Antibodies are very important in human health. They find and recognize foreign objects 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.
Figure 1. Antibody Molecule
Antibodies have an immunoglobulin form that looks like the Y shaped molecule above (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. We have more than 10^11 antibody forms because of this, 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. Our lab focuses on the fragment antigen binding (Fab) region, which contacts an antigen, and thus is important for vaccine design studies.
Rapidly evolving pathogens, such as human immunodeficiency (HIV) and influenza (flu) viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies (bnAbs) require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In the case of influenza, one must generally study a population of individuals to understand how antibody affinity maturation results in bnAbs against a particular hemagglutinin (HA) found on the virion surface. In HIV infected individuals, viruses and B-cells evolve together, creating a virus-antibody “arms race”. An HIV infected individual contains a microcosm of many different viruses and antibodies, and one can study affinity maturation in a single host to understand the development of bnAbs for a particular HIV epitope.
Broadly neutralizing antibodies (bnAbs) are especially important for vaccine development because they target many different variants of rapidly evolving pathogens, such as HIV and flu. BnAbs become potent through antibody affinity maturation (see below), the process by which B cells produce antibodies with increased affinity for antigen during the course of an immune response.
Figure 2. Schematic of Antibody Affinity Maturation
Antibody affinity maturation is the process by which B cells undergo extensive rounds of proliferation, somatic hypermutation, and antigen-affinity driven selection. B cells express B cell receptors (BCRs) on their cell membrane and allow the B cell to bind a specific antigen, against which it will initiate an antibody response (Fig. 2). Negative selection (failure to interact with antigen) results in apoptosis, or programmed cell death, which is thought to play a key role in the selection of high-affinity variants. The B cell progeny with the highest affinities for antigen will be selected to survive.
Over several rounds of selection, the resultant secreted antibodies produced will have effectively increased affinities for antigen. The result is the production of improved antibodies that effectively recognize infectious agents, and for the production of durable memory B cells. B cells may differentiate into memory B cells, which will respond more quickly to a second exposure to antigen, or antibody-secreting plasma cells.
In summary, in HIV infected individuals, as well as flu infected populations, viruses and B-cells evolve together, creating a virus-antibody “arms race”. The objective of our research is to analyze the arms race in donors who have developed antibodies of significant breadth, which would be informative for immunogen design. We use approaches from both biochemistry and structural biology to understand the interactions of macromolecules with one another at atomic resolution at different time points during infection (i.e. look at the evolution of their interactions).
Our Areas of Focus
Figure 5. Crystal Structure of an Fab (blue) in complex with a piece of the HIV Envelope (red). Green sticks represent glycans (sugars) found on the HIV envelope surface.
Figure 6. Negative Stain EM 2D Class Averages. Low resolution images can provide us with information on the conformation of HIV Env, as well as where the antibody is binding. Arrows in red are pointing to Fabs on HIV Env.
Figure 7. Negative Stain EM 3D Reconstruction. High resolution crystal structures (green and gray ribbon/cartoon) are fit into the low resolution EM map (gray surface). Arrows point to a sugar on HIV Env (at position 332) that is a requirement for the indicated antibody to bind.
The influenza hemagluttinin (HA), like HIV Env, is a trimeric assembly found on the outer surface of the virus (Fig. 8), and the target of broadly neutralizing antibodies. Our current work focuses on identifying how antibodies, produced from vaccinating mice, bind to HA. We are using negative-stain and cryo-electron microscopy for these studies.
Figure 8. Schematic of a flu virion (left) with a zoomed-in view of its HA spike (right). The flu spike has a globular head and stem region, both of which are targets of neutralizing antibodies. Image adapted from Lofano et. al 2015. Front. Immunol.
Major Goals, Questions and Approaches
- How do the antibodies bind to the antigen (e.g. HIV envelope, influenza HA)?
- Use binding studies, atomic resolution structure, etc.
- Electron Microscopy (negative stain & cryo-EM)
- X-ray crystallography
- Biolayer interferometry
- Use binding studies, atomic resolution structure, etc.
- How does the binding of antibody to antigen change over the course of infection/evolution?
- Mutational analysis, binding studies
- Analysis of antibodies and/or antigen from different time points of an infection
- Can we design an antigen to engage the ancestor antibody of a given lineage?
- Analyze sequence and/or structure data
- Cloning, binding studies
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.)
- Bacterial cell growth
- Mammalian tissue culture maintenance
- Pull downs
- Biolayer Interferometry
- X-ray Crystallography
- Negative Stain Electron Microscopy
- Cryo-electron Microscopy
We gratefully acknowledge support from:
- amfAR - The Foundation for AIDS Research
- Swarthmore College