The Sero-epidemiology Of Circovirus In Human And Development Of A Novel Vaccine Against Circovirus

WHO statistics show that over the last 40 years,about 75 % of recently emerging infectious diseases affecting humans are of animal origin,these pathogens including human immunodeficiency virus,Ebora virus,West Nile virus,Nipah virus and Hantaan virus,etc.The recent outbreaks of severe acute respiratory distress syndrome(SARS),Ebola hemorrhagic fever,highly pathogenic group A subtype H5N1,low pathogenic group A subtype H7N9 and swine influenza virus subtypes H1N1 avian influenza viruses have a negative impact on economic development and human health in both developing and industrial countries.The newly emerging and reemerging infectious diseases,and new cycles of pandemics,have emerged as a global public health threat and have posed a significant national burden on economies and public health in worldwide.Therefore,the emerging of human infectious diseases caused by animal pathogens has received more attentions.Porcine circovirus(PCV),one of the smallest known animal viruses,is a common virus of pigs found throughout the world with the prevalence rate of 52.8 %~100 %,which is strongly associated with the occurrence of postweaning multisystemic wasting syndrome(PMWS).It is generally believed that PCV has a narrow host range that is restricted to pigs,however,the antibodies reacting with PCV were found in sera of human in northeast China,suggesting that PCV may be spread from pigs to people by crossing species barrier and may has evolved into a new zoonotic agent.Annual vaccination is the most effective and important measure to prevent horizontal transmission of infections.However,due to the lack of understanding of the newly emerging viruses,it is incapable to rapidly develop safe and effective vaccines against viral infection,resulting in hard prevention and control of the infectious disease.ObjectiveThis research aimed to explore the possible risk factors for the circoviruses infection in humans and to develop an effective vaccine against PCV2 infection.The results of this study will provide scientific evidence to surveillance and response to continuing threats from emerging and re-emerging zoonoses,and development of a novel antiviral vaccine can serve as a model for the prevention and control of other viral infections.Methods1267 serum samples were randomly selected from 16831 blood samples to detect the serum-specific PCV Ig G antibodies to estimate PCV infection in the population of Jilin Province.The data on demography were summarized as frequencies.Univariate odds ratios(ORs)and 95% CIs were undertaken to examine the association between sero-positivity and the related variables using logistic regression model.Variables that were statistically significant at P < 0.05 in the univariate analysis were entered into multivariate logistic regression analyses to examine the variables independently associated with sero-positivity.To avoid induction of non-protective antibodies directed to the decoy epitope during PCV2 vaccine immunization,we constructed an immunogen PCV2 b capsid protein in which the decoy epitope was replaced with a neutralizing B cell epitope and expressed it in E.coli,named?CP.The ?CP was purified and dialyzed to assemble into virus-like particles and observed under transmission electron microscopy.The ? CP was formulated in MontanideTM ISA 35 or ISA 206 adjuvant to prepare a vaccine.The stability,safety and antigenicity of the vaccine were evaluated before vaccination.Mice and pigs were vaccinated with the ?CP vaccine.Antibodies in sera of mice or pigs were detected to evaluate the ability of inducing protective antibody responses in mice and pigs.At 56 days post immunization,pigs were challenged with wild type PCV2 b,lung and lymph node tissues of challenged pigs were assessed by microscopic examination,real time PCR was used to assess PCV2 b viral DNA in the tissues.Results1.The serological evidence of exposure to PCV was detected in 81 of the 1267 human serum samples in Jilin Province,northeast China,giving an overall(raw)human seroprevalence of 6.39 %.The detection rate of PCV Ig G antibody in rural population was significantly higher than that in urban population(P < 0.001).May be due to the prevalence of live pigs in rural areas,workers engaged in agricultural production have more opportunities to contact live pigs led to the virus infection.In addition,patients with cirrhosis and hypertension may have a higher risk of infection with circovirus,but the reason for this is not clear.2.Though achievements have been made in prevention of porcine circovirus associated diseases by application of existing vaccines,the infection,spread and prevalence of PCV2 cannot be controlled effectively.This status impelled researchers to study that besides the production of neutralizing antibodies,whether the existing vaccines could divert the immune response and promote the viral infection.In this study,we found that the decoy epitope may be exposed during the preparation of inactivated PCV2 b vaccines,preparation of PCV2 b capsid protein based recombinant subunit vaccines,or the natural infection of PCV2 b.The exposition of decoy epitope could divert the immune response away from protective epitopes and induce the production of nonneutralizing antibodies,thus allowing for continue PCV2 replication and the progression towards disease.To avoid the decoy effect,we designed and produced a recombinant PCV2 b CP(?CP)by replacing the decoy epitope with a neutralizing B cell epitope and expressed in E.coli as soluble proteins.The purified ? CP was assembled into virus-like particles(VLPs)and formulated in MontanideTM ISA 35 or ISA 206 adjuvant to prepare a vaccine.The physical properties,such as appearance,viscosity,stability and other indicators were evaluated.The safety of the vaccine was determined by a safety test,the antigen was extracted from the emulsified vaccines to monitor their stability and antigenicity during vaccine manufacture and storage.Although unable to form typical VLPs,? CP could increase the production of the anti-PCV2 b antibodies among which no antibody against the decoy epitopes.The immunized mice could generate PCV2-specific antibody and obtain sustained and effective protection,the polyclonal antibody titers can reach 1: 64000.The ?CP immunized pigs could generate specific antibody against PCV2.6/6 immunized piglets appeared seroconversion and PCV2 ELISA antibody titers in piglets can up to 1: 800.Viral DNA loads were significantly lower in pigs vaccinated with ?CP than in pigs injected with PBS.Lymphoid depletion and histiocytic-to-granulomatous inflammation in lymphoid tissues in the pigs immunized with ?CP were significantly less severe than that in PBS group.Conclusion1.The serological evidence of exposure to PCV was detected in 81 of the 1267 human serum samples in Jilin Province,northeast China,giving an overall(raw)human seroprevalence of 6.39 %.The detection rate of PCV Ig G antibody in rural population was significantly higher than that in urban population(P < 0.001).Patients with cirrhosis and hypertension may have a higher risk of infecting the circovirus.2.Replacing the decoy epitope of PCV2 b capsid protein with a protective epitope could enhance efficacy of PCV2 b vaccine to stimulate mice and piglets producing anti-PCV2 b specific antibodies,among which no antibody against the decoy epitopes,and therefore induces improved protective immune responses in pigs challenged with PCV2 b.These results provide an alternative strategy for development of a novel and effective antiviral vaccine and will produce scientific evidence and technical platform for future development of related vaccines to prevent and control of viral infection and epidemics.