A central focus of recent research aimed at developing a vaccine for HIV-1 is the identification of potent broadly-neutralizing antibodies (bNAbs).  Due to work from several laboratories, many such antibodies have now been identified, produced in quantity as monoclonal antibodies, and characterized with respect to key properties such as epitope specificity, affinity for the corresponding HIV-1 epitope, and neutralizing activity against many strains of varying susceptibility to antibody-mediated inactivation (important examples of these publications are: Scheid et al., 2009; Walker et al., 2009; Wu et al., 2010; Walker et al., 2011; Huang et al., 2012).  These successes notwithstanding, the scale of the challenge facing the vaccine developers is clarified by the following facts: 1) potent bNAbs only develop in 10-30% of infected individuals, 2) it typically takes between two and three or four years after initial infection for these antibodies to appear in the blood of these individuals, and 3) antibodies with the desired attributes often have extraordinary numbers of somatic mutations in the variable domains that mediate binding to the HIV-1 antigen (Klein et al., 2013a).   A study (Klein et al., 2013b) published earlier this year from the laboratory of Michel Nussenzweig both illuminates one possible factor accounting for the impressive length of time and number of mutations associated with the generation of potent bNAbs and provides an extraordinary example of the power of intense selection to confound expectations arising from previously observed associations.  In this instance, the undermined expectations related to the well-established functional correlates of hypervariable and framework regions within antibody variable domains.

Antibody variable (V) domains, found on both of the constituent polypeptide chains composing antibody molecules, i.e. heavy and light chains, are the structures containing the sites that bind to antigens.  Within the V domains, polypeptide sequences are classified into hypervariable (HV) regions and framework (FW) regions based on the extent of amino acid sequence variability.  As implied by the names, HV regions exhibit more amino acid sequence variability from one antibody to the next than do the FW regions.  Well-supported conventional wisdom has it that the great majority of amino acid residues that make physical contact with antigen are in the HV regions while the FW regions serve primarily to provide a stable scaffold from which to suspend the HV loops.  In most of the many known antibody-antigen complexes for which three-dimensional structures are known, only an occasional FW residues is seen to engage in direct van der Waals contact with antigen residues.

While most antibodies against other antigens typically exhibit 15-20 somatic mutations in the heavy chain variable domain (VH), antibodies with weak neutralizing activity against HIV-1 can display up to 40 VH mutations from germline sequences.  Impressively, potent bNAbs specific for HIV-1 can exhibit as many as 80-100 VH mutations.  Even more unexpectedly, Nussenzweig and colleagues show that many of these residues are in FW regions.  Some of the bNAbs they study have thirty or more VH somatic mutations located in FW regions.

Klein et al. further show that reverting all of the FW mutations to their germline amino acids greatly reduces neutralization breadth (fraction of tested strains that suffer reduced infection ability) and potency (reciprocal of antibody concentration required to neutralize half of a virus population) for most of the bNAbs but has little effect on antibodies with weaker neutralization activity.  Additionally, the authors demonstrate that germline reversion only for FW residues that do not directly contact antigen also reduces potency and breadth of neutralization.

Based on all of their data, Nussenzweig and colleagues conclude that FW residues can contribute substantially to the neutralization activity of bNAbs through both direct contact with antigen and through influencing the positioning (in some sense) of contact residues.  They argue that this unique pattern of V domain somatic mutation may require multiple rounds of mutation and selection of B lymphocytes in the germinal centers, the anatomical structures within secondary lymphoid organs that are the main sites for somatic hypermutation and affinity maturation of immunoglobulin genes in B cells.  Such iterative mutation and selection are likely the result of the ability of HIV-1 to escape the antibodies generated by initial rounds of somatic hypermutation due to the exceptionally high mutation rate of the HIV-1 genome.  This process is a genetic arms race with a vengeance played out within the confines of a single host.

The broader lesson is that the reasonable assumption, based on inductive reasoning, that amino acids in the antibody FW regions were functionally important primarily as scaffolding permitting the propitious display of the HV loops is clearly not necessarily correct.  I have previously noted the Principle of Radical Evolutionary Indifference, which suggests that evolution by natural selection is unconstrained by our category boundaries and classification schemes.  The work of Nussenzweig and his associates provides a powerful illustration of this principle at work in a medically relevant context of great import.  It also illustrates the ease with which intense selection can sunder even firmly established associations between structures and functions and suggests that biologists and biomedical scientists should exercise caution when tempted to universalize structure-function correlations made in a limited number of experimental contexts.


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