Construction of a recombinant vaccine expressing Nipah virus glycoprotein using the replicative and highly attenuated vaccinia virus strain LC16m8

Abstract

Nipah virus (NiV) is a highly pathogenic zoonotic virus that causes severe encephalitis and respiratory diseases and has a high mortality rate in humans (>40%). Epidemiological studies on various fruit bat species, which are natural reservoirs of the virus, have shown that NiV is widely distributed throughout Southeast Asia. Therefore, there is an urgent need to develop effective NiV vaccines. In this study, we generated recombinant vaccinia viruses expressing the NiV glycoprotein (G) or fusion (F) protein using the LC16m8 strain, and examined their antigenicity and ability to induce immunity. Neutralizing antibodies against NiV were successfully induced in hamsters inoculated with LC16m8 expressing NiV G or F, and the antibody titers were higher than those induced by other vaccinia virus vectors previously reported to prevent lethal NiV infection. These findings indicate that the LC16m8-based vaccine format has superior features as a proliferative vaccine compared with other poxvirus-based vaccines. Moreover, the data collected over the course of antibody elevation during three rounds of vaccination in hamsters provide an important basis for the clinical use of vaccinia virus-based vaccines against NiV disease. Trial Registration: NCT05398796.

Fig 1. Genome structure of the recombinant LC16m8 expressing NiV surface glycoproteins.

Schematic diagrams of the procedure for constructing recombinant LC16m8 (upper) and genome structures of the foreign genes inserted into the LC16m8 genome (lower). Plasmid pRecB5R.1 expresses the mCherry and XGPRT genes under the vaccinia early and late promoter (P) and contains the homologous sequences that flank the B5R gene (UP and DOWN). The genes of interest (GOIs), which were cloned into the pRecB5R.1, were transferred to the LC16m8 genome through homologous recombination. The selection marker genes XGPRT and mCherry were self-excised from the intermediate recombinant viruses by intragenomic homologous recombination between the UP and shorter sequence homologous to UP (U’). The dotted lines indicate the expected locations of the homologous recombination sites. The final recombinant viruses express the NiV G or F gene, while the complementary genome DNA expresses the EGFP gene.

Fig 2. Expression and function of the envelope glycoproteins of NiV.

(A and B) RK-13 cells were infected with LC16m8-G or LC16m8-F at an MOI of 0.01. At 40 hpi, the cells were fixed and stained with a polyclonal antibody against NiV G protein (A) or F protein (B), followed by incubation with Alexa Fluor 488-conjugated anti-rabbit secondary antibody. The stained cells were observed under a phase-contrast or fluorescence microscope. (C) RK-13 cells were transfected with an expression plasmid driven by the vaccinia early and late promoter (pRecB5R.1-NiV-G, pRecB5R.1-NiV-F, or pRecB5R.1). At 24 h posttransfection, the transfectant cells were infected with LC16m8-G or LC16m8-F at an MOI of 0.01. At 24 hpi, the formation of syncytia was observed under a fluorescence microscope.

Fig 3. Induction of neutralizing antibodies against NiV.

(A) Experimental design and schedule of inoculations with LC16m8 and the recombinant LC16m8s. Seventeen hamsters were intramuscularly inoculated with 1×10⁶ PFU of the LC16m8. At 4 weeks, three groups of animals consisting of 4–7 hamsters each were inoculated with 5×10⁶ PFU of the LC16m8-G, 2.5×10⁶ PFU each of LC16m8-G and LC16m8-F, or the growth medium (negative control) as a prime immunization. Half of each group (2–4 hamsters) were further inoculated with the same recombinant viruses as a boost immunization. Two weeks after the final dose of the recombinant LC16m8, all hamsters were euthanized, which was followed by serum collection. Neutralizing titers were analyzed using live NiV (B) or pseudo-typed VSV expressing secreted alkaline phosphatase (SEAP) bearing NiV G and F proteins (VSV-NiV-SEAP) (C). (D) Experimental design and schedule of inoculations with the recombinant LC16m8s. Single recombinant virus (LC16m8-G or LC16m8-F, 5×10⁶ PFU/hamster) or a mixture of the viruses (2.5×10⁵ PFU each) was intramuscularly administered to hamsters once or twice at a 2-week interval without pre-immunization of LC16m8. Two weeks after the last inoculation with the recombinant virus, sera were collected from all hamsters. Neutralization titers were determined based on the assay using live NiV (E) or the VSV-NiV-SEAP (F). Bars show the mean values.

Fig 4. Effective induction of neutralizing antibodies by three immunization cycles with the recombinant LC16m8s in the VV-preimmunized hamsters.

(A) Experimental design and schedule of inoculations with LC16m8 and the recombinant LC16m8s. Twenty-seven hamsters were intramuscularly inoculated with 1×10⁶ PFU of the LC16m8. At 4 weeks, three groups of hamsters (n = 8, 9 or 10) were inoculated with 2×10⁶ PFU of the LC16m8-G (Group 1: VV-G-single-site), 2×10⁶ PFU of the LC16m8-F (Group 2: VV-F-single-site) or both viruses (1×10⁶ PFU each, onto two sites) (Group 3: VV-G-F-separate-sites). Two weeks after infection with the recombinant LC16m8s, one-third of the hamsters in each group were inoculated once or twice at a 2-week interval with the same recombinant viruses as the prime inoculation. Two weeks after the last shot, a serum sample was collected from each hamster. All groups of hamsters are shown with roman numerals in parentheses. Neutralizing titers were determined using live NiV (B). Each circled dot represents the titer (ND₅₀) of each serum sample. Dotted line and bars show mean ± SEM. A one-tailed Student’s t test assuming unequal variance was used for analyzing the data. ND₅₀ values below the LOD (<2) were assigned a value of 1. Antibody titers were also determined using the IFA method (C). BHK cells were transfected with the expression plasmid encoding NiV-G or NiV-F. At 24–36 hpi, the cells were fixed and reacted with dilutions of the hamster serum, followed by incubation with Alexa Fluor 488-conjugated anti-hamster secondary antibody. The stained cells were observed under a fluorescence microscope. IFA titers were defined based on the maximum serum dilution in which a fluorescence signal was detected, and the titers measured in BHK cells expressing NiV-G (BHK-G) or BHK cells expressing NiV-F (BHK-F) are indicated by a triangle or circle dot, respectively.