David Ho: Characterizing and isolating antibodies from convalescent patients (CUMC COVID-19 Symposium)
Summary by: Arooba Ahmed ('23)
Dr. Ho’s group specializes in isolating and neutralizing antibodies from patients recovering from illnesses. In addition, they screen and construct entry inhibitors, which are a class of antiretroviral drugs used in combination therapy for the treatment of HIV infection. Currently they wish to focus on understanding the spiked protein that is at the surface of the coronavirus. Its structure was recently solved, showing that the Receptor Binding Domain (RBD) binds to the receptor on epithelial cells in the airways. They are looking for antibodies that will bind to the receptor binding domain and mediate virus neutralization.
They have multiple approaches that they are using in parallel. But to conduct these studies they need to identify the right patients. Using specific patient samples from mainland China professors, they constructed reagents such as the spike primer and s-1 portion of spiked gene on the virus and found that binding of primer was better than the s-1 protein.
One treatment option being utilized is plasma therapy, but this is a long process which can be ineffective, as demonstrated by the minimal neutralizing responses a few weeks after recovery from infection. Looking at the blood sample data from the first case admitted to New York Presbyterian shows that the patient was trying to neutralize the COVID virus, but only two weeks into the illness. As of now, they are working with three cases to obtain neutralizing antibodies.
In the first strategy, the group takes the blood samples and sorts for memory B cells that are CD27 positive. Memory B cells are important because they can repeatedly generate an accelerated and robust antibody-mediated immune response in the case of re-infection. CD27 is a protein currently of interest to immunologists as a co-stimulatory immune checkpoint molecule. They then performed RT-PCR on these cells to measure the amount of RNA for this protein and removed the heavy and light chain genes (which create specific polypeptide sequences of different molecular weights on antibodies). They then displayed these genes on a yeast library so they could screen out any receptor they wished to use. Yeast library is a protein engineering technique that uses the expression of recombinant proteins incorporated into the cell wall of yeast for isolating and engineering antibodies. The yeast library technique is beneficial because it is a renewable source.
The second strategy looks at sorting through cells. They wanted CD19 (a biomarker for normal and neoplastic B cells) positive and CD20 (B-lymphocyte antigen) negative immature blood cells. These cells are subject to single cell PCR sequencing, which examines the sequence information from individual cells to provide a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment.
In their final approach, they took the blood serum with high concentration of the neutralizing antibody, absorbed that with the trimer formed from the RBD, eluted the bound antibodies and subjected it to mass spectrometry analysis. This technique has been used in the past to identify antibodies in HIV infected serum, but in that case databases were readily available for sequencing. Here, there are no databases available so they must identify specific amino acid sequences and from the DNA sequencing and sort out antibody species to generate a database.