Table 1.
The combined modes of plasmids for pseudotyped recombinant lentivirus production.
Fig 1.
Schematic illustration and theoretical structure of pseudotyped lentiviral particles.
A. Control pseudotyped lentiviral particles VSV-G/LV-eGFP, which was constructed with envelope plasmid pCMV-VSV-G, core plasmid pLV-eGFP and packaging plasmid psPAX2. B. Pseudotyped lentiviral particles VSV-G/LV-RABV-G, which was constructed with envelope plasmid pCMV-VSV-G, core plasmid pLV-RABV-G and packaging plasmid psPAX2. C. Pseudotyped lentiviral particles RABV-G/LV-eGF, which was constructed with envelope plasmid pCMV-RABV-G, core plasmid pLV-eGFP and packaging plasmid psPAX2. D. Pseudotyped lentiviral particles RABV-G/LV-RABV-G, which was constructed with envelope plasmid pCMV-RABV-G, core plasmid pLV-RABV-G and packaging plasmid psPAX2.
Table 2.
Grouping and immunity of BALB/c mice.
Fig 2.
Construction and Characterization of pseudotyped lentiviral vectors/particles with rabies virus glycoprotein.
A. Restrction enzyme digestion analysis of pLV-RABV-G. M: DL2000 DNA marker; 1 and 2 line: Double enzyme digestion product of pLV-RABV-G with Nhe I + Mlu I. B. Restrction enzyme digestion analysis of pCMV-RABV-G. M: 10000 bp DNA marker, 1 and 2 line: Double enzyme digestion product of pCMV-RABV-G with Nhe I and Mlu I. C. Analysis of Rabies virus RABV-G expression in recombinant pseudotyped lentiviral particles packaged by HEK293T cells by western blot. D. The specific reaction between recombinant pseudotyped lentiviral particles and serum detected by ELISA; ***p < 0.001. E. Fluorescence images of HEK293T cells at 72h post-transfected with recombinant lentivirus particles. A:Control; b:VSV-G/LV-eGFP; c:VSV-G/LV-RABV-G; d: RABV-G/LV-eGFP; e: RABV-G/LV-RABV-G. F. The expression of RABV-G mediated by recombinant pseudotyped lentivirus in HEK293T cells.
Fig 3.
Neutralization assay with the RABV G-pseudotyped recombinant lentivirus.
The immunized mouse sera were two fold serially diluted with PBS and incubated with the indicated pseudotyped recombinant lentivirus. The mixture was added to 293T cells. After incubation for 48hs, the serum neutralizing activity against RABV was assessed by the decrease in GFP expression. A.Neutralization assay using RABV-G/LV-eGFP and VSV-G/LV-eGFP recombinant lentivirus. B. Correlation analysis of recombinant pseudotyped lentivirus neutralization test and FAVN for serum neutralizing antibody titers from vaccinated mice, A high rs value (rs = 0.825, **p < 0.01) indicated a strong positive linear correlation.
Fig 4.
Purification and titers determination of RABV G-pseudotyped recombinant lentivirus.
After transfection with plasmids for 48 hours, purify the viral particles packaged by HEK293T cells using sucrose density gradient centrifugation and measure their concentration by qPCR.
Fig 5.
Evaluation of anti-RABV-G immunogenicity of pseudotyped recombinant lentivirus in mice after one-dose immunization.
A. Detection of immunized serum anti-RABV-G IgM antibody levels in inoculated mice at 3th and 7th day. B. Detection of immunized serum anti-RABV-G IgG antibody levels in inoculated mice at 1st, 2nd and 3rd week. C. Detection of sustained serum anti-RABV-G IgG antibody titers in different groups. D. Detection of immunized serum neutralizing antibody titers in different groups. ***p < 0.001, **p < 0.01, *p < 0.05, #: The antibody titers did not reach detectable levels.
Fig 6.
Evaluation of anti-RABV-G immunogenicity of each pseudotyped recombinant lentivirus in mice with different immunization mode.
A. Detection of serum anti-RABV IgG antibody levels in different groups of mice after one or two-dose immunization. B. Detection of neutralizing antibody titers in serum from different groups after one or two-dose immunization at 3nd and 4th week. ***p < 0.001, **p < 0.01.