Background
Our Team of Biomolecules:
Our Team of Biomolecules:
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Antibodies are very important in human health. They find and recognize foreign objects (called "antigens") in our bodies and help us fight off infection, especially when we become sick. They work by either preventing viruses or harmful bacteria or other foreign antigens from entering our cells or they actually activate the cells responsible for killing viruses or other pathogens.
Antibodies have an immunoglobulin form that looks like a Y shaped molecule (Fig. 1). The fragment antigen binding (Fab) region contacts an antigen, such as a bacteria or virus, through its variable domains, which are highly variable. These are the regions that are recombined and mutated to produce a large repertoire of antibodies, which allows the antibodies to recognize so many different foreign antigens. The tips of these molecules have loops, called complementarity determining regions (CDRs), that directly make contact with an antigen. |
Figure 1. Antibody Molecule
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RNA virus spikes are recognized by antibodies, and thus are important for vaccine design studies. They are located on the outer surface of a virus (Fig. 2), and help the virus latch onto and enter a target host cell. Viruses, such as those from human immunodeficiency (HIV), influenza (flu), and severe acute respiratory syndrome (SARS) viruses, escape immune defenses in a variety of ways, making vaccine development challenging. HIV and flu are rapidly evolving pathogens, and so they accumulate lots of mutations in their surface spikes. Theses viruses also cover their spikes with sugars that the immune system considers "self" and won't produce antibodies against. |
Figure 2. Schematic of an HIV virion (left) with a zoomed-in view of its spike (right). The orange and blue ovals represent variable loops on the HIV surface. Both the red and green portions represent the HIV spike. |
Protein kinases that we study function inside the cell, unlike the antibodies that prevent viruses from host-cell entry. There are ~500 known human kinases, highlighting their importance in life. Protein kinases play a critical role in antibody production by regulating the signaling pathways that control the development, activation, proliferation, and differentiation of B cells, which produce antibodies. Some kinases regulate survival and growth signals. Additionally, kinase activity is essential for recombination mutation events that enhance antibody diversity and efficacy. Kinases are enzymes that act as molecular on/off switches by transferring a phosphate (negatively charged ion) from one molecule to another. This can turn on other molecules, which then can go on to perform some action, including the expression of certain genes. In the case of B cell activation, B cells proliferate and can become plasma cells which secrete antibodies.
Our Areas of Focus
1. Signaling Pathways That Influence Antibody Production.
B cells produce antibodies against both foreign antigens, such as viruses, as well as self antigens. In the case of self antigens, there are processes in the body that indicate that the antibodies are binding self and the B cells that produce these antibodies are destroyed. If they are not destroyed, the result is autoimmune disease.
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The message that an antigen has bound to receptors on the outer surface of B cells gets transmitted into the cell through signaling pathways involving kinases and other molecules (Fig. 3). B cells have a receptor (called the B cell receptor or "BCR") on their outer surface, that bind to an antigen. Upon antigen binding to the BCR, the Src-family kinase Lyn is one of the first kinases activated. Lyn phosphorylates other biomolecules which then creates docking sites for other kinases. These early events activate multiple pathways, including the RAS–RAF–MEK–ERK pathway. We are determining models of how these kinases bind to their targets and how they themselves are regulated to ensure activity when needed.
Understanding these pathways is important for understanding how B cells function properly to produce antibodies against foreign antigens and also how disruptions in these kinase signaling networks makes them fail in eliminating antibodies that bind self antigens. |
Figure 3. Antigen binding to B cell receptor (BCR) activates intracellular proteins via biochemical modifications, such as phosphorylation. This turns on kinases, such as Lyn and ERK. Downstream events can lead to the activation of transcription factors that help turn on genes to lead to cell proliferation, differentiation, etc.
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2. Analyzing Interactions between Viral Spikes and Antibodies.
Viral entry into host cells can cause a range of issues. Additionally, while there are drugs that help with treatment against viruses, sometimes the development of resistance or lack of access to medication can be an issue. The development of therapeutic antibodies, and/or vaccines to trigger antibodies that prevent viral entry into host cells, would help control and reduce the burden of disease.
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Towards this goal, we are analyzing how antibodies bind to the spikes of a variety of viruses (Fig. 4). Much of our focus is on the spikes of HIV, but we also focus on those from other RNA viruses, including influenza (flu) and SARS-CoV-2. In particular, we study antibodies from infected individuals who have managed to fight off or control these viruses, as well as antibodies from animals that have been immunized with some form of the spike.
Analyzing antibodies from infected individuals will provide insight on features of the antibodies that are good to elicit by vaccination, or ones that can be used therapeutically. Moreover, analyzing antibodies from immunization trials will provide insight on how good the vaccine design strategy was and suggest further improvements. |
Figure 4. Antibodies binding to spikes on viral surface
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We use approaches from both biochemistry and structural biology to understand the interactions of macromolecules with one another at atomic resolution. We expect that these studies will help us better understand the processes that result in antibodies, and inform the design of vaccines and antibody-based treatments.
We use approaches from both biochemistry and structural biology to understand the interactions of macromolecules with one another at atomic resolution. We expect that these studies will help us better understand the processes that result in antibodies, and inform the design of vaccines and antibody-based treatments.
Techniques Employed
Our lab employs a variety of techniques (the list below indicates some of the main techniques used):
- Sequence Analysis
- DNA recombinant technology
- Protein expression and purification
- Various forms of chromatography (affinity, size exclusion, etc.)
- Electrophoresis
- Bacterial cell growth
- Mammalian tissue culture maintenance
- Pull downs
- Biolayer Interferometry
- Phosphorylation Assays
- X-ray Crystallography
- Negative Stain Electron Microscopy
- Cryo-electron Microscopy
- Computational Modeling
FUNDING
We gratefully acknowledge support from:
- NIH NIAID
- The Camille & Henry Dreyfus Foundation
- The Pittsburgh Foundation
- Research Corporation for Science Advancement
- amfAR - The Foundation for AIDS Research
- Swarthmore College