Exam 4: Antigen Recognition by B-Cell and T-Cell Receptors

Some species, like camels, alpacas, and llamas, have evolved variant forms of immunoglobulin proteins that retain the ability to bind to antigens. While overall the antibodies made by these animals are simpler than human or mouse antibodies, an important feature conserved among all of these antibodies is:

Individuals or mice with defects in the biochemical pathways needed for loading peptides onto MHC molecules show greatly increased susceptibility to virus infections. Experiments examining the MHC molecules present on the surface of host cells in these individuals would show:

Antibody binding to a pathogen surface is greatly enhanced when both antigen-binding sites of the antibody are engaged at once, a feature known as bivalent binding. It is possible for antibodies to bind bivalently to a wide variety of components on many different pathogen surfaces due to the flexibility in the protein at the hinge region and at the V-C junction.

Amino acid sequence analysis of all of the peptides found in a single IgG antibody would reveal unique peptide sequences totaling ~600-700 amino acids. Using this estimate, the predicted molecular weight of an antibody protein would be ~70-75 kDa. Yet, an intact antibody protein has a molecular weight of ~150 kDa. The explanation for this discrepancy is:

MHC genes are the most polymorphic genes in the human genome. This means that, within the population, few individuals share exactly the same sequences for all of their MHC proteins. What deleterious outcome might occur if all humans shared exactly the same sequence for their MHC proteins?

MHC class I molecules generally bind peptides that are 8-10 amino acids. Each allelic variant has preferences for the amino acid residues at key anchor positions, but will not bind every possible peptide containing the correct anchor residues.

Each immunoglobulin (Ig) domain is composed of a structure known as a ' $\beta$ -sandwich,' which consists of two $\beta$ sheets covalently linked by a disulfide bond. Only a subset of the ~110 amino acids in each domain are required to establish this overall structure, and it is these amino acids that are highly conserved when comparing Ig domains to each other. What might be the advantage of this structure for use as antibody variable domains?

T cells expressing $\gamma$ : $\delta$ TCRs are distinct from those expressing $\alpha$ : $\beta$ TCRs in that they do not generally recognize host cell responses to infections or tissue damage; rather they recognize components of the pathogen directly.

Like innate sensors of infections (TLRs, NLRs, RLRs), antibodies frequently recognize nucleic acids of pathogenic organisms.

Once expressed on the surface of host cells, an MHC protein remains stably associated with its bound peptide for several days. This highly stable peptide binding behavior is important because:

$\alpha$ : $\beta$ TCRs are membrane-bound proteins comprised of two polypeptides linked by a disulfide bond. Both polypeptide components of the $\alpha$ : $\beta$ TCR are members of the immunoglobulin superfamily, and each of their domains share structural similarity with regions of antibody proteins. However, due to the different functions of TCRs versus antibodies, the overall domain organization of the TCR is not the same as for an antibody. In the cartoon in Figure Q14), describe three features that are incorrect illustrations of the $\alpha$ : $\beta$ TCR.

Synthesis question: Several vaccines against viral infections are made by isolating purified surface proteins of the viral particle, mixing them with an adjuvant to stimulate an innate immune response, and injecting the mixture into people. Two examples of this are the vaccine against Hepatitis B virus, and the vaccine against Human Papilloma Virus (the 'cervical cancer' vaccine). One interesting property of vaccines of this type (known as 'subunit vaccines') is that there is a requirement for a CD4 T cell response to the vaccine antigen in order to generate antibodies to the innocuous protein in the vaccine. In the case of the Hepatitis B vaccine, the viral protein included in the vaccine is the Hepatitis B surface antigen (HepB-SAg), a protein that is approximately 200 amino acids in length. The graph in Figure shows the data from immunizing individuals with this vaccine, and monitoring their production of protective antibody responses to the viral protein. a) What results would be predicted if experiments were performed to examine the CD4 T cell responses to the HepB-SAg in these same individuals? In particular, indicate whether all individuals would show similar responses. Explain your reasoning. b) Most vaccines, particularly those made by immunizing individuals with purified pathogen components, are designed to elicit robust antibody responses to the immunizing antigens. For vaccines against viral infections (like the HepB vaccine), the immune responses generated are only protective when administered prophylactically, i.e., before the individual is ever exposed to the pathogen. Why is it essential to vaccinate against viral infections before the individual is ever exposed to the virus?

In Figure Q8), which close-up view of these two V domains has the amino acid sequences most important for antigen-binding highlighted correctly in red?

Synthesis question: For the last five years, the seasonal flu vaccine has contained a mixture of two Influenza A strains and one Influenza B strain. The Influenza A strains were categorized as H1N1 and H3N2 subtypes. This nomenclature refers to the sequences of the two surface glycoproteins on the Influenza A virus particle, the hemaglutinin (H) and the neuraminidase (N). Antibodies specific for these glycoproteins are known to be effective at preventing flu infection. a) The highly pathogenic Asian avian flu causes a fatal infection in about 60% of the individuals infected. The seasonal flu vaccine does not provide protection against this strain of Influenza. Is the highly pathogenic Asian avian flu likely to be an H1N1 or H3N2 strain of influenza? Why or why not? In the seasonal vaccine, the two strains of Influenza A, H1N1 and H3N2, are both required to provide protection to individuals exposed to one or the other of the viral strains. Currently, research efforts are focused on trying to generate protective CD8 T cell responses to Influenza A, with the goal of generating a 'universal' or broadly neutralizing vaccine that would provide protection against multiple strains of the virus, even those not included in the vaccine. b) Based on the information provided in the cartoon of the two viral strains shown in Figure , what is the reasoning for expecting CD8 T cell responses to be protective against multiple different strains of Influenza A? From year to year, the Influenza A strains circulating in the population undergo a process known as 'antigenic drift' in which mutations accumulate in the viral genes, leading to modest changes in the amino acid sequences of the viral proteins. Due to this antigenic drift, different isolates of the H1N1 or H3N2 strains are included in the annual flu vaccine. Shown in Figure are some of the regions of viral proteins that often undergo antigenic drift from year to year. c) What is the minimum number of amino acids that needs to change for a neutralizing antibody to the neuraminidase of the H1N1 strain on the left to no longer bind to the neuraminidase of the H1N1 strain on the right?

Early studies analyzing the antibody protein fragments generated after proteolytic cleavage revealed important information about the overall structure of the antibody molecule. Which cleavage pattern (indicated by the red triangles in Figure) yields a fragment that has the same antigen-binding avidity as the intact antibody, but is unable to activate complement after binding to a pathogen?

Which of the two antibodies shown in Figure are most likely to have the same antigen-binding specificity? Explain your reasoning.

One striking feature of TCR interactions with peptide:MHC complexes is that amino acid residues in the MHC protein are as important to the TCR binding strength as are amino acid residues in the pathogen-derived peptide. This feature is in contrast to antigen recognition by antibodies, which is a direct interaction that is independent of other host proteins. Based on the different functions of T cells versus antibodies in the adaptive immune response, the fact that TCRs recognize components of both the MHC and the bound peptide exists to:

When a mixture of different IgG antibody proteins are treated with the enzyme papain, each antibody is cleaved into three roughly equal size fragments. From each original antibody, two of the three fragments are identical to each other, and represent the 'arms' of the antibody 'Y'. These fragments are known as Fab fragments. The third fragment is known as the Fc region, because this fragment will crystallize when purified. The reason a mixture of Fc fragments will crystallize is because:

The antibody protein has two functional domains, one for antigen binding and a second to confer specific effector functions. These two functional domains are encoded by the antibody light chain and antibody heavy chain polypeptides, respectively.

The cellular distribution of MHC class I versus MHC class II molecules is quite different, with MHC class II molecules generally expressed on a very limited set of cell types. This is because: