Long-term Immunity Of Smallpox Vaccine In China In Deterring Potential Monkeypox Virus Transmission

Background:Monkeypox(Mpox)is a viral zoonosis,which mainly occurs in West and Central Africa.In May 2022,multiple cases of monkeypox were detected in several non epidemic countries,which has attracted increasing attention worldwide.The clinical manifestations of monkeypox are similar to those of smallpox,with fever,rash and swollen lymph nodes,and can lead to a number of complications.The smallpox vaccine protects against monkeypox.The World health organization(WHO)announced that smallpox had been eradicated in 1980,and most countries discontinued the routine Vaccinia virus(VACV)immunization.Therefore,individuals who were born after 1980 do not have VACV-elicited immunity,and cannot be protected from monkeypox virus(MPXV)infection.With the first Mpox case imported from an European country to the Chinese mainland on September 16,2022[1],the investigation of residual immunity to VACV in the Chinese population has become urgent.Before 1980,millions of people were were inoculated with the replicative TianTan Vaccinia virus strain(VTT)as part of the smallpox eradication campaign[2,2].Previous studies have found that the protection rate of smallpox vaccine based on VACV against Mpox is about 85%.Vaccines against monkeypox mainly include modified Vaccinia virus(MVA),LC16m8,ACAM2000 and DryVAX.It has been reported that two commercially available smallpox vaccines MVABN and ACAM2000 are expected to have strong cross-immune reaction with MPXV[4].However,there are no large-scale studies in China to confirm whether VTT,like MVABN and ACAM2000,can effectively exert its long-term protective effect in the Chinese population.In order to better cope with the international situation of Mpox epidemic,and the potential outbreak of Mpox epidemic in China,the residual immunity level of the Chinese population to VACV was determined to assess the susceptibility of the Chinese population to MPXV,which can provide important basic data for the development of protection strategies against MPXV infection.Objective:In the face of the current international pattern of Mpox outbreaks,it is crucial to accurately assess the potential impact of residual immunity in the Chinese population on predicting Mpox outbreaks.Therefore,in order to evaluate the susceptibility of China population to MPXV and provide basic data and new ideas for formulating appropriate protection strategies against MPXV infection in the future,we have carried out systematic and large-scale studies on humoral and cellular immune responses and immune persistence against VACV in the Chinese population.Methods:(1)The primers targeting Cas9 sgRNA in TK region annealed and the Glu gene amplified by PCR were inserted into its vector,respectively,to construct Cas9dNLS-gRNA plasmid and Glu homologous recombinant plasmid.Two plasmids were cotransfected into 293T cells to obtain recombinant rTV-Gluc virus through homologous recombination.(2)We conducted a systematic immunological study on 1070 healthy volunteers of different ages from different regions in China.The enrolled healthy volunteers ranged in age from 6 to 84 years.They are from Beijing,Shanxi,Heilongjiang,Hubei and Shenzhen.All serum samples collected in this study were inactivated at 56℃ for 30 min before use,and the serum VTT-specific Immunoglobulin G(IgG)antibody level was determined by enzyme-linked immunosorbent assay(ELISA)method.Neutralizing antibody(NAbs)levels in serum were measured by three neutralizing assay methods:Plaque reduction neutralization test(PRNT)and luciferase-based Microneutralization assay(MNA).Then VTT-specific memory B cell and T cell immune responses of Peripheral blood mononuclear cells(PBMCs)were detected by Enzyme-linked immunospot assay(ELISpot).Results:(1)After PCR identification,the construction of recombinant rTV-Gluc strain was correct.(2)More than 50%participants born from 1930 to 1969 have VTT-specific IgG,whereas VTT-IgG positivity rates are 29.9%and 4.8%for those born in the periods from 1970 to 1979 and from 1980 to 1989,respectively,indicating a dramatic decline in the younger population.(3)Among individuals born before 1980,only 28.7%showed detectable NAb responses against VTT at low levels,and the majority(71.3%)had a VTT neutralizing antibody titer lower than 1/4,and only a few(1.9%)had a VTT neutralizing antibody titer higher than 1/32.(4)Cross-sectional analysis of serum IgG antibody showed that the positive rate of serum VTT-specific IgG increased with age.(5)The prevalence of serum neutralizing antibody in individuals born before 1950 was higher than that in those born between 1950 and 1980.(6)According to ELISpot analysis,most people maintain VTT memory B(in 68.6%participants)and memory T(in 65.7%participants)cells,which are absent in individuals born from 1980 to 2008.(7)VTT-specific memory B cell and memory T cell immune responses in individuals born before 1980 had little effect with age.Conclusion:The research on the long-term immunity of VTT in China is the first largescale and systematic immunological study of VTT immunity in the Chinese population for more than 5 decades(1940-2008),including humoral and cellular immunity.We found that more than 65%individuals who were born before 1980 exhibited memory B cell and memory T cell responses.However,the low prevalence and titers of neutralizing antibody against VTT in the Chinese population suggest for the first time the target group that needs to prioritize vaccination.To protect the population from monkeypox virus and other related pathogenic orthopoxviruses(OPXV),effective and safe vaccine strategies need to be developed to establish herd immunity.