Influence of temperature on the antigenic changes of virus-like particles

Abstract

Purpose: In this study, we investigated whether the antigenic changes of the virus-like particles (VLPs) are affected by the temperature during storage.

Materials and methods: After exposing the recombinant influenza VLPs to various temperatures for a period, antigenic changes were examined through in vitro hemagglutination receptor binding assay and in vivo mouse experiments.

Results: Influenza VLPs were exposed at three different temperatures of low, middle, and high on a thermo-hygrostat. High temperature exposed influenza VLPs were showed significantly reduced HA activity and immunogenicity after mouse single immunization over time compared low and middle. When the VLPs exposed to the high temperature were inoculated once in the mice, it was found that the immunogenicity was significantly reduced compared to the VLPs exposed to the low temperature. However, these differences were almost neglected when mice were inoculated twice even with VLPs exposed to high temperatures.

Conclusion: This study suggests that similar protective effects can be expected by controlling the number of vaccination and storage conditions, although the antigenic change in the VLP vaccines occurred when exposed to high temperature.

Keywords: Antigenicity; Temperature; Virus-like particle vaccines.

Fig. 1. Characterization of influenza PR8 VLPs. (A) Schematic diagram of influenza PR8 VLPs expressed HA and M1 proteins. (B) Negative stain electron microscopy of influenza PR8 VLPs (C) Western blot of HA (75 kDa) and M1 (25 kDa) proteins expressed in PR8 VLPs. VLPs, virus-like particles; HA, hemagglutinin.

Fig. 2. Experimental design to determine antigenic change according to the storage temperature of influenza VLPs. Influenza VLPs were stored in low, middle, and high temperature conditions, and after 1 day (D1), 7 days (D7), and 1 month (M1), the in vitro antigenicity was measured by HA assay, and in vivo immunogenicity was measured through animal experiments. VLPs, virus-like particles; HA, hemagglutinin.

Fig. 3. Measurement of HA activity change of influenza VLPs stored at different temperature conditions. PR8 VLPs of the same amount and volume were stored for 1 day (A), 7 days (B), and 1 month (C) under low (4℃), middle (25℃), and high (42℃) temperature conditions and HA activity was measured by HA assay. VLPs, virus-like particles; HA, hemagglutinin. ^*p<0.05 and ^**p<0.01; significant differences (Student t test).

Fig. 4. Influenza A/PR8 virus specific IgG responses. Group of mice (n=6) were intramuscularly immunized with 2.6 µg of PR8 VLPs stored at low (A), middle (B), and high (C) temperature conditions. Blood sample were collected from PR8 VLP immunized mice at 3 weeks after immunization. VLPs, virus-like particles; HA, hemagglutinin; IgG, immunoglobulin G; OD, optical density. ^**p<0.01; significant differences (Student t test).

Fig. 5. Influenza A/PR8 virus specific IgG responses after boost immunization with PR8 VLPs stored at different temperature conditions. Mice were intramuscularly immunized on days 0 and 21 with PR8 VLPs stored at low (A), middle (B), and high (C) temperature conditions. Sera were collected from PR8 VLP immunized mice at 3 weeks after last immunization. VLPs, virus-like particles; HA, hemagglutinin; IgG, immunoglobulin G; OD, optical density.

Fig. 6. Influenza A/PR8 virus specific immunoglobulin G responses after boost immunization with PR8 VLPs stored at different temperature conditions. Mice were intramuscularly immunized on days 0 and 21 with PR8 VLPs stored at low (A), middle (B), and high (C) temperature conditions. Sera were collected from PR8 VLP immunized mice at 3 weeks after last immunization. VLPs, virus-like particles.