Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins.

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.

Immunogenetic and structural analysis of a class of HCV broadly neutralizing antibodies and their precursors.

Elicitation of broadly neutralizing antibodies (bnAbs) is a leading strategy in rational vaccine design against antigenically diverse pathogens. Here, we studied a panel of monoclonal antibodies (mAbs) from mice immunized with the hepatitis C virus (HCV) envelope glycoproteins E1E2. Six of the mAbs recognize the conserved E2 antigenic site 412-423 (AS412) and cross-neutralize diverse HCV genotypes. Immunogenetic and structural analysis revealed that the antibodies originated from two different germline (GL) precursors and bind AS412 in a ß-hairpin conformation. Intriguingly, the anti-HCV activity of one antibody lineage is associated with maturation of the light chain (LC), whereas the other lineage is dependent on heavy-chain (HC) maturation. Crystal structures of GL precursors of the LC-dependent lineage in complex with AS412 offer critical insights into the maturation process of bnAbs to HCV, providing a scientific foundation for utilizing the mouse model to study AS412-targeting vaccine candidates.

Probing the antigenicity of hepatitis C virus envelope glycoprotein complex by high-throughput mutagenesis.

The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen.

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

Unique Structural Features of Influenza Virus H15 Hemagglutinin.

Influenza A H15 viruses are members of a subgroup (H7-H10-H15) of group 2 hemagglutinin (HA) subtypes that include H7N9 and H10N8 viruses that were isolated from humans during 2013. The isolation of avian H15 viruses is, however, quite rare and, until recently, geographically restricted to wild shorebirds and waterfowl in Australia. The HAs of H15 viruses contain an insertion in the 150-loop (loop beginning at position 150) of the receptor-binding site common to this subgroup and a unique insertion in the 260-loop compared to any other subtype. Here, we show that the H15 HA has a high preference for avian receptor analogs by glycan array analyses. The H15 HA crystal structure reveals that it is structurally closest to H7N9 HA, but the head domain of the H15 trimer is wider than all other HAs due to a tilt and opening of the HA1 subunits of the head domain. The extended 150-loop of the H15 HA retains the conserved conformation as in H7 and H10 HAs. Furthermore, the elongated 260-loop increases the exposed HA surface and can contribute to antigenic variation in H15 HAs. Since avian-origin H15 HA viruses have been shown to cause enhanced disease in mammalian models, further characterization and immune surveillance of H15 viruses are warranted.IMPORTANCE In the last 2 decades, an apparent increase has been reported for cases of human infection by emerging avian influenza A virus subtypes, including H7N9 and H10N8 viruses isolated during 2013. H15 is the other member of the subgroup of influenza A virus group 2 hemagglutinins (HAs) that also include H7 and H10. H15 viruses have been restricted to Australia, but recent isolation of H15 viruses in western Siberia suggests that they could be spread more globally via the avian flyways that converge and emanate from this region. Here we report on characterization of the three-dimensional structure and receptor specificity of the H15 hemagglutinin, revealing distinct features and specificities that can aid in global surveillance of such viruses for potential spread and emerging threat to the human population.

DEF pocket in p38α facilitates substrate selectivity and mediates autophosphorylation.

Signaling processes are primarily promoted by molecular recognition and corresponding protein-protein interactions. One of the key eukaryotic signaling pathways is the MAP kinase cascade involved in vital cellular processes such as cell proliferation, differentiation, apoptosis, and stress response. The principle recognition site of MAP kinases, the common docking (CD) region, forms selective interactions with substrates, upstream activators, and phosphatases. A second docking site, defined as the DEF site interaction pocket (DEF pocket), is formed subsequent to ERK2 and p38α activation. Both crystal structures of p38α in its dually phosphorylated form and of intrinsically active mutants showed the DEF pocket, giving motivation for studying its role in substrate activation and selectivity. Mutating selected DEF pocket residues significantly decreased the phosphorylation levels of three p38α substrates (ATFII, Elk-1, and MBP) with no apparent effect on the phosphorylation of MK2 kinase. Conversely, mutating the CD region gave the opposite effect, suggesting p38α substrates can be classified into DEF-dependent and DEF-independent substrates. In addition, mutating DEF pocket residues decreased the autophosphorylation capability of intrinsically active p38α mutants, suggesting DEF-mediated trans-autophosphorylation in p38α. These results could contribute to understanding substrate selectivity of p38α and serve as a platform for designing p38α-selective DEF site blockers, which partially inhibit p38α binding DEF-dependent substrates, whereas maintaining its other functions intact. In this context, preliminary results using synthetic peptides reveal significant inhibition of substrate phosphorylation by activated p38α.

Systematic discovery of antibacterial and antifungal bacterial toxins.

Microorganisms use toxins to kill competing microorganisms or eukaryotic cells. Polymorphic toxins are proteins that encode carboxy-terminal toxin domains. Here we developed a computational approach to identify previously undiscovered, conserved toxin domains of polymorphic toxins within 105,438 microbial genomes. We validated nine short toxins, showing that they cause cell death upon heterologous expression in either Escherichia coli or Saccharomyces cerevisiae. Five cognate immunity genes that neutralize the toxins were also discovered. The toxins are encoded by 2.2% of sequenced bacteria. A subset of the toxins exhibited potent antifungal activity against various pathogenic fungi but not against two invertebrate model organisms or macrophages. Experimental validation suggested that these toxins probably target the cell membrane or DNA or inhibit cell division. Further characterization and structural analysis of two toxin-immunity protein complexes confirmed DNase activity. These findings expand our knowledge of microbial toxins involved in inter-microbial competition that may have the potential for clinical and biotechnological applications.

What has been will be again-The story of viral pathogens.

The emergence of novel avian-origin influenza viruses to circulate in humans has remained a significant threat to public health that might lead to the next pandemic. Studying the receptor specificity of novel influenza subtypes was a crucial milestone in my career path as a young scientist.

Conformational bias imposed by source microseeds results in structural ambiguity.

The p38 MAP kinase pathway is an essential component of numerous cellular signalling networks which are usually activated in response to extracellular environmental stress conditions. In addition to the canonical activation, several alternative activation pathways have been identified for p38; one of these, in which p38 is initially phosphorylated on Tyr323 and consequently autoactivated, is exclusive to T cells and is induced by TCR activation. Intrinsically active and inactive mutants at position 323 have been developed in order to evaluate the structural changes that occur upon TCR-induced activation. In order to promote crystal growth, cross streak-seeding techniques were utilized. This technique has gained popularity in promoting crystal growth when spontaneous nucleation induces critical defects or is being entirely hindered. The crystal characteristics of some mutants were highly similar to those of the wild-type source seeds (form A). In contrast, other mutants crystallized spontaneously with a different space group and molecular packing (form B). One of the active mutants (Y323T) crystallized in both crystal forms, displaying different packing characteristics and significant differences in molecular conformation that were clearly dictated by the source seeds. This implies that the source seeds used in cross streak-seeding could, in some cases, impose bias on the structural outcome of the studied molecule. Such incidents could occur when the conformational freedom permits crystal packing while not reflecting the authentic structure.

From Structural Studies to HCV Vaccine Design.

Hepatitis C virus (HCV) is a serious and growing public health problem despite recent developments of antiviral therapeutics. To achieve global elimination of HCV, an effective cross-genotype vaccine is needed. The failure of previous vaccination trials to elicit an effective cross-reactive immune response demands better vaccine antigens to induce a potent cross-neutralizing response to improve vaccine efficacy. HCV E1 and E2 envelope (Env) glycoproteins are the main targets for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. Therefore, a molecular-level understanding of the nAb responses against HCV is imperative for the rational design of cross-genotype vaccine antigens. Here we summarize the recent advances in structural studies of HCV Env and Env-nAb complexes and how they improve our understanding of immune recognition of HCV. We review the structural data defining HCV neutralization epitopes and conformational plasticity of the Env proteins, and the knowledge applicable to rational vaccine design.

Proof of concept for rational design of hepatitis C virus E2 core nanoparticle vaccines.

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are responsible for cell entry, with E2 being the major target of neutralizing antibodies (NAbs). Here, we present a comprehensive strategy for B cell-based HCV vaccine development through E2 optimization and nanoparticle display. We redesigned variable region 2 in a truncated form (tVR2) on E2 cores derived from genotypes 1a and 6a, resulting in improved stability and antigenicity. Crystal structures of three optimized E2 cores with human cross-genotype NAbs (AR3s) revealed how the modified tVR2 stabilizes E2 without altering key neutralizing epitopes. We then displayed these E2 cores on 24- and 60-meric nanoparticles and achieved substantial yield and purity, as well as enhanced antigenicity. In mice, these nanoparticles elicited more effective NAb responses than soluble E2 cores. Next-generation sequencing (NGS) defined distinct B cell patterns associated with nanoparticle-induced antibody responses, which target the conserved neutralizing epitopes on E2 and cross-neutralize HCV genotypes.

An alternate conformation of HCV E2 neutralizing face as an additional vaccine target.

To achieve global elimination of hepatitis C virus (HCV), an effective cross-genotype vaccine is needed. The HCV envelope glycoprotein E2 is the main target for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. E2 is structurally flexible and functions in engaging host receptors. Many nAbs bind to the "neutralizing face" on E2, including several broadly nAbs encoded by the VH1-69 germline gene family that bind to a similar conformation (A) of this face. Here, a previously unknown conformation (B) of the neutralizing face is revealed in crystal structures of two of four additional E2-VH1-69 nAb complexes. In this conformation, the E2 front-layer region is displaced upon antibody binding, exposing residues in the back layer for direct antibody interaction. This E2 B structure may represent another conformational state in the viral entry process that is susceptible to antibody neutralization and thus provide a new target for rational vaccine development.

VH1-69 antiviral broadly neutralizing antibodies: genetics, structures, and relevance to rational vaccine design.

Broadly neutralizing antibodies (bnAbs) are potential therapeutic molecules and valuable tools for studying conserved viral targets for vaccine and drug design. Interestingly, antibody responses to conserved epitopes can be highly convergent at the molecular level. Human antibodies targeting a number of viral antigens have often been found to utilize a restricted set of immunoglobulin germline genes in different individuals. Here we review recent knowledge on VH1-69-encoded antibodies in antiviral responses to influenza virus, HCV, and HIV-1. These antibodies share common genetic and structural features, and often develop neutralizing activity against a broad spectrum of viral strains. Understanding the genetic and structural characteristics of such antibodies and the target epitopes should help advance novel strategies to elicit bnAbs through vaccination.

Genetic and structural insights into broad neutralization of hepatitis C virus by human VH1-69 antibodies.

An effective vaccine to the antigenically diverse hepatitis C virus (HCV) must target conserved immune epitopes. Here, we investigate cross-neutralization of HCV genotypes by broadly neutralizing antibodies (bNAbs) encoded by the relatively abundant human gene family V H 1-69. We have deciphered the molecular requirements for cross-neutralization by this unique class of human antibodies from crystal structures of HCV E2 in complex with bNAbs. An unusually high binding affinity is found for germ line-reverted versions of VH1-69 precursor antibodies, and neutralization breadth is acquired during affinity maturation. Deep sequencing analysis of an HCV-immune B cell repertoire further demonstrates the importance of the V H 1-69 gene family in the generation of HCV bNAbs. This study therefore provides critical insights into immune recognition of HCV with important implications for rational vaccine design.

N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses.

A species barrier for the influenza A virus is the differential expression of sialic acid, which can either be α2,3-linked for avians or α2,6-linked for human viruses. The influenza A virus hosts also express other species-specific sialic acid derivatives. One major modification at C-5 is N-glycolyl (NeuGc), instead of N-acetyl (NeuAc). N-glycolyl is mammalian specific and expressed in pigs and horses, but not in humans, ferrets, seals, or dogs. Hemagglutinin (HA) adaptation to either N-acetyl or N-glycolyl is analyzed on a sialoside microarray containing both α2,3- and α2,6-linkage modifications on biologically relevant N-glycans. Binding studies reveal that avian, human, and equine HAs bind either N-glycolyl or N-acetyl. Structural data on N-glycolyl binding HA proteins of both H5 and H7 origin describe this specificity. Neuraminidases can cleave N-glycolyl efficiently, and tissue-binding studies reveal strict species specificity. The exclusive manner in which influenza A viruses differentiate between N-glycolyl and N-acetyl is indicative of selection.

The Neutralizing Face of Hepatitis C Virus E2 Envelope Glycoprotein.

The high genetic variability of hepatitis C virus, together with the high level of glycosylation on the viral envelope proteins shielding potential neutralizing epitopes, pose a difficult challenge for vaccine development. An effective hepatitis C virus (HCV) vaccine must target conserved epitopes and the HCV E2 glycoprotein is the main target for such neutralizing antibodies (NAbs). Recent structural investigations highlight the presence of a highly conserved and accessible surface on E2 that is devoid of N-linked glycans and known as the E2 neutralizing face. This face is defined as a hydrophobic surface comprising the front layer (FL) and the CD81 binding loop (CD81bl) that overlap with the CD81 receptor binding site on E2. The neutralizing face consists of highly conserved residues for recognition by cross-NAbs, yet it appears to be high conformationally flexible, thereby presenting a moving target for NAbs. Three main overlapping neutralizing sites have been identified in the neutralizing face: antigenic site 412 (AS412), antigenic site 434 (AS434), and antigenic region 3 (AR3). Here, we review the structural analyses of these neutralizing sites, either as recombinant E2 or epitope-derived linear peptides in complex with bNAbs, to understand the functional and preferred conformations for neutralization, and for viral escape. Collectively, these studies provide a foundation and molecular templates to facilitate structure-based approaches for HCV vaccine development.

The 150-Loop Restricts the Host Specificity of Human H10N8 Influenza Virus.

Adaptation of influenza A viruses to new hosts are rare events but are the basis for emergence of new influenza pandemics in the human population. Thus, understanding the processes involved in such events is critical for anticipating potential pandemic threats. In 2013, the first case of human infection by an avian H10N8 virus was reported, yet the H10 hemagglutinin (HA) maintains avian receptor specificity. However, the 150-loop of H10 HA, as well as related H7 and H15 subtypes, contains a two-residue insert that can potentially block human receptor binding. Mutation of the 150-loop on the background of Q226L and G228S mutations, which arose in the receptor-binding site of human pandemic H2 and H3 viruses, resulted in acquisition of human-type receptor specificity. Crystal structures of H10 HA mutants with human and avian receptor analogs, receptor-binding studies, and tissue staining experiments illustrate the important role of the 150-loop in H10 receptor specificity.

A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors.

In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.

HMGB1 Activates Proinflammatory Signaling via TLR5 Leading to Allodynia.

Infectious and sterile inflammatory diseases are correlated with increased levels of high mobility group box 1 (HMGB1) in tissues and serum. Extracellular HMGB1 is known to activate Toll-like receptors (TLRs) 2 and 4 and RAGE (receptor for advanced glycation endproducts) in inflammatory conditions. Here, we find that TLR5 is also an HMGB1 receptor that was previously overlooked due to lack of functional expression in the cell lines usually used for studying TLR signaling. HMGB1 binding to TLR5 initiates the activation of NF-κB signaling pathway in a MyD88-dependent manner, resulting in proinflammatory cytokine production and pain enhancement in vivo. Biophysical and in vitro results highlight an essential role for the C-terminal tail region of HMGB1 in facilitating interactions with TLR5. These results suggest that HMGB1-modulated TLR5 signaling is responsible for pain hypersensitivity.

Structure and receptor binding of the hemagglutinin from a human H6N1 influenza virus.

Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and human receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. This binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics.