| Influenza,or flu,is a disease caused by influenza virus.According to a report from WHO,in each flu epidemic season,about 5 to 15 percent of people suffer from flu.In the United States,the extra deaths toll caused by flu is about 25,000 each year.Presently,vaccination is recognized as the most effective and economical means for prevention of influenza,while influenza vaccine(split virion),including two type A strains,H1N1 and H3N2,and one type B strain,are the most widely used in the world.Considering that influenza viruses are liable to variation,the strains for production of influenza vaccines need to be changed dependent on the epidemic situations,and people need to be vaccinated every year accordingly.However,as pandemic influenza occurs,relevant vaccines may not be marketed timely because strain for vaccine production need to be prepared,and relevant references standards need to be developed,,thus it is difficult to provide effective protection for people at the early stage of viral prevalence.In addition,evaluation on influenza vaccine's quality is to be improved.On the surface of influenza virus envelope,there are two types of glycoproteins,i.e.hemagglutinin(HA)and neuraminidase(NA).Antibodies for both protect the body effectively,while the effect from HA is more significant.Therefore,at present HA content is taken as an indicator for active ingredients of influenza vaccine(split virion),which is detected by the method of Single Radial Immune Diffusion(SRID).But for NA antigen,there is only an activity test to prove its presence as lack of effective quantitative detection method,and also,there is no generally acceptable criterion for influenza vaccines.Thus,development of universal antibody against influenza virus became research hotspot in recent years.A universal antibody against several subtypes of influenza virus may protect the human body at first time as an important measure for prevention and control of influenza outbreaks.Furthermore,it may be used for quality control of both present influenza vaccines and newly developed influenza vaccines.In this study,new strategy was used for preparation of universal antibody against influenza virus.First,through a bioinformatics analysis,conservative sequences were screened out from almost all HA and NA amino acid sequences for influenza viruses as target sites,and then a HA and NA monoclonal universal antibody was prepared by immunization of animals with a synthetic polypeptide.All the HA and NA monoclonal universal antibodies can bind several influenza virus subtypes in vitro,and have a protective effect in the protection model of mice against influenza virus challenge,which indicated the potential applications of universal antibodies in influenza treatment.Additionally,considering that there are three NA subtypes in an influenza vaccine,the study further analyzed NA subtypes,screened out the conservative peptide sequences in the subtypes but specific in other types,and so prepared universal NA subtype specific antibodies.A test method for respectively quantifying each of the three NA subtypes in the influenza vaccine(split virion)was successfully established.1 Preparation and identification of HA and NA universal antibodiesBased on the results of previous bioinformatics analysis,conservative peptide Uni1 for HA and conservative peptide HCA2 for NA,were synthesized respectively,and coupled with carrier proteins.Then,monoclonal antibodies were prepared by immunization of mice.Total 6 strains of monoclonal antibodies against Uni1 site of HA were obtained,and then variable region sequencing and subtype identification were performed for those antibodies.All the 6 strains of monoclonal antibodies can bind different subtypes of influenza viruses in the ELISA test.In a Western Blot test,5 strains of monoclonal antibodies can bind H1N1,H3N2 and H7N9,while another strain can not only bind above three strains of influenza virus HA,but also bind type B influenza virus HA,which showed a more broad spectrum binding effect.Total 13 stains of HCA2 monoclonal antibodies against NA were obtained and 4 strains of HCA2 monoclonal antibodies with the highest titer in ELISA may bind different subtypes of influenza viruses.The neutralization against viruses by the antibodies was investigated through a plaque neutralization test.In the test,Uni1-1 monoclonal antibody reduced the size and number of plaques,which indicated neutralizing activity of the antibody.Also,a membrane fusion inhibition test was used for exploring action mechanism of this universal antibody for inhibiting virus proliferation.2 Protective effect of HA and NA universal antibodies in miceThe protective effect of the 6 strains of HA monoclonal antibodies was tested by infection models of mice.The results showed that all of the mice challenged by PR8 virus were protected,but different in effect.The protective effect of Uni1-1 antibodies was the best.After dosing as per 50mg/kg,among mice challenged by 5 times LD50 of PR8 virus,60 to 80% survived,while among mice challenged by the same dosage for Uni1-5,the protective rate was only 10%.As tested with NA monoclonal antibodies,the protective rates from 4 strains of monoclonal antibodies are lower than that of Uni1-1antibodies.HCA2-2 monoclonal antibodies had the highest protective rate,and after dosing as per 100mg/kg,the protective rate for the mice was 67%,which is comparable to the protective rate of 50mg/kg dosage for Uni1-1.The protective rate of HCA2-3 and HCA2-8 was 33% under the same dosage,while there is no protective effect of HCA2-7 monoclonal antibodies.For Uni1-1 monoclonal antibodies,several types of influenza viruses were used for a challenge test,so as to verify their universality.The viruses used for the test include H1N1,H3N2,H5N1 and H7N9.After dosing as per 100mg/kg,the protective rate against H1N1 was 100%,and on the third day after challenge,the influenza virus titer for lungs of mice in the test group was 1% of that in the control group;the protective effect against H5N1 influenza virus was 50%,and the virus titer for lungs in the test group was 7% of that in the control group;the protective effect against H3N2 influenza virus was 70%,and the virus titer for lungs in the test group was 4% of that in the control group;the protective effect against H7N9 influenza virus was 60%,and the virus titer for lungs in the test group was 7% of that in the control group.Additionally,a comparison between the test group and the control group for body weight change of mice was conducted.Above results showed that Uni1-1 monoclonal antibodies had a broad spectrum of protective effect in vivo for several subtypes of influenza viruses,could significantly reduce the influenza virus titer for lungs,and increase the survival rate of mice.3 Preparation and application of universal NA subtype universal antibodiesThree NA subtype universal antibodies for influenza vaccines were prepared through bioinformatics analysis and screening the conservative peptide sequences in the NA subtypes but specific in different types.N1 to N9 and type B influenza viruses were used for specificity verification of the antibodies prepared,which proved that the three antibodies reacted only with corresponding subtypes of NA,without cross reaction with other subtypes of NA.In this study,the standards of H1N1,H3N2 and type B influenza viruses for NA assay were also prepared,and through optimization of experimental conditions,the Slot-Blot method for determining the NA content in trivalent influenza vaccine was established,and influenza vaccines from several manufacturers were tested.The results showed that different sources of vaccines were different in content of the same subtype of NA.For example,the content for N1 was 6080ng/ml at maximum,and 145ng/ml at minimum.For the same manufacturer,the difference in NA content between different subtypes was dramatic,e.g.the content for N1 was the highest,while for type B was generally lower.To summarize,HA and NA monoclonal universal antibodies,as well as NA subtype universal antibodies,were successfully prepared in the study.Their broad spectrum neutralization was proved by in vitro method,and a preliminary discussion on relevant action mechanism was conducted;a broad spectrum protective effect against virus challenge was proved in mice;a test method was established with NA subtype universal antibodies,and the method filled the void for quality control of influenza vaccines. |
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