Bio Patrika interviews Dr. Datta on her thoughts about “method to rapidly identify target sites of HIV virus neutralizing antibodies”

Dr. Rohini Datta’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Datta is currently a Postdoctoral Research Fellow in the lab of Jin Billy Li at Department of Genetics, Stanford School of Medicine, Stanford University, USA. Work presented here is done as part of her PhD thesis in the lab of Prof. Raghavan Varadarajan at Molecular Biophysics Unit, Indian Institute of Science, Bangalore. Dr. Rohini published a paper entitled “A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing” as a first author in Proc Natl Acad Sci USA journal (2020).

How would you explain your paper’s key results to the non-scientific community?

Despite decades of research, the quest for an effective HIV vaccine that can stimulate the production of neutralizing antibodies is still ongoing. Neutralizing antibodies not only bind to the virus but also prevent further infection. Our recent paper describes an exciting new method to rapidly identify target sites (‘epitopes’) of these neutralizing antibodies on HIV viruses.

We focused our epitope mapping efforts on the HIV envelope glycoprotein (Env), the major surface protein of HIV. We mutated the virus to introduce several cysteine molecules on the surface of the Env protein. We then added a chemical label that binds specifically to the cysteines and treated mutant viruses with neutralizing antibodies. The addition of the cysteine label at target sites blocks the binding of neutralizing antibodies, and as a result, these viruses survive and cause infection. Sequencing genes of the surviving viruses were then used to rapidly identify epitopes of these neutralizing antibodies (Figure 1).

Figure 1. Simplified schematic of our epitope mapping technique.

Identification of epitopes in sera (the portion of blood containing mixed antibodies) using conventional approaches is quite challenging – since different antibodies in the mixture have different epitopes. By leveraging our technology, we successfully identified multiple epitopes targeted by sera from an HIV-infected patient. This patient can naturally withstand HIV infection in the absence of anti-retroviral therapy on account of having potent neutralizing antibodies. Such individuals are extremely rare (less than 1%) and are called ‘elite neutralizers’. Knowledge of epitopes targeted by antibodies from elite neutralizers, obtained using our approach, can be used to design better vaccines against HIV to end the global HIV/AIDS epidemic. The method can also be straightforwardly extended to other emerging viruses such as SARS-CoV-2.

Figure 2. Engineered cysteines (in red) mapped on the surface of the HIV Env protein showing (A) top view and (B) side view. The Env protein is an assembly of three gp120 molecules (green) and three gp41 molecules (blue). The cysteines that are introduced are uniformly distributed on the surface of the Env to maximize the coverage of epitopes.

“[…] enables identification of epitopes directly on infectious HIV viruses.”

What are the possible consequences of these findings for your research area?

Our method enables the identification of epitopes directly on infectious HIV viruses. This is a significant improvement over other indirect epitope mapping methods (which require purified proteins or cell surface display) and represents a major advance in the field.

Using our approach, neutralizing antibody epitopes can be accurately mapped in a highly parallelizable manner, without the need for advanced and expensive instrumentation used in techniques such as X-ray crystallography and cryo-electron microscopy. Besides, a significant advantage of our method is the ability to distinguish neutralizing antibodies from non-neutralizing ones. It is the former that is the focus of vaccine design efforts, and the ability to differentiate between the two can aid in the development of better vaccines against HIV.

What was the exciting moment (eureka moment) during your research?

The discovery of epitopes targeted by antibodies of the HIV-infected ‘elite neutralizer’ was the most exciting part of the study. Neutralizing antibodies from this patient’s sera target two distinct epitopes on the HIV Env protein – we believe that this contributes to the observed neutralization activity. The presence of highly potent neutralizing antibodies targeting these distinct epitopes makes the patient naturally resistant to HIV infection in the absence of anti-retroviral therapy. These experiments were made possible by the patient sera provided by our collaborator and co-author, Dr. Luke Elizabeth Hanna from NIRT, Chennai. The epitopes identified by us can inform future vaccine design regimens.

What do you hope to do next?

I hope that the technology described in this work will be utilized widely to identify neutralizing antibody epitopes in emerging viral infections such as SARS-CoV-2. In my current role, I am exploring the biology and regulation of RNA editing by ADAR deaminases. My long-term goal is to elucidate the mechanistic basis of the perturbation of cellular pathways in viral infections, centering on the role of regulatory RNA molecules. This would integrate my educational background, academic training, and current postdoctoral research.

Where do you seek scientific inspiration?

I derive scientific inspiration from advisors, teachers, mentors, and peers. Being advised by Prof. Varadarajan during my PhD has been a learning experience – I have felt constantly motivated to carry out productive research under his able guidance. My postdoctoral advisor Prof. Li, through his creative problem-solving skills and affable mentorship, makes research enjoyable and provides an ideal environment for me to work.

I have also sought inspiration from reading biographies of influential female scientists like Marie Curie, Lise Meitner, Rachel Carson, Dorothy Hodgkin, Rita-Levi Montalcini, to name a few. It is humbling to know how these illustrious women dealt with tough working conditions, sexism, exile, skepticism and not always getting the recognition they deserved. The remarkable achievements of these women have changed science as we know it!

How do you intend to help Indian science improve?

At IISc, I have been actively involved with several scientific outreach programs throughout my PhD, including high school teachers’ training from underprivileged government schools in Karnataka. During my PhD, I have mentored several interns in their research projects. Some of them are now pursuing PhD programs in India. I am an author for the The Wire Science, where I recently wrote about the frontrunner COVID vaccines and my personal take on them. In the future, I will continue to work for the advancement of Indian science through scientific outreach, mentorship, and effective scientific communication.

Reference

Datta R, Roy Chowdhury R, Manjunath K, Hanna LE and Varadarajan R. A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing. Proc Natl Acad Sci USA. 117(47):29584-29594 (2020).

Current Affiliation: Postdoctoral Research Fellow, Department of Genetics, Stanford School of Medicine, Stanford University, USA

Postdoctoral advisor: Prof. Jin Billy Li

Website: https://med.stanford.edu/profiles/rohini-datta

Twitter: https://twitter.com/dattarohini

The Biophysics PhD program at the Indian Institute of Science provided me an exciting opportunity to work at the interface of cutting-edge structural biology and viral pathogenesis. The primary focus of my PhD was on the co-evolution between the HIV-1 virus and neutralizing antibodies – the result of an arms race between the host immune system and the rapidly evolving virus. At IISc, I was fortunate to have Prof. Raghavan Varadarajan as my doctoral advisor. The Varadarajan laboratory has been at the forefront of vaccine design efforts against HIV, influenza and more recently, SARS-CoV-2 (http://mbu.iisc.ac.in/~rvgrp/research.html).

Working on the formidable pathogen HIV, which hijacks the RNA editing machinery of host cells to drive viral replication, piqued my interest in the biology and regulation of RNA editing. I am currently pursuing postdoctoral research in the laboratory of Prof. Jin Billy Li at Stanford University, a leader in the field of RNA editing by ADAR deaminases. ADARs carry out highly targeted Adenosine-to-Inosine modifications (‘edits’) in double-stranded RNA substrates. These modifications play key roles in a plethora of biological functions, including the regulation of innate immunity in response to viral infections. In the Li lab, I am working on identifying novel dsRNA substrates of ADAR that play a role in cancer and autoimmune diseases. This research will provide important clues on the role of RNA editing in immune regulation and the mechanisms of autoimmunity.

Email: rohini.datta89@gmail.com; rohinid@stanford.edu

Learn more about research interests of Prof. Varadarajan lab http://mbu.iisc.ac.in/~rvgrp/home.html

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