assembling ha-cov-2 particles by cotransfection of hek293t cells for quantification of sars-cov-2 variants

SARS-CoV-2 변이체에서 백신 유도 중화항체 정량화

As of May 2023, there have now been more than 766 million COVID-19 cases1. Despite worldwide vaccination campaigns, SARS-CoV-2 continuously circulates and infects people, largely due to the emergence of new variants such as Delta (B.1.617.2) and Omicron (B.1.1.529) that drive new waves of infection2,3,4. Given that SARS-CoV-2 is constantly evolving, it is important to develop quick assays that can accurately measure the infectivity of emerging variants and the effectiveness of vaccine-induced neutralizing antibodies against these variants. These assays are essential for pandemic surveillance and for determining the efficacy of vaccines and their variant-specific boosters.
Due to the highly contagious nature of SARS-CoV-2, the Center for Disease Control and Prevention (CDC) requires that the study of SARS-CoV-2 and its variants is conducted in biosafety level (BSL) 3 facilities5,6. This BSL-3 requirement limits the use of live viruses for quantifying the infectivity of viral variants and their neutralizing antibodies in common research and clinical laboratories. In addition, traditional SARS-CoV-2 neutralization assays, such as the plaque- or cytopathic effect-based assays using replication-competent live viruses, are time-consuming and require long incubation periods7. Several spike (S) protein-pseudotyped SARS-CoV-2 pseudoviruses have been developed to quantify the effectiveness of neutralizing antibodies8,9,10,11,12. In SARS-CoV-2, the S protein is the major protein that mediates viral entry13, and is the main antigen used in SARS-CoV-2 vaccines9,10,14,15,16. The S protein-pseudotyped virions, such as those of the vesicular stomatitis virus (VSV-G) or lentivirus, have been used for the quantification of neutralizing antibodies17,18,19. Nevertheless, the lentivirus-based pseudovirus normally requires 2 to 3 days of infection in order to quantify reporter signals. VSV-based pseudovirus systems often contain residual VSV viruses, which can result in high rates of false-positive results and typically require 24 h of infection20.
A novel SARS-CoV-2 pseudovirus system, the hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2), has been recently developed by Hetrick et al12. Ha-CoV-2 provides a new tool for the rapid quantification of virus infectivity and virus sensitivity to neutralizing antibodies in common BSL-2 laboratories. Structurally, Ha-CoV-2 resembles the SARS-CoV-2 virion particle, consisting of SARS-CoV-2 structural proteins including the S protein (S), the membrane (M), the nucleocapsid (N), and the envelope (E), and there is no structural protein from other viruses. Additionally, Ha-CoV-2 particle contains a rapid-expressing RNA genome from an alphavirus for fast reporter expression in cells. Ha- CoV-2 has been shown to quickly measure the neutralizing activity of antibodies in the sera of vaccinated and convalescent individuals12. As demonstrated by Hetrick et al., when compared with lentivirus-based SARS-CoV-2 pseudovirus in a time course assay, Ha-CoV-2 expressed the Luc reporter as early as 2-4 h post-infection while the lentivirus-pseudovirus expressed Luc after 24 h12. In addition, the potential application of Ha-CoV-2 variants for quantifying neutralizing antibodies is further demonstrated by using a&#160; standard monoclonal neutralizing antibody, 27BV (See Supplementary Figure 1)12. This work details the use of the Ha-CoV-2 platform for rapid quantification of the infectivity of SARS-CoV-2 variants, using the Delta (B.1.617.2) and the Omicron (B.1.1.529) variants as examples. In addition, the potential application of Ha-CoV-2 variants for quantifying neutralizing antibodies is further demonstrated by using a standard monoclonal neutralizing antibody, 27BV12.

저자 스포트라이트: SARS-CoV-2 연구에서 오미크론 하위 변이체 평가 및 중화 항체를 위한 유사형 바이러스 시스템

SARS-CoV-2 변이체 및 중화 항체의 신속한 정량화를 위한 Ha-CoV-2 슈도바이러스 적용

We have established two main findings in this protocol. First, our Ha-CoV-2 platform demonstrates that the newly emerged Omicron subvariant is more infectious than the original Wuhan strain. Additionally, Omicron subvariants are more resistant to neutralizing antibodies induced by previous strains.SARS CoV-2 research requires BSL-3 facilities, making it difficult for common laboratories to monitor SARS-CoV-2 variants and to quantify neutralizing antibodies. Our Ha-CoV-2 system is a pseudotype virus that can be used in common laboratories and provides a faster way to quantify viral variants and their sensitivity to neutralizing antibodies. Ha-CoV-2 is a virus-like particle that has all the structural proteins of SARS-CoV-2 and resembles SARS-CoV-2.Also, Ha-CoV-2 uses and alpha viral vector for robust and rapid gene expression, producing fast results and a high signal to noise ratio for great reproducibility.

SARS-CoV-2 변이체의 정량화를 위한 HEK293T 세포의 공형질주입에 의한 Ha-CoV-2 입자 조립

T75 플라스크에서 열 비활성화 FBS, 페니실린 및 스트렙토마이신을 포함하는 HEK293T 세포와 DMEM을 유지하는 것으로 시작합니다. 세포 계수를 위해 20 마이크로리터의 세포 현탁액과 20 마이크로리터의 트리판 블루 용액을 혼합합니다. 20마이크로리터의 염료 혼합 세포 현탁액을 세포 계수 챔버에 추가하고 밀리리터당 세포 수를 계산합니다.10cm 세포 배양 페트리 접시에 HEK293T 세포와 10mL의 완전한 DMEM을 시드합니다. 섭씨 37도의 이산화탄소 인큐베이터에서 밤새 세포를 배양합니다. 다음 날, 전체 배지를 제거하고 9mL의 DMEM 무혈청 배지로 교체합니다.주어진 바이러스 입자 조립 화합물을 사용하여 cotransfection 혼합물을 준비합니다. 혼합물을 페트리 접시에 천천히 넣고 섭씨 37도에서 6시간 동안 배양합니다. 배양 후 무혈청 DMEM을 완전한 DMEM 배지로 교체하고 최대 48시간 동안 cotransfection을 위한 배양을 계속합니다.그런 다음 단층 표면을 반복적으로 피펫팅하여 세포를 분리하고 현탁액을 15밀리리터 튜브로 옮깁니다. 셀 현탁액을 400G에서 5분 동안 원심분리합니다. 상층액을 모아 0.22미크론 필터를 통과시킵니다.Ha-CoV-2 유사 바이러스가 포함된 여과액을 섭씨 80도에서 보관합니다.

assembling-ha-cov-2-particles-cotransfection-hek293t-cells-for

Research

JoVE Journal

Immunology and Infection

Assembling Ha-CoV-2 Particles by Cotransfection of HEK293T Cells for Quantification of SARS-CoV-2 Variants

247 조회수.  George Mason University. T75 플라스크에서 열 비활성화 FBS, 페니실린 및 스트렙토마이신을 포함하는 HEK293T 세포와 DMEM을 유지하는 것으로 시작합니다. 세포 계수를 위해 20 마이크로리터의 세포 현탁액과 20 마이크로리터의 트리판 블루 용액을 혼합합니다. 20마이크로리터의 염료 혼합 세포 현탁액을 세포 계수 챔버에 추가하고 밀리리터당 세포 수를 계산합니다.10cm 세포 배양 페트리 접시에 HEK293...

비디오: SARS-CoV-2 변이체의 정량화를 위한 HEK293T 세포의 공형질주입에 의한 Ha-CoV-2 입자 조립

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

연구

면역학 및 감염병학

Begin by maintaining HEK293T cells and DMEM containing heat inactivated FBS, Penicillin, and Streptomycin in a T75 flask. For cell counting, mix 20 microliters of cell suspension with 20 microliters of trypan blue solution. Add 20 microliters of dye-mixed cell suspension to the cell counting chamber and count the number of cells per milliliter.Seed HEK293T cells and 10 milliliters of complete DMEM in a 10 centimeter cell culture Petri dish. Incubate the cells overnight in a carbon dioxide incubator at 37 degrees Celsius. The next day, remove the complete medium and replace it with nine milliliters of DMEM serum-free medium.Using the given compounds of viral particle assembly, prepare a cotransfection mixture. Slowly add the mixture to the Petri dish, and incubate at 37 degrees Celsius for six hours. After incubation, replace the serum-free DMEM with a complete DMEM medium and continue incubation for cotransfection for up to 48 hours.Then detach the cells by repeatedly pipetting over the surface of the monolayer and transfer the suspension into a 15 milliliter tube. Centrifuge the cell suspension at 400G for five minutes. Collect the supernatant and pass it through a 0.22 micron filter.Store the filtrate containing Ha-CoV-2 pseudovirus at 80 degrees Celsius.

Quantification of Ha-CoV-2 Variants and 27BV Neutralization Activity Against Ha-CoV-2(Luc) Variants

Begin by maintaining HEK-293T ACE2 TMPRSS2 cells in DMEM containing heated activated FBS, penicillin, and streptomycin in a T75 flask. For cell counting, mix 20 microliters of cell suspension with 20 microliters of Trypan Blue solution. Add 20 microliters of dye-mixed cell suspension to the cell counting chamber, and count the number of cells per milliliter.Seed 2.5 times 10 to the power of four HEK-293T ACE2 TMPRSS2 cells in 50 microliters of complete DMEM medium into each well of a 96-well plate. Incubate the cells in a carbon dioxide incubator overnight at 37 degrees Celsius. The next day, replace the 50 microliters of DMEM with 50 microliters of virus suspension, and incubate for 18 hours at 37 degrees Celsius.After incubation, add 7.5 microliters of cell lysis buffer to each well, and mix on an orbital shaker for two minutes. Once the cells are lysed, add freshly prepared firefly luciferase assay solution, and mix them on an orbital shaker for one minute. Analyze the luciferase activity by using a commercial luciferase microplate reader.For neutralizing antibody assay, seed HEK-293T cells in 50 microliters of complete DMEM as demonstrated previously. In a 96-well plate, add eight microliters of 27 BV neutralizing antibody, and perform serial dilutions with six microliters of serum-free DMEM. Add 54 microliters of Ha-CoV-2 virus particle to the serially diluted antibody, and mix the suspension.Incubate for one hour at 37 degrees Celsius in 5%carbon dioxide. Then add 50 microliters of virus suspension to the wells containing HEK-293T cells. For controls, add 50 microliters of complete DMEM medium into the wells containing only HEK-293T cells.Place the plate in an incubator at 37 degrees Celsius for 18 hours. After lysing the cells as demonstrated previously, add freshly prepared firefly luciferase assay solution, and mix by orbital shaking for one minute. Analyze the luciferase activity by using a commercial luciferase microplate reader.Ha-CoV-2 Omicron and Omicron variants generated four to tenfold higher signal than the Wild-type Ha-CoV-2, suggesting higher infectivity. The antibody 27 BV demonstrated neutralizing activity against both Delta and Omicron variants. The ID50 of 27 BV for Omicron was approximately 10 times less potent than the ID50 for Wild-type and Delta.