Immunogenicity and Neutralization of Recombinant Vaccine Candidates Expressing F and G Glycoproteins against Nipah Virus

Abstract

Nipah virus (NiV), of the Paramyxoviridae family, causes highly fatal infections in humans and is associated with severe neurological and respiratory diseases. Currently, no commercial vaccine is available for human use. Here, eight structure-based mammalian-expressed recombinant proteins harboring the NiV surface proteins, fusion glycoprotein (F), and the major attachment glycoprotein (G) were produced. Specifically, prefusion NiV-F and/or NiV-G glycoproteins expressed in monomeric, multimeric (trimeric F and tetra G), or chimeric forms were evaluated for their properties as sub-unit vaccine candidates. The antigenicity of the recombinant NiV glycoproteins was evaluated in intramuscularly immunized mice, and the antibodies in serum were assessed. Predictably, all homologous immunizations exhibited immunogenicity, and neutralizing antibodies to VSV-luciferase-based pseudovirus expressing NiV-GF glycoproteins were found in all groups. Comparatively, neutralizing antibodies were highest in vaccines designed in their multimeric structures and administered as bivalent (GMYtet + GBDtet) and trivalent (Ftri + GMYtet + GBDtet). Additionally, while all adjuvants were able to elicit an immunogenic response in vaccinated groups, bivalent (GMYtet + GBDtet) and trivalent (Ftri + GMYtet + GBDtet) induced more potent neutralizing antibodies when administered with oil-in-water nano-emulsion adjuvant, AddaS03. For all experiments, the bivalent GMYtet + GBDtet was the most immunogenic vaccine candidate. Results from this study highlight the potential use of these mammalian-expressed recombinant NiV as vaccine candidates, deserving further exploration.

Keywords: Nipah virus; antigenicity; pseudotype neutralization assay; recombinant vaccine.

Figure 1

Production and confirmation of recombinant NiV proteins. The recombinant NiV F and G glycoproteins were expressed from the mammalian expression system. (a) Schematic representation of the plasmid constructs for NiV recombinant protein expression. (b) SDS-PAGE of recombinant NiV F and G glycoproteins. Image was created in Biorender.com with publication and licensing rights. (c) Western blot of recombinant NiV F and G glycoproteins. M, molecular size marker; P, multiple-tag positive control (GenScript, Cat. No. M0101, China); R, reducing condition; NR, non-reducing condition; Fm, F glycoprotein monomer; Ftri, F glycoprotein trimer; GMYm, G glycoprotein derived from Malaysia strain monomer; GBDm, G glycoprotein derived from Bangladesh strain monomer; GMYtet, G glycoprotein derived from Malaysia strain tetramer; GBDtet, G glycoprotein derived from Bangladesh strain tetramer; c, chimeric.

Figure 1

Production and confirmation of recombinant NiV proteins. The recombinant NiV F and G glycoproteins were expressed from the mammalian expression system. (a) Schematic representation of the plasmid constructs for NiV recombinant protein expression. (b) SDS-PAGE of recombinant NiV F and G glycoproteins. Image was created in Biorender.com with publication and licensing rights. (c) Western blot of recombinant NiV F and G glycoproteins. M, molecular size marker; P, multiple-tag positive control (GenScript, Cat. No. M0101, China); R, reducing condition; NR, non-reducing condition; Fm, F glycoprotein monomer; Ftri, F glycoprotein trimer; GMYm, G glycoprotein derived from Malaysia strain monomer; GBDm, G glycoprotein derived from Bangladesh strain monomer; GMYtet, G glycoprotein derived from Malaysia strain tetramer; GBDtet, G glycoprotein derived from Bangladesh strain tetramer; c, chimeric.

Figure 2

Immunogenicity of the NiV-F and NiV-G vaccine candidates in BALB/c mice. (a) Schematic timeline of the immunization and serum collection schedule in mice. Mice were vaccinated intramuscularly twice at two-week intervals, and the serum was collected two and five weeks post-booster vaccination to determine humoral response by ELISA. Image was created in Biorender.com with publication and licensing rights. Specific IgG antibodies for (b) NiV-F, (c,d) NiV-G, and (e) NiV-F/G. The specific antibodies in the serum (n = 8) were measured by ELISA, and the values are presented as mean absorbance at 450 nm (OD 450 nm) ± SD. Statistical significance between groups for each time point collection (2 and 5 weeks post-booster) was performed using unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Immunogenicity of the NiV-F and NiV-G vaccine candidates in BALB/c mice. (a) Schematic timeline of the immunization and serum collection schedule in mice. Mice were vaccinated intramuscularly twice at two-week intervals, and the serum was collected two and five weeks post-booster vaccination to determine humoral response by ELISA. Image was created in Biorender.com with publication and licensing rights. Specific IgG antibodies for (b) NiV-F, (c,d) NiV-G, and (e) NiV-F/G. The specific antibodies in the serum (n = 8) were measured by ELISA, and the values are presented as mean absorbance at 450 nm (OD 450 nm) ± SD. Statistical significance between groups for each time point collection (2 and 5 weeks post-booster) was performed using unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Neutralization of VSV-luciferase-NiV-pseudovirus. Neutralization assays were performed on individual mouse sera collected at five weeks post-booster vaccination from mice vaccinated with recombinant NiV vaccine harboring NiV-pre-F and NiV-G glycoprotein in (a) monomeric structure, (b) multimeric structure, and (c) chimera, either administered as monovalent or multivalent. The IC50 for each sample were calculated by curve fitting and non-linear regression in GraphPad Prism, and indicated in Log10 values. Statistical significance between monovalent and multivalent groups for each vaccine structure (monomer, multimer, and chimera) was performed using one-way ANOVA and Tukey’s multiple comparison test. Each shape within group represent individual values of mouse sera, bars indicate mean values of the group, and error bars are ± standard deviation. ** p < 0.001, *** p < 0.001.

Figure 4

Immunogenicity of vaccines with different adjuvants. (a) Schematic timeline of the immunization and serum collection schedule in mice. Mice were vaccinated intramuscularly twice with bivalent (GMYtet + GBDtet) and trivalent (Ftri + GMYtet + GBDtet) in different adjuvants at two-week intervals, and the serum was collected two weeks post-booster vaccination to determine humoral response by ELISA. Image was created in Biorender.com with publication and licensing rights. Specific IgG antibodies for (b) NiV-G and (c) NiV-F. The specific antibodies in the serum (n = 6) were measured by ELISA at two weeks post-booster immunization, and the values are presented as mean absorbance at 450 nm (OD 450nm) ± SD. Statistical significance between groups was performed using one-way ANOVA, and Tukey’s multiple comparison test was used to compare significance compared to the control (++ p < 0.01; +++, p < 0.001) and between groups (* p < 0.05). (d) Neutralization assays were performed on sera collected at two weeks post-booster vaccination from vaccinated mice. Each shape within group represent individual values of mouse sera, and error bars are ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.