This research experience was at The Scripps Research Institute (TSRI) in La Jolla, California.
The Scripps Research Institute (TSRI) — one of the world’s largest, private, non-profit research organizations — stands at the forefront of basic biomedical science, a vital segment of medical research that seeks to comprehend the most fundamental processes of life.(www.scripps.edu)
In here I had the experience of working in the International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Center under the expertise of Dr. Richard Wyatt and Dr. Shailendra Kumar in HIV-1 gp140 trimer design for protein-based vaccine approach.
Abstract
The Human Immunodeficiency Virus (HIV) is a lentivirus that infects vital immune-cells that regulate immunological response against viruses, bacteria, pathogens and substances that appear harmful for the human body. HIV entrance into permissible cells is mediated by its surface exposed envelope glycoprotein complex or gp160 (Env) and is the sole target for broadly neutralizing antibodies. For a successful HIV vaccine, a soluble, native-like and stable Env mimic will likely be required to induce high-titer broadly neutralizing antibodies(bNAbs). In this study, we employed the newly discovered cleavage-independent Native Flexible Linker (NFL) platform for the design and development of a native-like, soluble and stable Env immunogen. This platform replaces the furin cleavage site in gp140 with a flexible “Glycine-Serine” peptide linker (G4S) and has a HR1 helix-destabilizing substitution (I559P) to achieve highly stable and covalently linked gp120-gp41 trimers in native-like association/structure. DU422 is a Tier-2, Clade-C HIV-1 isolated in South Africa. The DU422 Env gene was modified according to the NFL platform, expressed in 293F cells and affinity-purified by lectin column. After lectin purification we got >90% pure DU422NFL gp140 Env. Immunoprecipitation by trimer preferring bNAbs and non-NAB suggested the presence of native-like, soluble trimers along with small proportion of aberrant Env trimeric/dimer/monomer species. Further purification by SEC gave a homogenous sharp peak composed entirely of DU422NFL trimers and a small dimer/monomer peak. In this study we have successfully designed, expressed and purified a native-like Clade-C DU422 Env trimer in a NFL platform with high yield, which upon further investigation could be used as a vaccine candidate for immunogenicity studies.
In this research experience I gained deep knowledge of laboratory techniques including and not limited to :
- Bacteria transformation using heat shock and competent cells
- Cell Transfection for protein expression
- Site-directed mutagenesis
- Liquid bacteria culture inoculation
- Inmunoprecipitation
- SDS-PAGE
- Plasmid DNA purification (Maxi-Prep and Mini-prep)
- Engineering of mutation inducing primers
Also encountering troubleshooting in the following areas:
- DNA from bacteria cell transformation without desired mutation due to Dpn1 treatment not being sufficient
- Low or no protein production from transfection due to insufficient use of transfection agent
- Genomic contamination after Plasmid DNA purification due to excess of Lysing time
This research experience received funding by NIH grants: HIV Vaccine Research and Design (HIVRAD) Program (P01): PO1 AI104722, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID): AI100663 and The Scripps Research Institute SURF Program
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