Construction and immunogenicity evaluation of a recombinantnewcastle disease virus expressing SARS-CoV spike protein

ABSTRACT

Severe Acute Respiratory Syndrome (SARS) is a zoonotic disease that is acute, feverish and accompanied by respiratory system and even multiple organ infections. Although no SARS infection cases have been reported since 2004, the outbreak of new coronavirus pneumonia (COVID-19) in Wuhan, Hubei Province, my country in December 2019 indicates that SARS coronavirus (SARS-CoV) or SARS-like coronavirus (SARSL-CoV) is highly likely to become widespread in the population again. In this study, a full-length cDNA clone pBRN-FL-SARS-CoV-S expressing the SARS-CoV spike protein (S protein) was first constructed, and the Newcastle Disease Virus (NDV) LaSota vaccine strain reverse genetic operating system was used to rescue The recombinant virus rLa-SARS-CoV-S expressing SARS-CoV S protein was identified. After rLa-SARS-CoV-S was infected with BHK-21 cells at a dose of MOI 0.01 for 36 hours, the SARS-CoV S protein was detected by western blot and laser confocal test. The results showed that the S protein was correct in the infected cells. Expressed and accurately located on the cell membrane. After inoculating 10-day-old SPF chicken embryos with rLa-SARS-CoV-S at a dose of 1x104 EID50, allantoic fluid was collected at different time points and the EID50 was determined. The growth kinetic curve of the virus showed that rLa-SARSCoV-S could The chicken embryo grows at high titer, consistent with the parental virus. Dilute the rLa-SARS-CoV-S and parent virus by 10-fold ratio and inoculate 10-day-old SPF chicken embryos and record the death time of each chicken embryo. Calculate the average chicken embryo death time according to the highest dilution of the virus. Lethal time (MDT), the results showed that the MDT of rLa-SARS-CoV-S was 112.8 h and the MDT of NDV LaSota was 96 h, indicating that the recombinant virus still maintains the low pathogenicity characteristics of the NDV LaSota vaccine strain. RLa-SARS-CoV-S and NDV LaSota were injected intramuscularly with 6-week-old BALB/c mice at a dose of 5x106 EID50 and boosted on the 21st day. At the same time, a PBS control group was set up. The results of the mouse safety test showed that all the mice vaccinated with rLa-SARS-CoV-S survived without any clinical symptoms, and the weight gain was consistent with that of the NDV LaSota group and the control group;the mice were treated on 21 d and 42 d after immunization Blood was collected to prepare serum, and the level of IgG antibodies against SARS-CoV S protein in mice was detected by ELISA. The results showed that the recombinant protein can induce mice to produce higher levels of specific IgG antibodies after initial immunization and booster immunization. The level can be maintained for a longer period of time. The results of this study indicate that rLa-SARS-CoV-S has potential value as a SARS-CoV vaccine candidate, and at the same time provides ideas for the development of SARS-CoV-2 vaccines.