Very Few Substitutions in a Germ Line Antibody Are Required To Initiate Significant Domain Exchange

2G12 is a broadly neutralizing anti-HIV-1 monoclonal human IgG1 antibody reactive with a high-mannose glycan cluster on the surface of glycoprotein gp120. A key feature of this very highly mutated antibody is domain exchange of the heavy-chain variable region (V_H) with the V_H of the adjacent Fab of the same immunoglobulin, which assembles a multivalent binding interface composed of two primary binding sites in close proximity. A non-germ line-encoded proline in the elbow between V_H and C_H1 and an extensive network of hydrophobic interactions in the V_H/V_H′ interface have been proposed to be crucial for domain exchange. To investigate the origins of domain exchange, a germ line version of 2G12 that behaves as a conventional antibody was engineered. Substitution of 5 to 7 residues for those of the wild type produced a significant fraction of domain-exchanged molecules, with no evidence of equilibrium between domain-exchanged and conventional forms. Two substitutions not previously implicated, A^H14 and E^H75, are the most crucial for domain exchange, together with I^H19 at the V_H/V_H′ interface and P^H113 in the elbow region. Structural modeling gave clues as to why these residues are essential for domain exchange. The demonstration that domain exchange can be initiated by a small number of substitutions in a germ line antibody suggests that the evolution of a domain-exchanged antibody response in vivo may be more readily achieved than considered to date.Protein oligomers are able to exchange or swap an element of their secondary structure or an entire protein domain. The functional unit in domain-exchanged proteins thereby stays preserved, as only the linking hinge loop changes conformation significantly (4, 17, 27). Analogous to other domain-swapped proteins, antibodies can exchange an entire domain, in this case the heavy-chain variable region (V_H), with an equivalent heavy-chain variable region of an adjacent Fab (V_H′) within the same immunoglobulin (Ig) molecule (11). The advantages of domain-exchanged proteins, including antibodies, are higher local concentrations of active sites, a larger binding surface, and a potential secondary active site at the new subunit interface (27, 45). The one and only antibody shown to be domain exchanged to date is 2G12 (7, 11), but this arrangement is potentially possible for any Ig and could have been overlooked at least in some instances.2G12 is one of only a few high-affinity monoclonal antibodies with broad neutralizing activity against different subtypes of HIV-1 (5, 30, 40, 43). The antibody binds a dense cluster of N-linked high-mannose glycans (Man_8-9GlcNAc₂) on the envelope surface glycoprotein gp120 (10, 35, 36, 41). The domain-exchanged arrangement forms a multivalent binding site composed of two primary binding sites in close proximity and a proposed secondary binding site formed by the novel V_H/V_H′ interface (11). 2G12 provides protection against infection in animal models (19, 31) and has been shown to induce neutralization escape following passive immunization in humans (39).Consensus has grown that a successful HIV-1 vaccine will need to include a component that elicits broadly neutralizing antibodies (8, 18, 21, 26, 32, 42). All attempts to elicit 2G12-like antibodies with the desired specificity and neutralization activity have failed to date (22, 29, 44), conceivably due to difficulties in generating adequate mimicry of the glycan cluster and tolerance mechanisms or, very likely, the inability to induce domain exchange (1). Unraveling the mechanism of domain exchange and how this conformation might have evolved is highly desirable to direct future HIV-1 vaccine design to elicit 2G12-like antibodies.By comparison with other domain-exchanged proteins (27), the following three mechanisms have been proposed to contribute to the unique structure of 2G12 compared to the structure of a conventional antibody: destabilization of the “closed” V_H/V_L interface, conformational change in the elbow between V_H and C_H1, and an energetically favorable “open” V_H/V_H′ interface (11). Key residues involved in promoting domain exchange were predicted based on examination of interacting residues at the two interfaces and by the effects of alanine substitutions on the binding of wild-type 2G12 to gp120. However, the importance of these key residues for domain exchange was not directly demonstrated experimentally (11).Here, we explored the minimal requirements for domain exchange of 2G12, starting with a germ line version of the antibody that adopts a conventional antibody structure. Although wild-type 2G12 is heavily somatically mutated, only five to seven substitutions in the germ line version of the antibody were shown to produce a significant fraction of domain-exchanged molecules. The results suggest the evolution of domain-exchanged antibody responses may be more facile than considered to date.     

Structure,Receptor Binding,and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 Å resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.Influenza (flu) is an infection of the respiratory tract that affects millions of people every year. In addition to the seasonal toll, three flu pandemics in the past century caused millions of deaths worldwide in relatively short time periods (27). In April 2009, a novel strain of influenza A virus H1N1 (S-OIV) with swine origin emerged in North America and has become the first influenza pandemic in 4 decades. To date, this new H1N1 pandemic has spread globally and caused at least 7,800 deaths (World Health Organization, http://www.who.int).Hemagglutinin (HA) is the major surface envelope glycoprotein on influenza virus, and responsible for essential viral functions, such as binding to host receptors, viral entry, and membrane fusion (31). A key factor that determines the host range, restriction, and transmission of influenza virus is the specificity of HA for binding glycan receptors comprising terminal sialic acids linked to a vicinal galactose residue. HAs in avian viruses are specific for sialic acids with an α2,3-linkage, whereas in humans, the specificity is for sialic acids with an α2,6-linkage (Fig. ​(Fig.1a).1a). This simple linkage difference likely contributes to the inability of most avian influenza viruses to become established and transmit in the human population (26). Influenza pandemics in humans are generally associated with nonhuman viruses of novel antigenicity acquiring specificity for human receptors. HA is also the principal antigen of influenza viruses and the main target for neutralizing antibodies.Open in a separate windowFIG. 1.Crystal structure of H2 HA. (a) Chemical structures of α2,3- and α2,6-linked glycans, with the terminal sialic acid and galactose shown here. (b) Overview of the 1957 H2 trimer. One of the monomers is highlighted in green (HA1) and blue (HA2), respectively. Five potential glycosylation sites are found on each monomer (as labeled). Glycans in the density map are shown in orange. (c) Receptor binding site of H2. Residues involved in receptor binding, as suggested by the H3 structures, are shown in sticks. Aromatic residues comprising the base of the binding site are absolutely conserved in various HA subtypes. Residues from the 220 loop and position 190 are critical for the receptor specificity switch in H1, H2, and H3.Although future influenza pandemics seem inevitable, predicting the potential HA subtypes that will emerge remains a daunting task (41). To date, 16 HA subtypes have been identified and classified based on their antigenic properties (1). Theoretically, all influenza viruses new to the immune system of the human population today possess the potential to initiate a flu pandemic if their ability to enter human cells and transmit efficiently evolves. Historically, however, only viruses of three HA subtypes have acquired the ability to efficiently transmit from human to human, and these were responsible for the influenza pandemics of the last century: 1918 (H1N1), 1957 (H2N2), 1968 (H3N2), and 2009 (H1N1). In recent years, viruses of other HA subtypes (H5, H7, and H9) of avian origin have infected humans in sporadic cases and occasionally with very high mortality, such as H5N1 (2, 4, 10). A key barrier to avian flu becoming a human pandemic is its inefficient human-to-human transmission, which requires a switch of receptor specificity from α2,3- to α2,6-linked receptors. Although the H2 subtype has disappeared from the human population since 1968, it has reemerged in swine in the United States (19). Preparedness for future pandemics can be best addressed by rigorous characterization of the HA subtypes that have already caused pandemics, as well as development of therapeutic reagents that broadly target multiple influenza subtypes.Here, we present three crystal structures of human H2 HA from the 1957 pandemic at resolutions of 1.60, 1.73, and 1.75 Å. These structures, which differ only by one or two residues in the receptor-binding site, represent the evolution of binding specificity for human-like receptors of avian origin during the 1957 H2N2 pandemic. Structural comparisons among the structures, along with glycan array binding studies, have shed new light on the requirements for avian H2 HA to adapt for human transmission.     

Activation of the N-Terminally Truncated Form of the Stk Receptor Tyrosine Kinase Sf-Stk by Friend Virus-Encoded gp55 Is Mediated by Cysteine Residues in the Ecotropic Domain of gp55 and the Extracellular Domain of Sf-Stk

Friend virus induces an erythroleukemia in susceptible mice that is initiated by the interaction of the Friend virus-encoded glycoprotein gp55 with the erythropoietin (Epo) receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk, recruitment of a Grb2/Gab2/Stat3 signaling complex, and induction of Pu.1 expression by Stat3 are required for the development of the early stage of Friend disease both in vitro and in vivo. Here we demonstrate that the interaction of gp55 with Sf-Stk is dependent on cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk. Point mutation of these cysteine residues or deletion of these domains inhibits the ability of gp55 to interact with Sf-Stk, resulting in the inability of these proteins to promote the Epo-independent growth of erythroid progenitor cells. We also demonstrate that the interaction of gp55 with Sf-Stk does not promote dimerization of Sf-Stk but results in enhanced phosphorylation of Sf-Stk and the relocalization of Sf-Stk from the cytosol to the plasma membrane. Finally, we demonstrate that a constitutively active form of Sf-Stk (Sf-StkM330T), as well as its human counterpart, Sf-Ron, promotes Epo-independent colony formation in the absence of gp55 and that this response is also dependent on the cysteines in the extracellular domains of Sf-StkM330T and Sf-Ron. These data suggest that the cysteines in the extracellular domains of Sf-Stk and Sf-Ron may also mediate the interaction of these truncated receptors with other cellular factors that regulate their ability to promote cytokine-independent growth.Since Friend disease was first reported in 1957 (19), the acute erythroleukemia induced by the various strains of Friend virus have provided an excellent model to study multistage carcinogenesis (5). In the first stage, the virus infects erythroid progenitor cells and a viral glycoprotein, gp55, interacts with both the erythropoietin receptor (EpoR) and a naturally occurring truncated form of the stem cell-derived tyrosine kinase (Stk), Sf-Stk, resulting in the Epo-independent (Epo^ind) expansion of erythroid progenitor cells. The late stage of erythroleukemia in Friend disease is marked by inactivation of the p53 locus (6, 28, 38, 39, 51) and proviral integration into the Spi-1 locus (36, 43, 44), resulting in enhanced expression of Pu.1, which causes a block in erythroid differentiation and promoting the onset of acute erythroleukemia.Friend virus is a complex of two viruses, the spleen focus-forming virus (SFFV), which is a replication-defective C-type retrovirus, and the ecotropic Friend murine leukemia virus (F-MuLV). SFFV is responsible for the rapid splenomegaly and acute erythroleukemia induced by Friend virus infection (7, 64, 65, 67), while F-MuLV provides helper function and can be substituted for by other murine leukemia viruses (35). Specifically, the glycoprotein gp55, encoded by the SFFV env gene, acts as the transforming viral oncoprotein (2, 65).Several loci in the mouse genome that control Friend virus susceptibility have been identified. Fv1, Fv3, and Fv4 affect the ability of Friend virus to infect early erythroid progenitor cells. The Fv1 gene product inhibits Friend virus infection by interacting with the viral capsid protein (60). The Fv3 gene encodes cytidine deaminase Apobec3, which broadly inhibits retrovirus infection (42, 53, 57). The Fv4 gene product affects viral binding by competing for receptors on the cell membrane (59). Another set of genes, W, Sl, f, and Fv2, are required for the development or expansion of infected progenitor cells. Our previous work demonstrated that W, Sl, and f, which encode the kit receptor, its ligand SCF, and Smad5, respectively, also play key roles in the BMP4-dependent stress erythropoiesis pathway(46, 47, 55). Analysis of those mutants showed that Friend virus activates this pathway, leading to acute amplification of stress progenitors, which are targets of Friend virus in the spleen, and resulting in rapid onset of disease.The Friend virus susceptibility gene Fv2 encodes the stem cell-derived tyrosine kinase (Stk) receptor (48). A naturally occurring N-terminally truncated form of Stk, short-form Stk (Sf-Stk), is required for Friend virus susceptibility. Fv2^r/r mice, including C57BL/6, lack expression of Sf-Stk and are resistant to Friend virus infection, while full-length Stk expression is unaffected in these mice. An internal promoter within the Stk locus drives Sf-Stk expression, and Fv2^r/r mice harbor mutations in the internal promoter. Sf-Stk lacks the N-terminal ligand binding domain of full-length Stk but retains the transmembrane and tyrosine kinase domains. In vitro and in vivo expression of Sf-Stk in C57BL/6 bone marrow cells has been shown to confer Friend virus susceptibility to Fv2^r/r mice (18).Sf-Stk covalently interacts with gp55, resulting in constitutive activation of Sf-Stk (41). However, the mechanism by which this occurs is currently unknown. Here, we identify cysteines in the extracellular domains of Sf-Stk and gp55 that mediate this interaction. Furthermore, we demonstrate that while the association with gp55 is not required for the dimerization of Sf-Stk, the interaction of gp55 with Sf-Stk promotes tyrosine phosphorylation of Sf-Stk. In addition, while the extracellular cysteines in Sf-Stk promote retention of Sf-Stk in the cytoplasm in the absence of gp55, the interaction of Sf-Stk with gp55 through these cysteines results in enhanced cell surface localization of Sf-Stk. These changes in receptor activation and subcellular localization mediate the ability of Sf-Stk to induce gene expression and promote the Epo^ind growth of primary erythroblasts.     

Antibody 2G12 Recognizes Di-Mannose Equivalently in Domain- and Nondomain-Exchanged Forms but Only Binds the HIV-1 Glycan Shield if Domain Exchanged

The broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibody 2G12 targets the high-mannose cluster on the glycan shield of HIV-1. 2G12 has a unique V_H domain-exchanged structure, with a multivalent binding surface that includes two primary glycan binding sites. The high-mannose cluster is an attractive target for HIV-1 vaccine design, but so far, no carbohydrate immunogen has elicited 2G12-like antibodies. Important questions remain as to how this domain exchange arose in 2G12 and how this unusual event conferred unexpected reactivity against the glycan shield of HIV-1. In order to address these questions, we generated a nondomain-exchanged variant of 2G12 to produce a conventional Y/T-shaped antibody through a single amino acid substitution (2G12 I19R) and showed that, as for the 2G12 wild type (2G12 WT), this antibody is able to recognize the same Manα1,2Man motif on recombinant gp120, Candida albicans, and synthetic glycoconjugates. However, the nondomain-exchanged variant of 2G12 is unable to bind the cluster of mannose moieties on the surface of HIV-1. Crystallographic analysis of 2G12 I19R in complex with Manα1,2Man revealed an adaptable hinge between V_H and C_H1 that enables the V_H and V_L domains to assemble in such a way that the configuration of the primary binding site and its interaction with disaccharide are remarkably similar in the nondomain-exchanged and domain-exchanged forms. Together with data that suggest that very few substitutions are required for domain exchange, the results suggest potential mechanisms for the evolution of domain-exchanged antibodies and immunization strategies for eliciting such antibodies.The broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) human monoclonal antibody 2G12 recognizes a highly conserved cluster of oligomannose residues on the glycan shield of the HIV-1 envelope glycoprotein gp120 (9, 10, 36, 39, 44, 45). The antibody binds terminal Manα1,2Man-linked sugars of high-mannose glycans (Man_8-9GlcNAc₂) with nanomolar affinity using a unique domain-exchanged structure in which the variable domains of the heavy chains swap to form a multivalent binding surface that includes two conventional antigen-combining sites and a third potential noncanonical binding site at the novel V_H/V_H′ interface (10). gp120 is one of the most heavily glycosylated proteins identified to date, with approximately 50% of its mass arising from host-derived N-linked glycans (24). These glycans play an important role in shielding the virus from the host immune system (34). Carbohydrates are generally poorly immunogenic, and the dense covering of glycans is often referred to as the “silent face” (52). The oligomannose glycans on gp120 in particular are closely packed, forming a tight cluster, and the unique domain-exchanged structure of 2G12 has been proposed as a means to recognize this cluster (10).The attraction of 2G12 as a template for HIV-1 vaccine design has recently been highlighted in a study that showed the antibody can protect macaques against simian-human immunodeficiency virus (SHIV) challenge at remarkably low serum neutralizing titers (18, 30, 43). When using 2G12 as a template for design of a carbohydrate immunogen, some important considerations must be taken into account. First, 2G12 is unusual in its specificity (targeting host cell-derived glycan motifs presented in a “nonself” arrangement), and although the 2G12 epitope is common to many HIV-1 envelopes, 2G12-like antibodies are rarely elicited (5, 38). Second, due to inherently weak carbohydrate-protein interactions (49, 50), it can be assumed that in order for a carbohydrate-specific antibody to achieve the affinity required to neutralize HIV-1, the avidity of the interaction must be enhanced by both Fab arms of the IgG-contacting glycan motifs simultaneously on the HIV-1 envelope. Third, the unique domain-exchanged structure of 2G12 has not been described for any other antibody (10). These considerations raise a number of questions. Which antigen or sequence of antigens elicited 2G12? Is domain exchange the only solution for recognition of highly clustered oligomannoses? If so, can domain exchange be elicited by immunization with clustered oligomannose motifs (38)?Efforts to design immunogens that elicit responses to the glycan shield of HIV-1 and neutralize the virus have to date been unsuccessful (2, 3, 14, 20, 21, 28, 29, 32, 46-48). Immunogen design strategies that mimic the 2G12 epitope have focused on both chemical and biochemical methods to generate multivalent and clustered displays of both high-mannose sugars (Man_8-9GlcNAc₂) (13, 15, 20, 21, 27-29, 32, 47) and truncated versions of such sugars (Man₉ and Man₄ linked via a 5-carbon linker) (3, 46). These constructs typically bind 2G12 with a lower affinity (on the order of 1 to 3 logs) than recombinant gp120. Although mannose-specific antibodies have been elicited by these immunogens, no HIV-1-neutralizing activities have been described. In a study by Luallen et al., antibodies against recombinant gp120 were generated by immunization with yeast cells that had been mutated to display only Man₈GlcNAc₂ glycans (27, 29). However, no neutralization activity against the corresponding pseudovirus was noted. It was proposed that this was due to either the low abundance of the gp120-specific antibodies in the serum or the antibodies elicited being against carbohydrate epitopes that differed from the 2G12 epitope (27, 29).To gain a better understanding of the importance of domain exchange for glycan recognition and how 2G12 may have been induced, we analyzed the binding characteristics of a nondomain-exchanged (conventional Y/T-shaped) 2G12 variant antibody. This variant was generated by a single point mutation, I19R, that disrupts the V_H/V_H′ interface. We show that the mutant is still able to recognize the Manα1,2Man motif arrayed on yeast, synthetic glycoconjugates, and recombinant gp120 in enzyme-linked immunosorbent assay (ELISA) format but is unable to recognize the discrete, dense mannose clusters found on the surface of the HIV-1 envelope (as measured by neutralization activity and binding to HIV-1-transfected cells). We further show that a major conformational change in the elbow region between V_H and C_H1 in this nondomain-exchanged variant of 2G12 allows the variable domains to assemble in similar orientations with respect to each other, as in the 2G12 wild type (WT), with an identical primary binding site, although with dramatically different orientations with respect to the constant domains. Thus, we conclude that 2G12 recognizes Manα1,2Man motifs in an identical manner in both conventional and domain-exchanged configurations, and the 2G12 specificity for Manα1,2Man likely first arose in a conventional IgG predecessor of 2G12. Subsequent domain exchange was the key event that then enabled high-affinity recognition of the tight oligomannose clusters on HIV-1.     

Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis

The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly¹⁷⁶, Asp¹⁷⁷, Gly³²³, and Tyr³²⁴) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface.     

A new method for gravity correction of dynamometer data and determining passive elastic moments at the joint

Moments measured by a dynamometer in biomechanics testing often include the gravitational moment and the passive elastic moment in addition to the moment caused by muscle contraction. Gravitational moments result from the weight of body segments and dynamometer attachment, whereas passive elastic moments are caused by the passive elastic deformation of tissues crossing the joint being assessed. Gravitational moments are a major potential source of error in dynamometer measurements and must be corrected for, a procedure often called gravity correction. While several approaches to gravity correction have been presented in the literature, they generally assume that the gravitational moment can be adequately modeled as a simple sine or cosine function. With this approach, a single passive data point may be used to specify the model, assuming that passive elastic moments are negligible at that point. A new method is presented here for the gravity correction of dynamometer data. Gravitational moment is represented using a generalized sinusoid, which is fit to passive data obtained over the entire joint range of motion. The model also explicitly accounts for the presence of passive elastic moments. The model was tested for cases of hip flexion-extension, knee flexion-extension, and ankle plantar flexion-dorsiflexion, and provided good fits in all cases.     

Weighing risk factors associated with bee colony collapse disorder by classification and regression tree analysis

Colony collapse disorder (CCD), a syndrome whose defining trait is the rapid loss of adult worker honey bees, Apis mellifera L., is thought to be responsible for a minority of the large overwintering losses experienced by U.S. beekeepers since the winter 2006-2007. Using the same data set developed to perform a monofactorial analysis (PloS ONE 4: e6481, 2009), we conducted a classification and regression tree (CART) analysis in an attempt to better understand the relative importance and interrelations among different risk variables in explaining CCD. Fifty-five exploratory variables were used to construct two CART models: one model with and one model without a cost of misclassifying a CCD-diagnosed colony as a non-CCD colony. The resulting model tree that permitted for misclassification had a sensitivity and specificity of 85 and 74%, respectively. Although factors measuring colony stress (e.g., adult bee physiological measures, such as fluctuating asymmetry or mass of head) were important discriminating values, six of the 19 variables having the greatest discriminatory value were pesticide levels in different hive matrices. Notably, coumaphos levels in brood (a miticide commonly used by beekeepers) had the highest discriminatory value and were highest in control (healthy) colonies. Our CART analysis provides evidence that CCD is probably the result of several factors acting in concert, making afflicted colonies more susceptible to disease. This analysis highlights several areas that warrant further attention, including the effect of sublethal pesticide exposure on pathogen prevalence and the role of variability in bee tolerance to pesticides on colony survivorship.