B cell polytope in the HIV-2 envelope glycoprotein region. The HIV-2 sequence was obtained from the database. The study followed by 3D modelling, prediction of linear B-cell epitope mapping, antigenicity, allergenicity, peptide properties, and immune simulation was carried out via a webserver. The 3D structure of the peptide was displayed through molecular visualization software. The results showed that the 23-mer peptides E1 'HPRYTGVKNIRDITLTEPGRGSD', F1 'NFIENRKGTQHN' 12-mer, M1 'YLKDQARLNS' 10-mer, N1 'PWVNDSIQPNWNNMTWQQWEL QVRD' 25-mer, and O1 'KLQNSWNMGVQTO' can be used as a candidate polytope HIV-2 vaccine because it is recognized by B cells is an antigenic peptide with stable molecule, non-allergenic. The peptides trigger proliferation and activation of B cells to produces a humoral response and work as functionally protective antibody for neutralization of HIV-2.


Introduction
The first cases of human immunodeficiency virus type 2 (HIV-2) infection were identified in West Africa, affecting 2 million people in 1985, but then the case have also been found in France, Portugal, USA, and India. HIV-2 is predicted to be associated with viruses derived from the primate Sooty Mangabey, in contrast to the type 1 virus (HIV-1) originated from gorillas and chimpanzees 1 . New research reveals that HIV-2 has lower rates of transmission and virulence than HIV-1, but that HIV-2 also triggers acquired immune deficiency syndrome (AIDS). HIV-1 consists of viral groups M, N, O, P, with the M group consists of 12 subtypes and has the highest prevalence rate since the virus was first discovered in 1920. Since 2010, HIV-2 has been identified as having eight groups of AH 2 .
HIV-2 has a lower level of pathogenicity than HIV-1 and the mechanism that causes the low virulence of HIV-2 has not been found, and the antiretroviral drugs can be used only in HIV-1 3 . HIV-2 is resistant to non-nucleoside reverse transcriptase inhibitors (NNRTI) antiviral type by a unknow mechanism. The evidence base for HIV-1 and HIV-2 coinfection has not been found, but many scientists predict the possibility of this mechanism because the two viruses can have symbiosis to strengthen the pathogenicity and level of resistance to antiretroviral drugs 4 .
The development of HIV vaccines with various types such as DNA, virus-like particles, recombinant subunits, mRNA with clinical trials from phase 1 to 3, has been done since 1999 until present 5 . Research on HIV-1 vaccines such as Uhambo or HVTN 702 from Africa was discontinued in 2020 because they did not have effective efficacy in reducing viral load, but until now there are HIV-1 vaccine candidates such as the mRNA type (eOD-GT8 60mer) developed by Scripps Research & IAVI, Ad26.Mos4.HIV with envelope glycoprotein or viral vector vaccine type and subunit with the aim of increasing protection from global strain infection with HIV-1 strain from 2020 until now currently undergoing phase 3 clinical trial 6 . However, from various information on clinical trials of HIV vaccines, no research information was found aimed at developing an HIV-2 vaccine.
From the development progress of the HIV-1 vaccine to the clinical trial phase 3, there is also a subunit-type vaccine through the envelope glycoprotein, the use of the HIV-2 envelope glycoprotein in this study refers to the previous type of HIV-1 vaccine. Viroinformatics is a research approach that combines the fields of Bioinformatics with Virology to explore solutions that produce a prediction for solving problems related to viruses 7 . The combination of several peptides that make up the epitope that provides an extension of antibody neutralization and recognition of immune cells against pathogens is called a polytope 8 . The viroinformatics approach is possible as a one step in the development of the HIV-2 vaccine, in this study screening of predicted polytope or region to produce an effective vaccine candidate peptide and provide a protective immune response against HIV-2 infection

Sample Preparation
The HIV-2 envelope protein sequence with ID ADH04368.1 was obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/), the sequence consists of three regions, namely envelope, gp120, and transmembrane protein 9 . This study used the entire envelope of the HIV-2 virion, the sequence on the envelope having a length of 830-mer.

Protein modelling
The 3D structure of the HIV-2 gp120 envelope model construction and validation were carried out via the SWISS-MODEL server (https://swissmodel.expasy.org/). SWISS-MODEL works based on the identification of the similarity of the query sequence with the template to generate model construction. Homologous models were identified with a score of 20% and a validation score of the Ramachandran plot of 80% 10,11 .

B-cell linear epitope mapping
Recognition of B cell epitope on HIV-2 gp120 envelope via IEDB Analysis Resource server (http://tools.iedb.org/bcell/) using BepiPred 2, Kolaskar-Tongaonkar, and Emini Surfaces Accessibility methods. The B cell epitope probability score was generated by calculating the propensity scale, Hidden Markov Model, and predicting the physicochemical properties of the amino acids that make up the peptide. In the graph, the prediction results show that the peptide with the green area is a negative result as a B cell epitope while yellow is a positive prediction 12 .

Immune simulation of vaccine candidate
Stable peptides were simulated via C-ImmSim (http://kraken.iac.rm.cnr.it/C-IMMSIM/). Simulationbased on the ability of vaccine candidate peptides when triggering humoral immunity such as the amount of B cell production and antibody secretion in human. This study uses the default MHC allele with simulation step parameters 1000, 100 volume, Random Seed 12345 on C-ImmSim 14 .

Results
The Model Identification of Envelope gp120 HIV-2

The Model Identification of Envelope gp120 HIV-2
The 3D structure construction is carried out using the homology modelling method, the model with a similarity value of 20% is homologous to the template 15,16 . The homologous protein model has an abundance of conserved regions and allows 85% to 90% to be close to accurate, then the model is validated through the Ramachandran plot with consideration of a score of 80% for the favored region 17 . The results showed that the HIV-2 gp120 envelope protein modelling sequence (ID ADH04368.1) had a similarity of 37.04% and a score of 87.85% favored region and 0% bad bond. Visualization of the 3D envelope gp120 HIV-2 model was performed using EduPyMol software with cartoons structure and transparent surfaces (Figure 1).

Prediction of Peptide Vaccine Candidate of HIV-2
The HIV-2 envelope glycoprotein allows it to be recognized as an epitope and triggers B cell activation via a direct pathway. B cell epitope prediction using the BepiPred 2 method works based on the Random Forest algorithm calculation with a threshold of 0.500 to determine the probability of the epitope in the antigen query sequence 18 . The Emini Surfaces Accessibility method is used to predict B cell epitopes by calculating the probability of amino acids on the surface of the query antigen sequence with 1,000 as the threshold 19 . Prediction of B cell epitope through the Kolaskar-Tongaonkar method based on the calculation of the physicochemical probability of each amino acid making up the epitope with an accuracy of 75% 13 . The results showed that the peptides obtained from the predicted B cell epitope were twelve with label 1, five for peptides from Emini Surfaces Accessibility with label 2, and nine for peptides from Kolaskar-Tongaonkar with label 3, the total number of all peptides was twenty-six. with different lengths and positions on the HIV-2 gp120 envelope (Table 1). Positive predictions with the plot region being above the threshold as the epitope of cell B are shown in the graph in yellow and negative or the region is below the threshold (Figure 2).
Epitope composing peptides derived from positive predictions of the three methods consisting of BepiPred 2, Emini Surfaces Accessibility, and Kolaskar-Tongaonkar were identified as antigenic peptides through VaxiJen. The server works in determining antigen scores by calculating cross-covariance (ACC) on protein sequences with an accuracy of about 70% to 89%, the target organisms consist of bacteria, viruses, and tumors 20,21 . Then identification of allergenicity was carried out on antigenic peptides through AllerTop using the ACC method to explain the physicochemical properties of the amino acid composition to determine the categories of allergens and non-allergens. The results of this study show (Table 2).
Antigenic peptides with non-allergenic properties are identified through PortParam, as this server is used to calculate chemical and physical parameters on vaccine candidate peptides. The parameters used in this study are theoretical pi, aliphatic index, grand average of hydropathicity (GRAVY), and instability index 22 . The results of this study showed that there were five stable vaccine candidate peptides (Table 3.), then the 3D structure of the peptides was displayed using EduPyMol software with cartoons structure and transparent surfaces with publication standard staining (Figure 3).

Immune Simulation of HIV-2 Vaccine Candidate
When the HIV-2 viral entry mechanism occurs in host cells, the envelope glycoprotein or gp120 has an important role for attachment to the CD4+ receptor 23 . The design of the HIV-2 vaccine candidate with identification of the epitope on gp120 allows for the construction of antigenic peptides that trigger B cell recognition and immune response. The results showed that a polytope consisting of peptides E1, F1, M1, N1, and O1 could trigger the proliferation of B cell clones with IgM antibody isotypes and the activation process with a total population of 450 cells per mm3 (Figure 4). Polytope can be categorized as a good vaccine candidate because it can trigger a humoral response to neutralize HIV-2 infection.