Development of an Attenuated SARS-CoV-2 Strain Through Targeted Mutations in the Spike Protein
Creators: name: Hussain, Zahid
Publisher: name: Zenodo
Year: 2025
Subjects: Development
Attenuated
SARS-CoV-2 Strain
Targeted Mutations
in the Spike Protein

description:  

Abstract: 

The spike (S) protein of SARS-CoV-2 plays a pivotal role in viral infectivity and host immune recognition. Targeted mutations in the receptor-binding domain (RBD) and other functional regions of the spike protein can attenuate the virus by reducing its ability to infect human cells while preserving its immunogenicity. This study investigates specific mutations, including N501D and Q498L, designed to impair ACE2 receptor binding. The engineered strains were evaluated for infectivity, replication efficiency, and immunogenicity in vitro and in vivo. Results demonstrated significantly reduced infectivity and replication rates, coupled with robust immune responses. These findings highlight the potential of spike protein mutations to create attenuated viral strains for vaccine development and further research into SARS-CoV-2 immunology.

Introduction: 

SARS-CoV-2, the causative agent of COVID-19, utilizes its spike protein to bind to the ACE2 receptor, facilitating entry into host cells (Walls et al., 2020). The receptor-binding domain (RBD) of the spike protein is essential for this interaction, making it a prime target for interventions aimed at attenuating the virus. Attenuated viral strains have been critical tools in vaccine development, offering a balance between safety and efficacy (Plotkin, 2010).

This study aims to identify and evaluate spike protein mutations that attenuate SARS-CoV-2. By targeting the RBD and other functional regions, we hypothesize that the virus’s infectivity can be reduced while retaining its immunogenic properties. This approach may pave the way for the development of live-attenuated vaccines and contribute to the understanding of SARS-CoV-2’s pathogenesis.

 

 

 

 

Materials and Methods:

1. Design of Spike Protein Mutations



Mutations N501D and Q498L were selected based on computational modeling and prior studies indicating their potential to reduce ACE2 binding affinity (Lan et al., 2020).

Additional stabilizing mutations in non-RBD regions were introduced to ensure structural integrity.


2. Plasmid Construction and Viral Engineering



SARS-CoV-2 infectious clones were engineered using a reverse genetics system.

Mutations were introduced into the spike gene using site-directed mutagenesis.


3. In Vitro Assessment of Viral Properties



Cell Lines: Vero E6 and human lung epithelial cells (A549-hACE2).

Infectivity Assay: Plaque-forming units (PFU) were quantified after infection.

Replication Kinetics: Viral RNA levels were measured using RT-qPCR at multiple time points post-infection.


4. In Vivo Evaluation



Mouse models expressing human ACE2 were infected with the engineered strains.

Viral load was measured in lung tissues, and immune responses were assessed by ELISA for spike-specific antibodies.


5. Immunogenicity Testing



Immunogenicity was evaluated by quantifying neutralizing antibody titers and T-cell responses.


 

 

 

Results and Discussion:

1. Reduced Infectivity in Engineered Strains Mutant strains exhibited a 70% reduction in PFU compared to the wild-type virus in Vero E6 cells, indicating impaired infectivity. This reduction was consistent across RBD-targeted mutations (Fig. 1).

2. Attenuated Replication in Human Cells Replication kinetics showed a significant decline in viral RNA levels in A549-hACE2 cells infected with the mutant strains. Peak viral loads were reduced by 60% compared to the wild type (Fig. 2).

3. Immunogenicity and Immune Response Mice infected with mutant strains displayed robust antibody responses, with neutralizing titers comparable to those elicited by wild-type infection. Additionally, CD8+ T-cell activation was observed, indicating strong cellular immunity (Fig. 3).

4. Safety Profile No significant weight loss or clinical symptoms were observed in mice infected with the mutant strains, supporting their safety as vaccine candidates.

These findings demonstrate that targeted mutations in the spike protein can attenuate SARS-CoV-2 while maintaining immunogenicity. The N501D and Q498L mutations specifically impaired ACE2 binding, leading to reduced infectivity and replication. The robust immune responses elicited by these strains underscore their potential as live-attenuated vaccine candidates. Further studies should explore long-term immunity and efficacy in larger animal models.

 

 

 

 

 

 

 

 

References:



Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 181(2), 281-292.

Plotkin, S. A. (2010). Correlates of protection induced by vaccination. Clinical and Vaccine Immunology, 17(7), 1055-1065.

Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., ... & Wang, X. (2020). Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature, 581(7807), 215-220.