Research On Severe Fever With Thrombocytopenia Syndrome Virus Vaccines Based On Novel Vectors

Severe fever with thrombocytopenia syndrome virus(SFTSV)is an emerging zo-onotic pathogen first reported in 2009 in China with a fatality rate of 2–30%.SFTSV is a tick-borne bandavirus originally identified in Dabie mountain.Hu-man cases outside China were confirmed in South Korea,Japan,Vietnam,Myanmar,Thailand,and Pakistan in the subsequent decade.Similar to other bandaviruses,SFTSV is spherical with an envelope and a genome comprising large(L),medium(M),and small(S)fragments,in which the Gn and Gc proteins are encoded by M fragments.SFTSV Gn/Gc glycoprotein is responsible for receptor binding and membrane fu-sion and is the main target of neutralizing antibodies.SFTSV is mainly transmitted by the Haemaphysalis longicornis tick.Ferret model studies have revealed that SFTSV can be transmitted by body fluids such as urine and saliva.Recently,we found that hedgehogs are the major amplifying hosts.The symptoms of SFTSV infection include fever,muscle pain,vomiting,diarrhea,and thrombocytopenia.No licensed vaccines exist that prevent SFTSV infection.In our previous study,we developed a VSV-vector SFTSV vaccine by replacing the VSV glycoprotein(G)gene with the SFTSV Gn/Gc gene and named it rVSV-SFTSV/AH12-GP.This vaccine was highly attenuated and could induce strong and broad-spectrum protective immunity against SFTSV and Heartland virus(HRTV).However,rVSV-SFTSV/AH12-GP only propagated to 3×10~6 PFU/ml in Vero cells,limiting its further development.Here,we identified two mutations in the Gc protein that significantly enhance the titer to 7×10~7 PFU/ml without affecting the immunogenicity.This study mainly works through the following four aspects:1.To study the effect of these two mutations on virus reproduction,we introduced single or double mutations in rVSV-SFTSV/AH12-GP(named rVSV-WT,rVSV-C617R,rVSV-M749T,and rVSV-M749T+C617R,respectively).All mutants were successfully rescued in Vero cells and the characteristics of recombinant viral vector vaccines were studied.Western blot analysis of SFTSV Gc glycoprotein with polyclonal antibodies detected correct expression of recombinant virus Gc glycoprotein in both supernatant and cell lysates,and significantly higher Gc glycoprotein expression of mutant strain virus than wild-type virus was observed.Assays on growth curves also indicate higher titers of mutants than wild types.2.The attenuating effect,safety and immunogenicity of the vaccine were tested.In plaque formation experiments,rVSV-WT and mutants formed plaques in Vero cells were similar in size,but smaller than rVSV-HRTV and rVSV-G to form plaques.This shows that compared with rVSV-G and rVSV-e GFP-HRTV,both mutants and wild types have good attenuation effects.After vaccination,the immunodeficient mice did not lose weight and did not find VSV replication in the body.From this,the mutant virus can be considered safe for immunodeficient mice.There was no significant difference in the neutralizing antibody FRNT50 titer produced by wild-type and mutant vaccination,indicating that the mutation did not affect immunogenicity.3.Mutant strain vaccine has a protective effect against fatal SFTSV infection in immunodeficient mice.After 30 days of immunization,the immunized animals were attacked with 2×104 FFU lethal doses of SFTSV Wuhan strain,and the animals were observed for clinical signs and weight loss.As shown in Figure 3-15,mice inoculated with rVSV-WT or rVSV-M749T+C617R all survived,while control mice all died and viremia was detected within5 days of infection.4.Explore how mutant M749T and C617R increase the titer of rVSV-SFTSV by immunofluorescence and confocal microscopy.To determine the localization of mutant SFTSV glycoprotein,the Ds Red-KDEL fusion protein is expressed transiently in Vero cells.It was found that most of the mutated glycoproteins were localized to the endoplasmic reticulum,similar to the WT virus.Quantitative analysis showed that there was no significant difference in the expression levels of WT protein and mutant Gc protein.The effects of M749T and C617R on GC protein membrane localization were further studied,and it was found that M749T could significantly increase the level of Gc protein on the cytoplasmic membrane.