Somatic diversity in CDR3 loops allows single V-genes to encode innate immunological memories for multiple pathogens

The human Ab response to many common pathogens is oligoclonal, with restricted usage of Ig V-genes. Intriguingly, the IGVK3-11 and IGVH3-30 V-genes are repeatedly paired in protective Abs against the 23F polysaccharide of Streptococcus pneumoniae, as well as against the gB envelope protein of human CMV, where germline-encoded amino acids make key contacts with the gB protein. We constructed IgGs encoded by the germline IGVK3-11 and IGVH3-30 V-genes together with DNA encoding the respective CDR3 regions of the L chain and H chain found in a hypermutated anti-23F Ab. These IgGs encoded by germline V-genes bound specifically to 23F pneumococcal capsular polysaccharides with no reactivity to other serotypes of pneumococcal capsular polysaccharides or arrayed glycans and recognized L-rhamnose, a component of the 23F repeating subunit. IgGs encoded by this pair of germline V-genes mediated complement-dependent phagocytosis of encapsulated 23F S. pneumoniae by human neutrophils. Mutations in CDRL3 and CDRH3 had significant effects on binding. Thus, IGKV3-11 and IGHV3-30, depending on with which distinct DNA sequences encoding CDR3 they are recombined, can encode binding sites for protective Abs against chemically distinct Ags and thus, may encode innate immunological memory against human CMV and S. pneumoniae.     

Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp1663-Ser1669

We have identified the Factor VIII amino acid sequence Asp-Tyr-Asp-Asp-Thr-Ile-Ser (1663-1669) as the binding site of a Factor VIII activity neutralizing antibody (28 Bethesda units/mg). The binding site of another neutralizing antibody (10 Bethesda units/mg) overlapped only at Asp1663 and Tyr1664, whereas an antibody with minimal neutralizing activity (0.2 Bethesda units/mg) bound only at Asp1665-Ser1669. Residues comprising antibody binding sites were determined by blocking Factor VIII neutralization and/or binding to insolubilized Factor VIII with overlapping peptides, or with variant peptides in which a single amino acid was deleted or replaced with glycine. Eight additional antibodies to flanking sequences, and with similar affinities for Factor VIII, had little or no neutralizing activity (0-3.0 Bethesda units/mg). These studies suggest that Asp1663 and Tyr1664 may be structural features important to Factor VIII function.     

Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.     

A comparative analysis of the immunological evolution of antibody 28B4

In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K(d) = 25 microM) and 28B4 Fab (K(d) = 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95(H)Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.     

Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus.

Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Fourteen clonally isolated mutants demonstrated substantial resistance to multiple monoclonal antibodies, including K3-4C8-K3-2F2 and B5-B3. In addition, 13 mutants demonstrated a 10-fold or greater reduction in neutraliztion mediated by polyclonal human antibody. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development.     

Structural plasticity and the evolution of antibody affinity and specificity

The germline precursor to the ferrochelatase antibody 7G12 was found to bind the polyether jeffamine in addition to its cognate hapten N-methylmesoporphyrin. A comparison of the X-ray crystal structures of the ligand-free germline Fab and its complex with either hapten or jeffamine reveals that the germline antibody undergoes significant conformational changes upon the binding of these two structurally distinct ligands, which lead to increased antibody-ligand complementarity. The five somatic mutations introduced during affinity maturation lead to enhanced binding affinity for hapten and a loss in affinity for jeffamine. Moreover, a comparison of the crystal structures of the germline and affinity-matured antibodies reveals that somatic mutations not only fix the optimal binding site conformation for the hapten, but also introduce interactions that interfere with the binding of non-hapten molecules. The structural plasticity of this germline antibody and the structural effects of the somatic mutations that result in enhanced affinity and specificity for hapten likely represent general mechanisms used by the immune response, and perhaps primitive proteins, to evolve high affinity, selective receptors for so many distinct chemical structures.     

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.     

Germline antibody recognition of distinct carbohydrate epitopes

High-resolution structures reveal how a germline antibody can recognize a range of clinically relevant carbohydrate epitopes. The germline response to a carbohydrate immunogen can be critical to survivability, with selection for antibody gene segments that both confer protection against common pathogens and retain the flexibility to adapt to new disease organisms. We show here that antibody S25-2 binds several distinct inner-core epitopes of bacterial lipopolysaccharides (LPSs) by linking an inherited monosaccharide residue binding site with a subset of complementarity-determining regions (CDRs) of limited flexibility positioned to recognize the remainder of an array of different epitopes. This strategy allows germline antibodies to adapt to different epitopes while minimizing entropic penalties associated with the immobilization of labile CDRs upon binding of antigen, and provides insight into the link between the genetic origin of individual CDRs and their respective roles in antigen recognition.     

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.     

Study of the antigenic structure of the E1 glycoprotein of the Venezuelan equine encephalomyelitis virus using monoclonal antibodies]

The collection of eight rat and mouse hybridomas secreting the high affinity monoclonal antibodies to glycoprotein E1 of the Venezuelan equine encephalomyelitis has been obtained. The antigenic structure of E1 protein has been studied with the use of these antibodies for the strains Trinidad, TC-83 and 230 of the virus. Antigenic map of glycoprotein E1 based on competition radioimmunoanalysis is proposed. Five sites are mapped including eight epitopes binding monoclonal antibodies. Antibodies to sites E1-1, E1-3 and E1-5 are crossreactive in interaction with the virus of Venezuelan equine encephalomyelitis, while antibodies to site E1-5 interact also with the virus of tick-borne encephalitis. Antibodies to site E1-1 possess the protective effect and lack the neutralizing effect in tissue cultures. Antibodies to all sites of E1 protein are devoid of ability to neutralize the Venezuelan equine encephalitis virus.     

The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.     

Antibody humanization by molecular dynamics simulations—in‐silico guided selection of critical backmutations

Monoclonal antibodies represent the fastest growing class of biotherapeutic proteins. However, as they are often initially derived from rodent organisms, there is a severe risk of immunogenic reactions, hampering their applicability. The humanization of these antibodies remains a challenging task in the context of rational drug design. “Superhumanization” describes the direct transfer of the complementarity determining regions to a human germline framework, but this humanization approach often results in loss of binding affinity. In this study, we present a new approach for predicting promising backmutation sites using molecular dynamics simulations of the model antibody Ab2/3H6. The simulation method was developed in close conjunction with novel specificity experiments. Binding properties of mAb variants were evaluated directly from crude supernatants and confirmed using established binding affinity assays for purified antibodies. Our approach provides access to the dynamical features of the actual binding sites of an antibody, based solely on the antibody sequence. Thus we do not need structural data on the antibody–antigen complex and circumvent cumbersome methods to assess binding affinities. © 2016 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.     

Viral Escape from HIV-1 Neutralizing Antibodies Drives Increased Plasma Neutralization Breadth through Sequential Recognition of Multiple Epitopes and Immunotypes

Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies.     

Cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites

A number of flaviviruses are important human pathogens, including yellow fever, dengue, West Nile, Japanese encephalitis, and tick-borne encephalitis (TBE) viruses. Infection with or immunization against any of these viruses induces a subset of antibodies that are broadly flavivirus cross-reactive but do not exhibit significant cross-neutralization. Nevertheless, these antibodies can efficiently bind to the major envelope protein (E), which is the main target of neutralizing and protective antibodies because of its receptor-binding and membrane fusion functions. The structural basis for this phenomenon is still unclear. In our studies with TBE virus, we have provided evidence that such cross-reactive antibodies are specific for a cluster of epitopes that are partially occluded in the cage-like assembly of E proteins at the surfaces of infectious virions and involve-but are not restricted to-amino acids of the highly conserved internal fusion peptide loop. Virus disintegration leads to increased accessibility of these epitopes, allowing the cross-reactive antibodies to bind with strongly increased avidity. The cryptic properties of these sites in the context of infectious virions can thus provide an explanation for the observed lack of efficient neutralizing activity of broadly cross-reactive antibodies, despite their specificity for a functionally important structural element in the E protein.     

Idiotypic and fluorometric analysis of the antibodies that distinguish the lesion of the I-A mutant B6.C-H-2bm12

The serologic lesion of the I-A mutant mouse strain, bm12, was investigated with the use of monoclonal anti-Iab antibodies and anti-idiotypic (Id) reagents produced against these antibodies. In a fluorometric analysis, three different monoclonal anti-Iab antibodies (25-9-17, 34-5-3, 28-16-8) failed to bind bm12 cells, whereas two anti-Iab antibodies (25-5-16 and 17/227), which bound bm12 cells, showed about one-half the fluorescence intensity that they showed in binding to Iab antigens. Of the three monoclonal antibodies that failed to react with bm12 cells, two antibodies (25-9-17 and 34-5-3) were found to bind the same steric site on Iab molecules (cluster I). In contrast, the antibodies (25-5-16 and 17/227) that reacted with both Iab and Iabm12 antigens were found to bind a second distinct site (cluster II). The binding of antibody 28-16-8 to Iab antigens inhibited reciprocally the binding of cluster I and II anti-Iab antibodies, suggesting a possible third site, sterically located intermediate between the other two sites. To assess the relatedness of the antibodies defining the serologic lesion of bm12 mice, xenogeneic and syngeneic anti-Id reagents were produced against antibodies 25-9-17 and 28-16-8. By using these anti-Ids in a binding site-related inhibition assay, a cross-reactive idiotype was detected that is shared by 25-9-17 and 34-5-3 antibodies; thus these two monoclonal antibodies share several features, including 1) idiotypic determinants, 2) failure to bind bm12 cells, 3) binding the same spatial Iab site, and 4) having indistinguishable serologic fine specificity that corresponds with a previously defined predominant alloantigenic determinant recognized in the bm12 anti-Iab humoral response. Therefore, several parameters of antibody recognition of Ia can now be correlated with structural changes in Ia molecules. These findings will potentiate future studies of the T cell recognition of these same Ia epitopes.     

Selection of Apoptotic Cell Specific Human Antibodies from Adult Bone Marrow

Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC)-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.     

Topographical analysis of antigenic determinants on envelope glycoprotein V3 (E) of Japanese encephalitis virus, using monoclonal antibodies.

Ten monoclonal antibodies directed against envelope glycoprotein V3 (E) of Japanese encephalitis virus were obtained. They were characterized by hemagglutination inhibition (HI), neutralization, and enzyme-linked immunosorbent assay and divided into four types: flavivirus-cross-reactive HI and non-neutralizing antibody (group 1), subgroup-specific HI and non-neutralizing antibody (group 2), low HI and neutralizing antibody (group 3), and non-HI and neutralizing antibody (groups 4 and 5, respectively). Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay. The results of the competitive binding assay separated non-HI and neutralizing antibody into groups 4 and 5, respectively, and demonstrated the existence of at least five distinct antigenic determinants on V3. The site of group 1 was distinct from any other site. The sites of groups 2 and 3 seemed to be located close together. Our results suggest the following relationship between HI and neutralization: (i) The HI sites are separated from the neutralization sites, and (ii) there are two distinct HI sites, one of which is flavivirus cross-reactive, the other subgroup specific.     

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site.

The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during 'antigenic drift.'     

Improved induction of antibodies against key neutralizing epitopes by human immunodeficiency virus type 1 gp120 DNA prime-protein boost vaccination compared to gp120 protein-only vaccination

A major challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development is to elicit potent and broadly neutralizing antibodies that are effective against primary viral isolates. Previously, we showed that DNA prime-protein boost vaccination using HIV-1 gp120 antigens was more effective in eliciting neutralizing antibodies against primary HIV-1 isolates than was a recombinant gp120 protein-only vaccination approach. In the current study, we analyzed the difference in antibody specificities in rabbit sera elicited by these two immunization regimens using peptide enzyme-linked immunosorbent assay and a competitive virus capture assay. Our results indicate that a DNA prime-protein boost regimen is more effective than a protein-alone vaccination approach in inducing antibodies that target two key neutralizing domains: the V3 loop and the CD4 binding site. In particular, positive antibodies targeting several peptides that overlap with the known CD4 binding area were detected only in DNA-primed sera. Different profiles of antibody specificities provide insight into the mechanisms behind the elicitation of better neutralizing antibodies with the DNA prime-protein boost approach, and our results support the use of this approach to further optimize Env formulations for HIV vaccine development.     

Cluster and information entropy patterns in immunoglobulin complementarity determining regions

Previous studies of antibody binding domains have established many crucial features that include important structural positions, canonical formations, and the geometric correlations with the binding site nature and topography. In this work, position-specific frequency and hierarchical clustering analysis are used to explore the statistical pattern of the residues in the complementarity determining regions of human antibodies. In addition, Shannon's information entropy is computed for the entire heavy and light chains and compared with germline patterns to seek variability due to antibody clonal selection. Results are compared with reported analyses based on structural data and ligand-protein contact point computations based on Protein Data Bank records. Observations derived from the present sequence analysis are consistent with previous structural based methods. In the absence of structural data, methods used in this work can be effective and efficient computational tools used for identifying residues that are important for antigen targeting and predicting the probable amino acid distribution expected at these positions. The results in turn can be applied to help design or plan mutagenesis experiments to improve the binding properties of antibodies.