Design and Preliminary Immunogenicity Evaluation of Nipah Virus Glycoprotein G Epitope-Based Peptide Vaccine in Mice

Abstract

Background: The emergence of several paramyxoviruses, including Nipah virus (NiV), makes continued efforts in vaccine development as part of pandemic preparedness efforts necessary. Although NiV is a zoonotic pathogen with high case fatality, there is still no licensed vaccine. Methods: Herein, NiV attachment glycoprotein G (NiV-G), which is crucial to host cell receptor binding, was used to develop Nipah epitope-based peptide vaccines. A total of 39 B- and T-cell epitopes of NiV-G were shortlisted for peptide synthesis and evaluation using in silico analysis. Results: The in vitro antigenicity evaluation of the peptide candidates showed eight synthesized peptides (G7, stalk-domain epitopes) with relatively high binding to NiV-G antibody-positive serum (A_450nm: 1.39-3.78). Moreover, nine-mer (9-mer) peptides were found to be less reactive than their longer peptide counterparts (15-30 aa, G7-1, and G7-4), but 9-mer activity was enhanced with cyclization (NPLPFREYK, A_450nm: 2.66) and C-terminal amidation modification (NPLPFREYK-NH2, A_450nm: 1.39). Subsequently, in vivo validation in immunized mice revealed the immunogenicity potential of the G7-1 peptide vaccine (30 aa, NENVNEKCKFTLPPLKIHECNISCPNPLPF) to elicit a strong antigen-specific antibody response against their homologous peptide antigen (I.V., A_450nm: 1.48 ± 0.78; I.M., A_450nm: 1.66 ± 0.66). However, antibody binding to recombinant NiV-G protein remained low, suggesting limited recognition to the native antigen. Conclusions: This study focused on the preliminary screening and validation of peptide vaccines using single formulations with minimal modifications in the peptide candidates. Our findings collectively show the immunogenic potential of the NiV-G stalk-based epitope peptide vaccine as a novel therapeutic for NiV and underscores the need for strategic design, delivery, and formulation optimization to enhance its protective efficacy and translational application.

Keywords: Nipah virus; attachment glycoprotein G; epitopes; peptide vaccine; vaccine.

Figure 3

Preliminary antigenicity evaluation of synthesized NiV-G peptide vaccine candidates. The synthesized NiV-G candidate peptide candidates were used as capture antigens in microplates, and a routine ELISA was performed on NiV-G-positive serum. The specific binding of the antibody to the (a) G1, (b) G14, (c) G7, (d) G11, and (e) G17 synthesized peptides was measured in triplicates, and the data are presented as values of mean absorbance at 450 nm (A_450nm). ND: Not detected.

Figure 1

Schematic diagram of the overall methodology employed to design and develop NiV-G epitope-based peptide vaccines. The image was created in Biorender.com with publication rights.

Figure 2

In silico analysis of NiV-G sequence for epitope predictions. (a) B-cell epitope prediction score of NiV-G sequence performed by using BepiPred-2.0 at threshold of 0.5. Yellow colors indicate amino acid position over threshold value and indicate high antigenicity. (b) Individual BepiPred-2.0 residue scores of NiV-G amino acid sequence. B-cell epitope groups with over > 9 amino acid residues are highlighted in color and marked with their designated groups. Green colors indicate BepiPred residue score. (c) Location of B-cell and T-cell epitope groups in predicted NiV-G protein structure in Alphafold. Protein was visualized in Mol 3D viewer and adjusted. Ball and sticks in yellow indicate specific position of residues of groups. (d) HLA Class I immunogenicity scores and amino acid sequences of highly ranked 9-mers for each group. G1, Group 1; G7, Group 7; G11, Group 11; G14, Group 14; G17, Group 17.

Figure 2

In silico analysis of NiV-G sequence for epitope predictions. (a) B-cell epitope prediction score of NiV-G sequence performed by using BepiPred-2.0 at threshold of 0.5. Yellow colors indicate amino acid position over threshold value and indicate high antigenicity. (b) Individual BepiPred-2.0 residue scores of NiV-G amino acid sequence. B-cell epitope groups with over > 9 amino acid residues are highlighted in color and marked with their designated groups. Green colors indicate BepiPred residue score. (c) Location of B-cell and T-cell epitope groups in predicted NiV-G protein structure in Alphafold. Protein was visualized in Mol 3D viewer and adjusted. Ball and sticks in yellow indicate specific position of residues of groups. (d) HLA Class I immunogenicity scores and amino acid sequences of highly ranked 9-mers for each group. G1, Group 1; G7, Group 7; G11, Group 11; G14, Group 14; G17, Group 17.

Figure 4

Assessment of immunogenicity of NiV-G peptide vaccine candidates in BALB/c mice. (a) Illustration of immunization protocol timeline and sampling schedule for in vivo study in mice. Specific pathogen-free (SPF) mice received peptide vaccinations either intramuscularly (I.M.) with Alhydrogel^® adjuvant or intravenously (I.V.) on two occasions, spaced two weeks apart. Individual mouse serum was collected two weeks post booster vaccination to evaluate humoral immune response with ELISA. Image was generated in Biorender with publication license. (b) Mouse serum validation against epitope peptide antigens. Individual serum from vaccinated groups was analyzed for specific antibodies against their homologous NiV-G epitope peptide vaccine antigen. Data are presented as values of mean absorbance at 450 (A_450nm) ± SDs. +, p < 0.05 against group. Statistical significance between groups was analyzed by using Mann–Whitney test. (c) In vivo immunogenicity evaluation against NiV-G. Individual mouse serum (n = 4) was evaluated for specific antibody binding against NiV-G (REC31637). Results are expressed as values of mean absorbance at 450 (A_450nm) ± SDs. Statistical significance on all groups was determined by using Kruskal–Wallis test followed by Dunn’s multiple comparison test. *, p < 0.05 against negative control (NC); +, p < 0.05 against positive control (PC). Each data point represented by a shape within a group in ELISA figures corresponds to serum value from an individual mouse.

Figure 5

The location of the G7-1 peptide vaccine in the protein structure of NiV-G. The protein structure was retrieved from PDB (7TXZ), and the protein is visualized as its molecular surface representation. Highlighted in yellow is the specific location of G7 groups, and the specific ball and stick representation in yellow is the G7-1 peptide vaccine. The inset shows a simplified representation of protein in a cartoon representation with folding and specific amino acid residues with their specific position number.