Generation of SARS-CoV-2 virus-like particles in insect cells

The rapid spread of coronavirus 2 (SARS-CoV-2), a severe acute respiratory syndrome, caused a pandemic of COVID-19, pushing the world towards the development of a hasty but effective vaccine against the disease. .. Most of the COVID-19 vaccines currently in use rely on the principles of viral inactivation, adenoviral vectors, or the use of direct nucleic acids (mRNA / DNA).

In a new study recently published in the journal virus, Researchers have reported the development of promising COVID-19 vaccine candidates based on SARS-CoV-2 virus-like particles (VLPs) derived from co-expression of spikes (S), membranes (M), and envelopes. .. (E) Structural protein of baculovirus expression system.

In vitro Tests confirmed their antigenicity, and viral challenges in VLP-immunized hamsters showed their immunogenicity. Immunization with SARS-CoV-2 VLP did not prevent in vivo replication of the challenge virus. However, viral titers and disease pathological markers were reduced compared to the control group.

Virus-like particles (VLP)

VLPs correspond to virus-like structures. However, due to the lack of genomic DNA or RNA, replication is inadequate. VLPs can also be imagined as empty virus shells.These can interact directly antigen-Presentation cells (APCs), especially dendritic cells, which are the most powerful APCs.

Coronavirus M and E proteins (for co-expression) In a test tube It can lead to the formation of VLPs. The most abundant transmembrane protein among coronavirus envelope-related proteins-M mainly promotes the assembly of coronavirus particles. M is oligomerized to form a lattice that structures the conformation of the viral envelope, but mobilizes other structural proteins into the nascent viral particles to complete the assembly process. The small enveloped E protein is incorporated into the virion during development in very small amounts, but is essential for successful virus budding.

Spike S trimer proteins, which are involved in the interaction with the host receptor ACE2 and the promotion of membrane fusion, can also be co-expressed in cells expressing M and E. S is a highly immunogenic antigen, Neutralizing antibodyBlocks the interaction between S1 and ACE2.

VLP mimics the real viral structure and expresses trimer S proteins at multiple sites, thus appropriately stimulating humoral immunity, being easily recognized by antigen-presenting cells (APCs), and providing an appropriate T cell response. Generate. Also, co-administration of the adjuvant is not required due to the nature of their multimers.

Several VLP-based vaccines for other viruses produced by the baculovirus insect cell system are currently in use, demonstrating evidence of extensibility and acceptability, “the team emphasizes.

Construction and verification of SARS-CoV-2VLP

In the past, the team has constructed recombinant baculoviruses that express the S, E, and M structural proteins required for SARS-CoV VLP formation. Following a similar strategy, the current research team has developed a single recombinant baculovirus that expresses SARS-CoV-2 S, M and the previously used SARS-CoVE protein. The SARS-CoV and SARS-CoV-2 E proteins are functionally interchangeable for VLP production purposes. Therefore, the team used the existing SARS-CoVE gene insert to construct a new recombinant virus.

Western blotting (VLP competent construction) of insect cells infected with recombinant virus confirmed the expression of both S protein and M protein. It was also demonstrated that the S protein is present in the VLP structure as a trimer protein (natural form on the viral envelope). VLPs are purified on a sucrose gradient and form a clear band at a 35% sucrose gradient. Coomassie blue-stained SDS-PAGE of 35% gradient fractions showed three prominent bands of 10, 30, and 180 kDa corresponding to E, M, and S proteins, respectively.

Therefore, the team succeeded in producing a single recombinant baculovirus that can independently express all three structural proteins required for SARS-CoV-2VLP formation.

Transmission electron microscopy (TEM) of the fraction showed a VLP assembled as a vesicle-like structure about 100 nm in diameter showing coronal spikes characteristic of coronavirus particles.

SARS-CoV-2 VLP is both antigenic and immunogenic

Convalescent sera were used to examine the antigenicity of VLPs. Multiple convalescent sera strongly reacted with ELISA-immobilized VLPs, suggesting that VLPs showed S antigen in a form suitable for binding to the antibody.

The immunogenicity of VLP carrying the SARS-CoV-2S protein was tested using a Syrian hamster model showing disease pathology and neutralizing antibody response similar to human COVID-19. VLP candidate vaccination was given to 5 hamsters twice at 28-day intervals. Treatment controls were also not maintained.

Development of neutralizing antibodies to seroconversion and RBD was observed in 4 of 5 animals after the first dose and in all VLP-immune animals after the second dose. The control group showed no antibody activity.

Then, two weeks after the second VLP administration, hamsters were ly infected with the infectious dose of SARS-CoV-2 and antibody activity was observed. Neutralizing antibody titers increased faster than in the control group within 4 days of the challenge in VLP-immune animals.

The team also observed slow clearance of genomic RNA from the control oral swab compared to the VLP immune group. The peak viral load on day 2 of the challenge on the oral swabs in the treatment group was 1.4 x 10, much lower than in the control group.⁷ And 4.7 × 10^7, Each. However, the difference was not important. Nasal swabs showed a similar pattern.

On day 10, the lungs of VLP-immune animals had lower scores for inflammatory markers and fewer S-positive syncytial cells than unvaccinated controls.

All treated and control animals recovered by day 14 after the virus challenge, while immunized animals recovered significantly faster than controls.

The findings suggest that antibody levels are inadequate to protect against infection, but sufficient to reduce the severity of the disease.

In our study, VLPs are already immunogenic in the absence of an adjuvant and are already on both human and animal vaccines, providing expandability, an acceptable manufacturing process, and an established route to approval. Manufactured using the technology used, “the team concludes.