The Selection Of Candidate Vaccine Of Live-attenuated Respiratory Syncytial Virus

human respiratory syncytial virus is one of the most important pathogens that can cause viral lower respiratory tract illness in infants, children, elderly and the patients with immune deficiencies. The development of vaccine against respire- atory syncytial virus has been a first-class plan by wolde health organization. In all kinds of vaccines, a live attenuated RSV vaccine can provide the best prote- ction of respiratory tract. So we decided to make RSV mutant by cold-passing and mutagen. Second, clone a strain of virus by plaque purification. Finally, testing the ability of causing illness and inducing immune respond by inoculate- ing in animal model. At last, we can get the candidate vaccine with completely attenuated and enough immunogenicity. The research include two aspects.Basic researches about RSVIn this part, some characteristics of RSV were researched. At first, four kinds of cell that is used to inoculate RSV were compared. RSV can grow in KMB₁₇ cell,Vero cell,Hep-2 cell and Hela cell. We inoculated RSV into the these cell with the same conditions, then the pathological change of the cell is obserced and recorded during culturing. The result shows RSV cause the appearance of pathological changes on 6th day in KMB₁₇ cell, 75% of cell is observed pathological changes on 12th day. no clear morphology of syncytial cell can be observed with the infectious titer of 6.13 lg CCID₅₀/ml. At the same, RSV cause the appearance of pathological changes on 3th day in Vero cell, 75% of cell is observed pathological changes on 8th day. some clear morphology of syncytial cell can be observed with the infectious titer of 6.83 lg CCID₅₀/ml. the patho- logical changes appear on 5th day in Hep-2 cell and Hela cell, 75% of cell is observed pathological changes on 9th day. the morphology of syncytial cell can be observed is not signigicant as changes in KMB₁₇ cell. The infectious titer reach to 6.83 lg CCID₅₀/ml. So it is clear that RSV shows low sensitivity to cult- uring in KMB₁₇ cell whereas it can quickly cause pathological changes and reach to high level of infectious titer in Vero cell.Sometimes, the growth of RSV is observed a large variation correlated with the time of adsorption in inoculating and the pH of culture medium. This study discusses the two aspects. Firstly, RSV is cultured after adsorb 0min, 30min, 60min, 90min with four kind of cell. The effect of pathological changes in four kind of cell is observed. The result shows no difference at the time of pathol- ogical changes after adsorption. Then the features that RSV grows in different pH have been detected. In the series of pH6.4～ph7.6, it can be obserbed that RSV causes more rapid pathological changes with the rise of pH in some range, and reach to a high level of infectious titer as 6.63 lg CCID₅₀/ml in pH7.2. Meanwhile, it can be observed that cell become round and small to die as pH is higher than pH7.0, when cell appears obviouse syncytial morphology as pH is lower than pH7.0.Except using CPE test RSV, the electrical microscope slice and indirect immunofluorescence assay also were used to test RSV. The cell with obvious pathological changes was made to the electrical microscope slice. In high power field, the pathological changes of cell and some structure similar as RSV can be observed in the slice. With the immunofluorescence, the information about the distribution of pathogen can be presented, and the growth of RSV can be analy- sed from other sides. There is much immunofluorescence that can be detected in Vero cell.Animal model of RSV also was researched. One side, Inoculating RSV to the brain of sulking mouse, brain and lung is made as pathological section on the 14th day. Resulte demonstrate that sulking mouse were all survived after inocu- lating, and no inflammation in the brain and lung. It indicates that the brain of mice isn't the target organ attacked by the A₂ strain. On the other side, Inocul- ating RSV through the nasal tract of guinea-pig. Killed on the 7th and the 14th day. Heart, liver, spleen, lung and kidney are made as pathological section. Detecting the level of antibody against RSV in serum. Separating virus from the lung tissue of all guinea-pig. The result is that the weight of inoculated guinea -pig is lighter than the-control-group on the 14th day, their breath is abnormal. Interstitial pneumonia emerged in all the guinea-pig. Serum titer is 1:4. The average titer of virus isolated is 4.0 lg CCID₅₀/ml. it indicates that guinea-pig can be used as the animal model of virulence changes about RSV.Cold-passed and the detection of virulence changesThe research is based on the principle that temperature sensitivity feature can be formed by cold-passed inducing. So the developmental route is that passing RSV in KMB₁₇ cell at lower temperature. Detecting the temperature sensitivity feature by T rct. Up to now, RSV has been passed 5 passages at 37℃, the infectious titer fluctuate around 6.0 lg CCID₅₀/ml, these virus can be used at positive contrast in the future. At 32℃, RSV has been constantly passed 20 passages. The time history of pathological changes induced by per-passages virus also presente a large fluctuation, the infectious titer stay also around 6.0 lg CCID₅₀/ml, every passage doesn't have obvious temperature sensitivity feature in the T rct detection.Choosing cold-passed 6th and 11th passage virus to inoculate in guinea-pig. Detecting their virulence, then compared with the model by the primal virus inducing. The result shows that the weight of guinea-pig inoculated the 6th and 11th passages virus are similar to the-control-group, and their breath is nomal. Interstitial pneumonia emerged in all the guinea-pig. There is no significant difference among three passages. The 6th passage virus induces a serum titer of 1:8 while the 11th passage induces 1:4 as the prime virus. The average tite of virus isolated is also 4.0 lg CCID₅₀/ml. three passges virus have a same feature that the lung of guinea-pig is infiltrated by inflammatory cells as flake and nod- ules at 7th day, infiltrated by inflammatory cells as diffusion at 14th day. there is one guinea-pig, its'peribronchiolar mononuclear cell infiltrates accompanied by submucosal edema and bronchorrhea that lead to bronchiolar obstruction with patchy atelectasis and areas of compensatory emphysema. Maybe this condition is related to the difference between individuals. Generally, these three passages virus haven't been attenuated, RSV need to be cold-passed more passages.Above all, an the first aspect some basic research explored some features of RSV, we can optimize the culture condition based on these information for develop successful vaccine. The second aspect research mainly analysed the temperature sensitivity feature and virulence changes of cold-passed virus. And the results shows RSV dosen't have temperature sensitivity feature, and also haven't been attenuated after 20 passed. Contrast with some good candidates vaccine completely attenuated in the world, the virus need to be passed more passages at lower temperature, and the using of mutagen need to be considered. In this way, virus cloned by plaque purification will is suitable as candidate vaccine. So, the research only is a prime preparative for the development of RSV live attenuated vaccine. There are a lot of researchs to accomplish in the future.