Design and Synthesis of Peptide-conjugates as Vaccine Candidates for ZIKA Virus

Abstract

Zika virus was first identified in rhesus monkeys in 1947, but it has been known to infect humans since the 1960s. Due to its host, the Aedes mosquito, the virus circulating worldwide quickly, has lifted concerns over the safety of mankind. Any approved vaccine or drug is not available in the market against Zika infection. Microcephaly in newborns and Guillain-Barré syndrome in adults are major risks associated with Zika that mandate the development of vaccines to combat the virus. Hurdles in production, safety majors, insufficient financial resources, distribution and requirement of booster shots are prominent concerns in vaccine development. Therefore a vaccine strategy was needed to minimise these challenges along with a broad spectrum of protection from the Zika virus to worldwide population. Highly conserved peptide fragments related to critical viral proteins may be potent candidates for broadly reactive Zika vaccines that possess multiple immunogenic epitopes and the ability to interact with a wide range of HLA molecules. The peptide therapeutics usually confronts poor stability and insufficient antigen presentation obstacles, addressed by using adequate adjuvants with peptides. Polymer conjugates are promising vehicles for effective and safe vaccine development against infectious diseases. Antigen shelf stability, control release and presentation are prominent benefits of polymer conjugation with antigens. Due to biodegradability and hydrophilic, low-cost, flexible, and non-ionic properties, poly(ethylene glycol) (PEG) has attracted researchers for vaccine development strategies over the past few years. In addressing the above facts, the present study is oriented towards identifying highly conserved immunogenic peptides of the Zika virus precursor membrane protein (prM) using immunoinformatics techniques and in vitro validation of peptide and peptide conjugates for eliciting immune response. In silico approaches carried out includes different prediction algorithm in selecting peptides containing multiple epitopes having no undesirable response, and molecular docking and molecular dynamic simulation to analyse interaction of peptides with different HLAs. Further, the synthetically synthesized peptides were coupled with PEG to obtained prM peptide conjugates (MPC). Additionally, cell proliferation (MTT assay) and IFN-γ production (ELISA) of peripheral blood mononuclear cells (PBMCs), as a result of stimulation by peptides and peptide PEG conjugates, were performed. The phylogeny study demonstrated that there was conservation in the prM proteins during the years of the peak of the Zika virus outbreak (2015 – 2016). Mutational studies revealed less variation in the proteins except for position 17, where the mutational frequency was 0.209. Two highly conserved peptides were considered for epitope prediction. Three peptides (MP1, MP2 and MP3) which included both CD8+ and CD4+ T epitopes were selected with the use of different epitope predictions. The presence of B cell epitopes in the selected peptides was predicted, which was confirmed to contain two epitopes in MP1, three in MP2 and four epitopes in MP3 peptides. The peptides were found to be good antigenic along with being non-toxic and non-allergic. In addition, peptides had good physiochemical properties to be an antigen candidate. Population coverage analysis of selected peptides, in six different continents showed that population coverage was 60–99.8% (class I HLA) and 80–100% (class II HLA). Interaction analysis of HLA alleles with selected epitopes (HLA class I) and peptides (HLA class II) presented that most of the peptide-HLA (pHLA) complexes presented comparable docking scores and binding energies with natural bound peptide-HLA. Similarly, the bound pHLA complexes showed promising results on docking with TCR (T cell receptors) which were mostly found greater than natural bound peptide-HLA-TCR complexes which revealed good binding interactions of the peptides. Molecular dynamics simulation with best docked pHLA complexes showed that pHLA complexes were stable with RMSD <5.5A˚. Further, the selected peptides were conjugated with poly(ethylene glycol 400) (PEG 400) through coupling agent EDC. Conjugation of PEG 400 with peptides was confirmed by characterizing the obtained prM peptide conjugates (MPCs) by the Fourier transform infrared (FTIR) method due to the formation of ester bonding (C−O) between carboxylic and hydroxyl groups of peptides and PEG respectively. In in vitro validation, all peptides demonstrated influential results on cell proliferation and IFN-γ release. Peptides MP3 induced significant cell proliferation in eight samples, while MP2 and MP1 induced in six samples; similarly MP3 rendered significant IFN-γ release in seven samples, while MP2 and MP1 showed results in six samples out of 10 healthy volunteers’ blood samples. Additionally, all conjugates showed the significant upsurge in cell proliferation and cytokine production than their corresponding peptides. Conjugate MPC1 showed a significant increase in five samples (in cell proliferation) and six samples (in IFN-γ production) in comparison to MP1. Similarly, MPC2 and MPC3 showed differences in cell proliferation significantly in six samples and this difference in terms of IFN-γ release was found in five and seven of ten samples, respectively. Thus, peptides selected from the prM protein of the Zika virus through in silico and in vitro experimentations capable of eliciting immune response against the virus and conjugation of PEG with peptides could be an effective way to increase the peptide stability and immunogenicity.