Effects Of High Hydrostatic Pressure On Infectivity And Immunity Of Influenza A Virus

Influenza viruses belong to the family of Orthomyxoviridae. It can cause influenza. Influenza viruses are negative–strand RNA enveloped-viruses involving the strains of influenza A, B and C virus. Influenza is a common epidemical respiratory illness in humans and pandemic disease all over the world. The scale of influenza is close correlate with the antigenic variation. Up to now there is not effective therapeusis of influenza. Vaccination still is the major countermeasure against its spread. At present there are three types of influenza vaccines in use: whole-virion particles; split vaccines; subunit vaccines. But attenuated vaccines are still on the trial stage. There are some problems in these vaccines, such as serious sub-responds, low protection sufficiency, complicated process and safety, so it is necessary to develop new alternative influenza vaccine. High hydrostatic pressure (HHP) means the pressure surpass 100Mpa (Mpa pascal, 1Mpa=10 atmospheres), in which life activity is greatly restrained. HHP interferes with the infectivity of some virus through changing their structure and but preserving their immunological properties. Many results show the existence of different susceptibilities to pressure among the virus. The degree of inactivation depends on the pressure and incubation time. Moreover, low temperature and a subdenaturing urea concentration would lead to an enhancement of inactivation process. The investigation of pressured-treated viruses structure was measured by transmission electron microscopy, spectroscopic data and a sucrose density gradient, indicating that the size and structure of treated particles similar to the native virus. The reason why the pressured viruses maintain the immunogenic potential probably resides in the fact that the structural changes are very subtle, without changes in covalent chemistry, and there is no loss of the antigenic determinant. The reversion of displacement of the capsid subunit after a decompression cycle is not complete, i.e. the virus particles do not return to the conformation of the native one. Such particles are no longer able to infect susceptible cells as efficiently as the native ones do. However, they retain many of the epitopes necessary to elicit a satisfactory antibody response at the immune system. High hydrostatic pressure has been investigated as a potential method for viral inactivation and for feasible vaccine production. We are intent to confirm the possible applications of pressurization to preparation of influenza vaccines by our experiment. We study the effect of HHP on infectivity of influenza FM1 virus in the first section of the experiment. Firstly, we treated FM1 in different pressure and time. Secondly, we test the TCID50 of pressure-treated FM1, and according to the changes of TCID50 find the rules of FM1 inactivation. Our results suggest that when the pressure and incubation time are 300Mpa, 2.5h and 600Mpa, 0.5h respectively, the influenza viruses are completely inactivated. The cause of the inactivation of influenza viruses is that the pressure induces the fusion-active state of HA, and inhibits the virus infecting the sensitive cells. It is interesting to notice, however, that the major drop in virus titer occurs in the first 10min under pressure. The different susceptibilities for pressure may be due to a genetic heterogeneity, and/or by a conformational plasticity inherent to the capsid proteins in a virus particle. We study on the immunity of pressure-treated FM1 by inoculating mice. ICR mice were divided into several groups including normal group, adjuvantgroup, heated group, native virus group, attenuation group(300Mpa,2h) and inactivation group(300Mpa,2.5h) and the mice were inculated two times with corresponding immunogens. We test the humoral immunological function by enzyme-linked immunoadsorvent assay (ELISA) and microculture neutralization test; we test the cellular immunity function by MTT lymphodyte transformation test and assay of NK cell; we test immunological function by thymus indexes and spleen indexes; the test of immunoprotecive is evaluated by lung indexes and the histologic changes in the lungs of mice challenged with influenza virus. The method of statistical analysis use analysis of variance and t test. The results of indirectly ELISA and microculture neutralization test show: the effective special humoral immunity response were induced by high pressure-treated FM1 in the mice, and serum antibody titer is up to 1: 20000 after enhance immunization, it is notable difference compared with normal group and adjuvant group; but it is not notable difference compared with heated group and native virus group. The antibody has some neutralization ablity, and its titer is 1:32. The results of T lymphocyte transformation test and NK cell active detect test show: lymphocytic stimulation indexes and NK cell activity of pressure-treated FM1 groups were notable difference compared with normal group and adjuvant group; and were not notable difference compared with heated group and native virus group. The mice of pressure-treated FM1 groups produce an effective cellular immune response. The results of thymus indexes and spleen indexes show: the spleen indexes of pressure-treated FM1 groups were notable difference comparedwith normal group and adjuvant group; and were not notable difference compared with heated group and native virus group. There is an increase tendency in the thymus index, but it was not notable difference. The mice of pressure-treated FM1 groups have strong immunological function. The results of immunoprotecive test show: the mice lung indexes of pressure-treated FM1 groups were notable difference compared with virus control group and adjuvant group, and was not notable difference compared with heated group. The fact that the histologic lung of the mice in pressure-treated FM1 group were less damage confirm their protection .The mice of pressure-treated FM1 groups produce effective immune response. All results in mice immune test show: immunogenicity of pressure-treated FM1 is not changed. This is because that the structural changes are very subtle without changes in covalent chemistry and there is no loss of the antigenic determinant, which is necessary for the proper immune response. The surface structure has small change when the virus is treated by high pressure. The high pressure can change space structure of protein antigen material, primary structure is reserved, the covalent bond can not be destructed. Antigenic determinant of high pressure virus is not losed, so treated particles were able to elicit antibodies against the native particle as well as the native particle itself. The study on effects of the infectivity and immunity of pressure-treated FM1 suggest that HHP can be used to inactiviate influenza virus and result in particles that good immunogens. Pressure treatment is a pure physical approach, compressed at moment, well-distributed, and lower consumption. The employment of high pressure is a relatively safe and easy procedure for...