A common solution to group 2 influenza virus neutralization

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.Influenza viruses are a significant and persistent threat to human health worldwide. Annual epidemics cause 3–5 million cases of severe illness and up to 0.5 million deaths (1), and periodic influenza pandemics have the potential to kill millions (2). Inhibitors against the viral surface glycoprotein neuraminidase are widely used for the treatment of influenza infections, but their efficacy is being compromised by the emergence of drug-resistant viral strains (3). Vaccination remains the most effective strategy to prevent influenza virus infection. However, protective efficacy is suboptimal in the highest risk groups: infants, the elderly, and the immunocompromised (1). Furthermore, because immunity after vaccination is typically strain-specific and influenza viruses evolve rapidly, vaccines must be updated almost annually. The antigenic composition of the vaccine is based on a prediction of strains likely to circulate in the coming year, therefore, mismatches between vaccine strains and circulating strains occur that can render the vaccine less effective (4). Consequently, there is an urgent need for new prophylactic and therapeutic interventions that provide broad protection against influenza.Immunity against influenza viruses is largely mediated by neutralizing antibodies that target the major surface glycoprotein hemagglutinin (HA) (5, 6). Identification of antigenic sites on HA indicates that influenza antibodies are primarily directed against the immunodominant HA head region (7), which mediates endosomal uptake of the virus into host cells by binding to sialic acid receptors (8). Because of high mutation rates in the HA head region and its tolerance for antigenic changes, antibodies that target the HA head are typically only effective against strains closely related to the strain(s) by which they were elicited, although several receptor binding site-targeting antibodies with greater breadth have been structurally characterized (9–15). In contrast, antibodies that bind to the membrane-proximal HA stem region tend to exhibit much broader neutralizing activity and can target strains within entire subtypes and groups (16–25) as well as across influenza types (24). These stem-directed antibodies inhibit major structural rearrangements in HA that are required for the fusion of viral and host endosomal membranes and thus, prevent the release of viral contents into the cell (8). The stem region is less permissive for mutations than the head and relatively well-conserved across divergent influenza subtypes.Anti-stem antibodies are elicited in some, but not all, individuals during influenza infection or vaccination (20, 26) and thus, hold great promise as potential broad spectrum prophylactic or therapeutic agents and for the development of a universal influenza vaccine (27–29). The majority of the known heterosubtypic stem binding antibodies neutralize influenza A virus subtypes belonging to group 1 (17–20, 23, 25). Furthermore, two antibodies that target a similar epitope in the HA stem, like most heterosubtypic group 1 antibodies, are able to more broadly recognize both group 1 and 2 influenza A viruses (22) or influenza A and B viruses (24). Strikingly, group 2-specific broadly neutralizing Abs (bnAbs) seem to be rare, because only one has been reported to date (21). CR8020 uniquely targets a distinct epitope in the stem in close proximity to the viral membrane at the HA base and binds lower down the stem than any other influenza HA antibody (21).In the discovery process that led to the isolation of bnAb CR8020, we recovered additional group 2-specific bnAbs. Here, we describe one such bnAb, CR8043, which recognizes a similar but nonidentical footprint on the HA as CR8020 and approaches the HA from a different angle. Furthermore, these two bnAbs are derived from different germ-line genes and, consequently, use distinct sets of interactions for HA recognition. Thus, the human immune system is able to recognize this highly conserved epitope in different ways using different germ-line genes. Hence, this valuable information can be used for the design of therapeutics and vaccines targeting this site of vulnerability in group 2 influenza A viruses that include the pandemic H3N2 subtype.     

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.Seasonal influenza outbreaks across the globe cause an estimated 250,000–500,000 deaths annually (1). Current influenza vaccines need to be updated every few years because of antigenic drift (2). Despite intensive monitoring, strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past (2). Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift facilitating a potential pandemic outbreak. These concerns have expedited efforts toward developing a universal influenza vaccine.Neutralizing antibodies (nAbs) against hemagglutinin (HA) are the primary correlate for protection in humans and hence HA is an attractive target for vaccine development (3). The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the disulfide-linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit, whereas the stem domain predominantly comprises the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH-induced conformational rearrangement in the host-cell endosomes to adopt the virus–host membrane fusion-competent state (4, 5).The antigenic sites on the globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed nAbs (6). However, extensive efforts have resulted in the isolation of monoclonal antibodies (mAbs) that bind within the globular head and inhibit receptor attachment, which neutralize drifted variants of an HA subtype or heterosubtypic HA (7–16). The HA stem is targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes (17). The epitopes of these bnAbs in the HA stem are more conserved across different influenza HA subtypes compared with the antigenic sites in the HA globular head (18).During a primary infection, the immunodominant globular head domain suppresses the response toward the conserved stem. Several efforts have been made to circumvent this problem. Repeated immunizations with full-length, chimeric HAs (cHAs) in a protracted vaccination regimen have been shown to boost stem-directed responses in mice (19). Alternatively, full-length HA presented on nanoparticles (np) has been shown to elicit stem-directed nAbs (20). Attempts have also been made to steer the immune response toward the conserved HA stem by hyperglycosylating the head domain (21). Although the aforementioned strategies need to be further evaluated and provide novel alternatives, detrimental interference from the highly variable immunodominant head domain in eliciting a broad functional response cannot be completely evaded. A “headless” stem domain immunogen offers an attractive solution. However, early attempts at expressing the HA2 subunit independently in a native, prefusion conformation were unsuccessful. In the absence of the head domain, the HA2 subunit expressed in Escherichia coli spontaneously adopted the low-pH conformation (22) in which the functional epitopes of stem-directed bnAbs are disrupted. More recently, the entire HA stem region has been expressed in a prefusion, native-like conformation in both prokaryotic and eukaryotic systems adopting multiple strategies (23–26).Design of independently folding HA stem fragments which adopt the prefusion HA conformation presents another approach to elicit bnAbs against influenza (27, 28). The A helix of the HA2 subunit contributes substantial contact surface to the epitope of stem-directed bnAbs such as CR6261, F10, and others. Although multivalent display of A helix on the flock house virus as a virus-like particle platform elicited cross-reactive antibodies, it conferred only minimal protection (20%) against virus challenge in mice (29).We report the design and characterization of engineered headless HA stem immunogens based on the influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation-sensitive, stem-directed bnAbs such as CR6261 (30), F10 (31), and FI6v3 (32) with a high-affinity [equilibrium dissociation constant (K_D) of 10–50 nM]. The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both group 1 (H1 and H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD₉₀) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens confer robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous stem domain immunogens (23–25), the designed immunogens were purified from the soluble fraction in E. coli. The HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free, which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks.     

Cellular heterogeneity profiling by hyaluronan probes reveals an invasive but slow-growing breast tumor subset

Tumor heterogeneity confounds cancer diagnosis and the outcome of therapy, necessitating analysis of tumor cell subsets within the tumor mass. Elevated expression of hyaluronan (HA) and HA receptors, receptor for HA-mediated motility (RHAMM)/HA-mediated motility receptor and cluster designation 44 (CD44), in breast tumors correlates with poor outcome. We hypothesized that a probe for detecting HA–HA receptor interactions may reveal breast cancer (BCa) cell heterogeneity relevant to tumor progression. A fluorescent HA (F-HA) probe containing a mixture of polymer sizes typical of tumor microenvironments (10–480 kDa), multiplexed profiling, and flow cytometry were used to monitor HA binding to BCa cell lines of different molecular subtypes. Formulae were developed to quantify binding heterogeneity and to measure invasion in vivo. Two subsets exhibiting differential binding (HA^−/low vs. HA^high) were isolated and characterized for morphology, growth, and invasion in culture and as xenografts in vivo. F-HA–binding amounts and degree of heterogeneity varied with BCa subtype, were highest in the malignant basal-like cell lines, and decreased upon reversion to a nonmalignant phenotype. Binding amounts correlated with CD44 and RHAMM displayed but binding heterogeneity appeared to arise from a differential ability of HA receptor-positive subpopulations to interact with F-HA. HA^high subpopulations exhibited significantly higher local invasion and lung micrometastases but, unexpectedly, lower proliferation than either unsorted parental cells or the HA^−/low subpopulation. Querying F-HA binding to aggressive tumor cells reveals a previously undetected form of heterogeneity that predicts invasive/metastatic behavior and that may aid both early identification of cancer patients susceptible to metastasis, and detection/therapy of invasive BCa subpopulations.Breast tumors display substantial heterogeneity driven by genetic and epigenetic mechanisms (1–3). These processes select and support tumor cell subpopulations with distinct phenotypes in proliferation, metastatic/invasive proclivity, and treatment susceptibility that contribute to clinical outcomes. Currently, there is a paucity of biomarkers to identify these subpopulations (3–12). Although detection of genetic heterogeneity may itself be a breast cancer (BCa) prognostic marker (3, 13–15), the phenotypes manifested from this diversity are context-dependent. Therefore, phenotypic markers provide additional powerful tools for biological information required to design diagnostics and therapeutics. Glycomic approaches have enormous potential for revealing tumor cell phenotypic heterogeneity because glycans are themselves highly heterogeneous and their complexity reflects the nutritional, microenvironmental, and genetic dynamics of the tumors (16–18).We used hyaluronan (HA) as a model carbohydrate ligand for probing heterogeneity in glycosaminoglycan–BCa cell receptor interactions. We reasoned this approach would reveal previously undetected cellular and functional heterogeneity linked to malignant progression because the diversity of cell glycosylation patterns, which can occur as covalent and noncovalent modifications of proteins and lipids as well as different sizes of such polysaccharides as HA, is unrivaled (16, 17, 19). In particular, tumor and wound microenvironments contain different sizes of HA polymers that bind differentially to cell receptors to activate signaling pathways regulating cell migration, invasion, survival, and proliferation (19–22).More than other related glycosaminoglycans, HA accumulation within BCa tumor cells and peritumor stroma is a predictor of poor outcome (23) and of the conversion of the preinvasive form of BCa, ductal carcinoma in situ, to an early invasive form of BCa (24). HA is a nonantigenic and large, relatively simple, unbranched polymer, but the manner in which it is metabolized is highly complex (19, 25). There are literally thousands of different HA sizes in remodeling microenvironments, including tumors. HA polymers bind to cells via at least six known receptors (16, 19, 20, 26–32). Two of these, cluster designation 44 (CD44) and receptor for HA-mediated motility/HA-mediated motility receptor (RHAMM/HMMR), form multivalent complexes with different ranges of HA sizes (19, 29, 33), and both receptors are implicated in BCa progression (19–21, 23, 29, 30, 33–36). Elevated CD44 expression in the peritumor stroma is associated with increased relapse (37), and in primary BCa cell subsets may contribute to tumor initiation and progression (38–40). Elevated RHAMM expression in BCa tumor subsets is a prognostic indicator of poor outcome and increased metastasis (22, 33, 41). RHAMM polymorphisms may also be a factor in BCa susceptibility (42, 43).We postulated that multivalent interactions resulting from mixture of a polydisperse population of fluorescent HA (F-HA) sizes, typical of those found in remodeling microenvironments of wounds and tumors (19, 20, 29), with cellular HA receptors would uncover a heterogeneous binding pattern useful for sorting tumor cells into distinct subsets. We interrogated the binding of F-HA to BCa lines of different molecular subtypes, and related binding/uptake patterns to CD44 and RHAMM display, and to tumor cell growth, invasion, and metastasis.     

Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans

The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats.The emergence of novel influenza virus strains poses a continuous public health threat (1, 2). The World Health Organization estimates that influenza viruses infect one-billion people annually, with three- to five-million cases of severe illness, and up to 500,000 deaths worldwide (3). Following influenza virus infection, humoral immune responses against the viral hemagglutinin (HA) protein may persist for decades in humans (4). These anti-HA responses correlate strongly with protection against influenza infection (5). Serological memory is maintained by antibody-secreting long-lived plasma cells and reinforced by memory B cells, which can rapidly differentiate into antibody-secreting cells upon antigen reexposure (6).Influenza vaccine efficacy is constantly undermined by antigenic variation in the circulating viral strains, particularly in the HA and neuraminidase (NA) proteins. Current influenza vaccination strategies rely on changing the HA and NA components of the annual human vaccine to ensure that they antigenically match circulating influenza strains (7, 8). Developing an influenza vaccine that is capable of providing broad and long-lasting protective antibody responses remains the central challenge for influenza virus research.HA is a trimer, with each monomer comprised of two subunits: HA1, which includes the HA globular head, and HA2, whose ectodomain together with the N- and C-terminal parts of HA1 constitute the HA stem region (9). Phylogenetically, the 18 HA subtypes characterized so far are divided into two groups. Among strains that have recently caused disease in humans, H1 and H5 HAs belong to group 1, whereas H3 and H7 HAs belong to group 2 (10). Conventional anti-HA neutralizing antibodies primarily target a few immunodominant epitopes located in proximity to the receptor-binding domain within the globular head region of the molecule (11, 12). Although these antibodies are potentially protective, they are strain-specific because of the high variability of such epitopes, and thus lack, in general, the much-desired broad neutralizing activity. Recently, broadly neutralizing human (13–18) and murine (19) monoclonal antibodies (mAbs) directed against distinct epitopes within the HA stem region have been extensively characterized. These mAbs were shown to interfere with the influenza viruses’ life cycle in different ways (20). By generating monoclonal antibodies from plasmablasts isolated ex vivo, we demonstrated that these broadly neutralizing antibodies could be retrieved from patients infected with or vaccinated against the pandemic H1N1 2009 influenza virus (18, 21). Recent observations that HA stem epitopes are accessible on the majority of HA trimers on intact virions (22), and that a stable HA stem protein that is immunologically intact could be produced (23), provided further hope for the feasibility of a stem-based universal influenza vaccine (24).Notably, HA stem-specific mAbs isolated from humans showed a high degree of affinity maturation, suggesting a memory B-cell origin. These results raised two important questions that we address in the current study. First, what are the baseline levels of broadly cross-reactive stem-binding antibodies and memory B cells? Second, using current influenza vaccines, to what extent can HA stem-specific responses be boosted in comparison with those directed against the HA globular head?Structural studies have clearly demonstrated that the main neutralizing antibody epitopes within the HA stem region are conformation-dependent, and that the integrity of these epitopes requires the presence of the HA1 subunit in addition to the HA2 subunit, which constitute the bulk of the HA stem (16, 17). To be able to directly measure HA stem-reactive antibodies and memory B cells, we used a chimeric HA molecule that expresses the globular head of H9 HA on H1 backbone (25). Our data demonstrate that post-2009 trivalent inactivated vaccines (TIV) induced minimal stem-specific responses in comparison with head-specific responses. On the other hand, immunization with H5N1 generated relatively strong anti-HA stem responses, demonstrating that it is feasible to elicit broadly neutralizing responses in humans given the right immunogen design.     

Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HA_mg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HA_mg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HA_fg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HA_mg vaccine activated more diverse B-cell repertoires than the HA_fg vaccine and produced antibodies with cross-strain binding ability. In summary, the HA_mg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.HA glycoprotein on the surface of influenza virus is a major target for infectivity-neutralizing antibodies. However, the antigenic drift and shift of this protein mean that influenza vaccines must be reformulated annually to include HA proteins of the viral strains predicted for the upcoming flu season (1). This time-consuming annual reconfiguration process has led to efforts to develop new strategies and identify conserved epitopes recognized by broadly neutralizing antibodies as the basis for designing universal vaccines to elicit antibodies with a broad protection against various strains of influenza infection (2–6). Previous studies have shown that the stem region of HA is more conserved and able to induce cross-reactive and broadly neutralizing antibodies (7–9) to prevent the critical fusion of viral and endosomal membranes in the influenza lifecycle (10–14). Other broadly neutralizing antibodies have been found to bind regions near the receptor binding site of the globular domain, although these antibodies are fewer in number (15, 16).Posttranslational glycosylation of HA plays an important role in the lifecycle of the influenza virus and also contributes to the structural integrity of HA and the poor immune response of the infected hosts. Previously, we trimmed down the size of glycans on avian influenza H5N1 HA with enzymes and showed that H5N1 HA with a single N-linked GlcNAc at each glycosylation site [monoglycosylated HA (HA_mg)] produces a superior vaccine with more enhanced antibody response and neutralization activity against the homologous influenza virus than the fully glycosylated HA (HA_fg) (17). Here, to test whether the removal of glycans from HA contributes to better immune responses and possibly protects against heterologous strains of influenza viruses, we compared and evaluated the efficacy of HA glycoproteins with various lengths of glycans as potential vaccine candidates.     

Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

The fragmented nature of the influenza A genome allows the exchange of gene segments when two or more influenza viruses infect the same cell, but little is known about the rules underlying this process. Here, we studied genetic reassortment between the A/Moscow/10/99 (H3N2, MO) virus originally isolated from human and the avian A/Finch/England/2051/91 (H5N2, EN) virus and found that this process is strongly biased. Importantly, the avian HA segment never entered the MO genetic background alone but always was accompanied by the avian PA and M fragments. Introduction of the 5′ and 3′ packaging sequences of HA_MO into an otherwise HA_EN backbone allowed efficient incorporation of the chimerical viral RNA (vRNA) into the MO genetic background. Furthermore, forcing the incorporation of the avian M segment or introducing five silent mutations into the human M segment was sufficient to drive coincorporation of the avian HA segment into the MO genetic background. These silent mutations also strongly affected the genotype of reassortant viruses. Taken together, our results indicate that packaging signals are crucial for genetic reassortment and that suboptimal compatibility between the vRNA packaging signals, which are detected only when vRNAs compete for packaging, limit this process.The mechanisms by which animal viruses are introduced into and are disseminated through the human population remain to be addressed. In particular, emerging pathogenic influenza viruses, such as the highly pathogenic avian H5N1 virus and the 2009 “swine” H1N1 virus (H1N1pdm2009), pose major public health and scientific challenges (1, 2). Even though the natural reservoirs of influenza A viruses are wild aquatic birds, influenza A viruses exhibit a broad host range and a wide antigenic diversity, represented by combinations of 17 hemagglutinin (HA) and nine neuraminidase (NA) subtypes (3). Two subtypes of influenza A viruses, H1N1 and H3N2, currently are circulating in the human population.The genome of influenza A viruses is composed of eight single-stranded, negative-sense viral RNA (vRNA) segments. Each segment is associated with the heterotrimeric polymerase complex consisting of polymerase basic proteins 1 and 2 and polymeric acid (PB1/PB2/PA) and is covered by the viral nucleoprotein (NP) to form a viral ribonucleoparticle (vRNP). The fragmented nature of the genome allows the exchange of gene segments when two or more influenza viruses coinfect the same cell, in a process named “genetic reassortment” (4). Genetic reassortment is a major feature of influenza evolution and cross-species transmission and also is important for the generation of antigenically novel isolates by introducing novel HA segments in compatible genetic backgrounds (5–7). Future pandemic viruses most likely will carry different HA genes to which human populations are immunologically naive. The strains giving rise to the 1918 Spanish, 1957 Asian, and 1968 Hong Kong influenza pandemics all harbored an HA segment derived from an avian virus. The avian viruses circulating in the waterfowl are the source of the HA genes most likely to be introduced into the human population (8). Phylogenetic, epidemic, epizootic, and virology studies suggest that swine serve as “mixing vessels” for the generation of human–avian–swine reassortant viruses.When the reassortment process takes place between a human and an avian influenza virus, there are in theory 127 possible reassortant viruses harboring the avian HA segment. Two studies used forced reverse genetics (i.e., a minimal set of reverse genetic plasmids allowing no competition between segments) to generate all 127 reassortant viruses carrying the HA segment from an avian H5N1 virus in the genetic background of a human H3N2 or the HA segment from an avian H9N2 virus in the genetic background of the human 2009 pandemic H1N1 virus (9, 10). They showed that 49% (H5N1/H3N2) and 58% (H9N2/H1N1) of these reassortant viruses replicated efficiently in Madin–Darby canine kidney (MDCK) cells (9, 10). However, several reports indicated that the number of observed natural or experimental reassortant viruses is much smaller than 127, suggesting that reassortment is somehow restricted (4, 11, 12). When analyzing viruses from the nasal secretions of ferrets coinfected with human H3N2 and avian H5N1 viruses, Jackson et al. (13) observed that only 3.1% were reassortant viruses possessing the HA H5 gene, and they corresponded to only five distinct genotypes. Genetic reassortment between human H3N2 and an equine H7N7 virus has been studied using cotransfection (4). Only 1.6% of purified viruses, corresponding to two genotypes, were reassortant viruses possessing the HA H7 gene (4). In contrast, a high frequency of genetic reassortment was observed recently between swine-origin H1N1 and avian H5N1 viruses: 64% of purified viruses, corresponding to 20 different genotypes, were reassortant viruses possessing the HA H5 gene (14). In this case, the high reassortment rate was attributed to the triple reassortant internal gene cassette, consisting of the avian PA and PB2 genes, the nonstructural (NS), NP, and matrix (M) swine genes, and the human PB1 gene (15).The low number of reassortant genotypes usually generated from genetically diverse influenza viruses suggests incompatibilities at the protein and/or genomic level. Accumulating evidence indicates that protein incompatibility among the vRNP components is a limiting factor for reassortment between two viruses (4, 16–18), but little is known about genetic incompatibilities between the vRNA segments. Although incompatibility between proteins is expected to have similar effects in cotransfection or coinfection experiments and in forced reverse genetic experiments, genomic incompatibilities may have several possible effects, especially at the level of the vRNA-packaging signals. Some incompatibilities between packaging signals might reduce viral replication in the absence of competition (absolute incompatibility), whereas more subtle ones might be revealed only when vRNA segments from the two parental viruses compete for packaging (suboptimal compatibility). Reverse genetics-derived reassortant viruses (RGd-RV) that possess the H5N1 (H5) HA in an otherwise H3N2 genetic background show high replicative capacities in MDCK cells (10). Similarly, RGd-RV with the HA gene from H5N1 virus in the H1N1pdm2009 genetic background replicated efficiently in primary human respiratory epithelial cells and caused 100% mortality in mice (19). However, phylogenetic analyses of natural or experimental reassortant viruses have shown that the HA segment from avian, swine, or equine viruses was never incorporated alone in the genetic background of a human virus (13, 14, 20): The HA segment is packaged with additional groups of gene segments depending on the viral subtypes involved in the coinfection process (13, 14).The inability to obtain a virus containing a nonhuman HA gene in an otherwise human genetic background, in contrast with the ability to produce “7+1” RGd-RV with a high yield of replication, suggests that the reassortment process might be restricted by suboptimal compatibility between the vRNA-packaging signals (10).To predict how pandemic influenza viruses can emerge, the complex molecular mechanisms limiting or facilitating genetic reassortment must be deciphered. Using reverse genetics, cis-packaging signals of the human H1N1 WSN and PR8 strains were found to reside at both ends of each vRNA, including the UTRs, along with up to 80 bases of adjacent coding sequences (21–28). In this study, we generated reassortant viruses in vitro from avian H5N2 and human H3N2 viruses to identify incompatibilities between the two parental viruses arising at the vRNA level. Our experiments focusing on the generation of reassortant viruses containing the HA H5 gene segment in an H3N2 genetic background indicate that genomic suboptimal compatibility driven by the selective packaging mechanism limits the generation of HA H5 reassortant viruses in vitro.     

Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. Here, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.Influenza remains a major public health concern because seasonal influenza infects 600 million to 1.1 billion people annually, resulting in 3–5 million cases of severe disease, and 250,000–500,000 deaths (1). By comparison, the four influenza pandemics of the 20th century, caused by novel influenza strains infecting the immunologically naive human population, resulted in 50–100 million deaths (1–4). Influenza A immunity is principally mediated by the antibody response to the viral glycoprotein, hemagglutinin (HA) (5). HA is expressed as a preprotein, HA0, assembled as a trimer on the viral envelope, and cleaved by host proteases into HA1 and HA2. HA1 is a largely globular domain responsible for receptor binding, and HA2 is a rod-shaped helical bundle responsible for membrane fusion (Fig. 1A) (5). There are 18 genetically distinct subtypes of influenza A HA (H1–H18), of which only H1 and H3 currently circulate among humans (1, 6–9).Open in a separate windowFig. 1.Human monoclonal antibody H5.3 recognizes the H5 receptor-binding site. (A) H5.3-wt_H5hd complex overlaid on the VN/1203 H5 trimer (PDB ID code 2FK0) showing H5hd in gold, the H5.3 light chain in purple, the H5.3 heavy chain in teal, and the H5 trimer (2FK0) in gray. (B) A cartoon diagram of H5hd showing HA residues contacted by H5.3 as sticks. The structural elements of the RBS are highlighted: the 130 loop is cyan, the 140 loop is pink, the 150 loop is orange, the 190 helix is blue, and the 220 loop is green. Trp153 forms the base and denotes the approximate center of the receptor-binding site. (C) A surface representation of H5hd in the same orientation as in B, with the solvent inaccessible interface shown in gray. H5.3 contact residues are labeled and shown as sticks and colored by CDR, with CDRH1 in light blue, CDRH2 in blue, CDRH3 in teal, CDRL1 in light pink, CDRL2 in dark pink, and CDRL3 in purple.Despite the widespread presence of H5N1 influenza viruses in wild birds, the virus is not currently transmissible within the human population. Human-to-human transmission is inefficient and is partially restricted by the receptor specificity of the virus; human-type HAs preferentially recognize α2,6-linked sialic acid whereas avian-type HAs prefer α2,3-linked sialic acid (1, 10–12). However, there have been >600 human cases of H5N1 infection since 2004, resulting from the direct transmission of the virus from birds to humans, associated with an ∼60% mortality rate. There is the potential for a significant pandemic if H5 viruses develop the ability to spread efficiently between humans, which would necessitate specificity for α2,6-linked sialic acid (1–4, 13).Receptor binding occurs in a shallow depression on the HA globular head domain, the edges of which are formed by four structural elements, the 190 helix and the 130, 150, and 220 loops (Fig. 1B), and the receptor binding site (RBS) base, which includes invariant hydrophobic residues Tyr98, Trp153, and Leu194 (5, 14, 15). Receptor specificity is critically influenced by position 226 on HA; Gln226-containing H3 strains are specific for α2,3 sialic acid linkages, and Leu226-containing H3 strains are specific for α2,6 sialic acid linkages (5, 16). In H5 strains, Leu226 enhances binding to α2,6-linked sialic acid receptors, but H5 viruses isolated from humans contain mutations at other sites that also promote use of α2,6-linked sialic acid receptors (11, 17, 18). Recent influenza pandemics have been caused by the acquisition of mutations that change the receptor preference to α2,6 sialic acid linkages, and recent studies with multiply passaged laboratory strains indicated that only a small number of mutations are necessary to introduce preference for α2,6 linkages into H5 strains (1, 6–9, 18–23). These viruses, termed respiratory droplet transmissible (rdt), typically have three mutations in or near the receptor binding site on HA (21, 22).The most frequent potent neutralizing antibody response to HA arises from antibodies that target the receptor binding site and prevent virus attachment (5). Recent studies indicate that among RBS-directed antibodies, broad neutralization (across multiple isolates within a subtype or across subtypes) is achieved by insertion of a single complementary determining region (CDR) into the RBS to inhibit receptor binding (8). These broadly neutralizing antibodies (bnAbs) target conserved amino acids within the RBS and simultaneously avoid polymorphic sites on the ridges of the RBS. BnAbs may be relatively rare in human repertoires, and, as a consequence, current seasonal vaccine efforts focus on developing or boosting strain-specific responses to three or four currently circulating (“seasonal”) variants (2). Such strategies do not directly address the threat posed by noncirculating viruses with pandemic potential, such as H5 strains that circulate widely in wild bird populations and sporadically infect humans where they acquire mutations that enhance binding to human receptors (17). Instead, H5N1 vaccines against “prepandemic” strains have been developed commercially for future use in the case of a pandemic, illustrating that a prepandemic immunization program is feasible (24, 25).The immune response against H5N1 vaccines in healthy adults is less robust than for most seasonal influenza strains, typically resulting in a response restricted to the strain used in the vaccine and to closely related variants (26–29). Notwithstanding this observation, we recently described a panel of human anti-H5 antibodies induced in response to vaccination of volunteers with an experimental H5N1 subunit vaccine (30) and here describe the structure and characterization of a human monoclonal antibody, H5.3, bound to A/Vietnam/1203/2004 (VN/1203) H5 and to two H5 rdt variants. H5.3 is an RBS-directed antibody that recapitulates many of the electrostatic interactions of the natural receptor, sialic acid, as well as forming additional interactions to the periphery of the RBS that provide specificity. H5.3 is potent and specific despite containing only 11 mutations from its unmutated common ancestor (UCA) and maintaining the structural flexibility typically associated with unmutated antibodies, as evidenced by significant rearrangement of CDRH3 and CDRL3. The structures determined here offer a chemical explanation for the evident trade-off between breadth and potency, and the germ-line characteristics highlight the role of lightly mutated antibodies in neutralization of new viral strains.     

Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013–2014 influenza season

Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013–2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013–2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group.Seasonal H1N1 (sH1N1) viruses circulated in the human population for much of the last century and, as of 2009, most humans had been exposed to sH1N1 strains. In 2009, an antigenically distinct H1N1 strain began infecting humans and caused a pandemic (1–3). Elderly individuals were less susceptible to 2009 pandemic H1N1 (pH1N1) viruses because of cross-reactive antibodies (Abs) elicited by infections with older sH1N1 strains (3–7). pH1N1 viruses have continued to circulate on a seasonal basis since 2009. Influenza viruses typically cause a higher disease burden in children and elderly individuals (8) but pH1N1 viruses caused unusually high levels of disease in middle-aged adults during the 2013–2014 influenza season (9–12). For example, a significantly higher proportion of individuals aged 30- to 59-y-old were hospitalized in Mexico with laboratory-confirmed pH1N1 cases in 2013–2014 relative to 2011–2012 (11).Most neutralizing influenza Abs are directed against the hemagglutinin (HA) glycoprotein. International surveillance laboratories rely primarily on ferret anti-influenza sera for detecting HA antigenic changes (13). For these assays, sera are isolated from ferrets recovering from primary influenza infections. Seasonal vaccine strains are typically updated when human influenza viruses acquire HA mutations that prevent the binding of primary ferret anti-influenza sera. Our laboratory and others have demonstrated that sera isolated from ferrets recovering from primary pH1N1 infections are dominated by Abs that recognize an epitope involving residues 156, 157, and 158 of the Sa HA antigenic site (14, 15). The pH1N1 component of the seasonal influenza vaccine has not been updated since 2009 because very few pH1N1 isolates possess mutations in residues 156, 157, and 158. The majority of isolates from the 2013–2014 season have been labeled as antigenically similar to the A/California/07/2009 vaccine strain (9).It is potentially problematic that major antigenic changes of influenza viruses are mainly determined using antisera isolated from ferrets recovering from primary influenza infections. Unlike experimental ferrets, humans are typically reinfected with antigenically distinct influenza strains throughout their life (16). In the 1950s, it was noted that the human immune system preferentially mounts Ab responses that cross-react to previously circulating influenza strains, as opposed to new Ab responses that exclusively target newer viral strains (17). This process, which Thomas Francis Jr. termed “original antigenic sin,” has been experimentally recapitulated in ferrets (14, 18), mice (19–21), and rabbits (22). Our group and others recently demonstrated that the specificity of pH1N1 Ab responses can be shaped by prior sH1N1 exposures (14, 23–26). We found that ferrets sequentially infected with sH1N1 and pH1N1 viruses mount Ab responses dominated against epitopes that are conserved between the viral strains (14). These studies indicate that primary ferret antisera may not be fully representative of human influenza immunity.It has been proposed that increased morbidity and mortality of middle-aged adults during the 2013–2014 influenza season is primarily a result of low vaccination rates within these populations (27). An alternative explanation is that recent pH1N1 strains have acquired a true antigenic mutation that has been mislabeled as “antigenically neutral” by assays that rely on primary ferret antisera. Here we complete a series of experiments to determine if recent pH1N1 strains possess a mutation that prevents binding of Abs in middle-aged humans who have been previously exposed to different H1N1 strains.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Structural insights into the histone H1-nucleosome complex

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.Eukaryotic genomic DNA is packaged into chromatin through association with positively charged histones to form the nucleosome, the structural unit of chromatin (1–3). The nucleosome core consists of an octamer of histones with two copies of H2A, H2B, H3, and H4, around which ∼146 bp of DNA winds in ∼1.65 left-handed superhelical turns (4). At this level of the DNA packaging, chromatin resembles a beads-on-a-string structure, with the nucleosome core as the beads and the linker DNA between them as the strings (5). At the next level of DNA packaging, H1 histones bind to the linker DNA and the nucleosome to further condense the chromatin structure (6, 7). H1-mediated chromatin condensation plays important roles in cellular functions such as mitotic chromosome architecture and segregation (8), muscle differentiation (9), and regulation of gene expression (10, 11).Linker H1 histones typically are ∼200 amino acid residues in length, with a short N-terminal region, followed by a ∼70–80-amino acid structured globular domain (gH1) and a ∼100-amino acid unstructured C-terminal domain that is highly enriched in Lys residues. H1 stabilizes the nucleosome and facilitates folding of nucleosome arrays into higher-order structures (12–15). gH1 alone confers the same protection from micrococcal nuclease digestion to the nucleosome as the full-length H1 does (16). The N-terminal region of H1 is not important for nucleosome binding (16, 17), whereas the C terminus is required for H1 binding to chromatin in vivo (18, 19) and for the formation of a stem structure of linker DNA in vitro (17, 20, 21).The globular domain structures of avian H5 (22) and budding yeast Hho1 (23), which are both H1 homologs, have been determined at atomic resolution and show similar structures. In addition, numerous studies have indicated that gH1/gH5 binds around the dyad region of the nucleosome (14, 24), leading to many conflicting structural models for how the globular domain of H1/H5 binds to the nucleosome (SI Appendix, Fig. S1) (24–26). These models are divided into two major classes, symmetric and asymmetric, on the basis of the location of gH1/gH5 in the nucleosome. In the symmetric class, gH1/gH5 binds to the nucleosomal DNA at the dyad and interacts with both linker DNAs (16, 17, 27, 28). In the asymmetric class, gH1/gH5 binds to the nucleosomal DNA in the vicinity of the dyad axis and to 10 bp (27, 29–32) or 20 bp (19, 29, 33, 34) of one linker DNA, or is located inside the DNA gyres, where it interacts with histone H2A (35). In addition, Zhou and colleagues also characterized the orientation of gH5 in the gH5-nucleosome complex (29). The use of nonuniquely positioned nucleosomes and indirect methods may have contributed to the differences in these models (SI Appendix, Fig. S1).Multidimensional nuclear magnetic resonance (NMR), and in particular methyl-based NMR, provides a direct approach to the structural characterization of macromolecular complexes (36, 37). We have previously assigned chemical shifts of the methyl groups of the side chains of residues Ile, Leu, and Val in the core histones (38) and the backbone amides in the disordered histone tails (39), which provide the fingerprints for investigating the interactions between H1 and the nucleosome. Here, we used NMR, along with several other methods, to determine the location and orientation of the globular domain of a stable mutant of Drosophila H1 on a well-positioned nucleosome.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Predictive Value of Brain Natriuretic Peptides in Patients on Peritoneal Dialysis: Results from the ADEMEX Trial

Background and objectives: Natriuretic peptides have been suggested to be of value in risk stratification in dialysis patients. Data in patients on peritoneal dialysis remain limited.Design, setting, participants, & measurements: Patients of the ADEMEX trial (ADEquacy of peritoneal dialysis in MEXico) were randomized to a control group [standard 4 × 2L continuous ambulatory peritoneal dialysis (CAPD); n = 484] and an intervention group (CAPD with a target creatinine clearance ≥60L/wk/1.73 m²; n = 481). Natriuretic peptides were measured at baseline and correlated with other parameters as well as evaluated for effects on patient outcomes.Results: Control group and intervention group were comparable at baseline with respect to all measured parameters. Baseline values of natriuretic peptides were elevated and correlated significantly with levels of residual renal function but not with body size or diabetes. Baseline values of N-terminal fragment of B-type natriuretic peptide (NT-proBNP) but not proANP(1–30), proANP(31–67), or proANP(1–98) were independently highly predictive of overall survival and cardiovascular mortality. Volume removal was also significantly correlated with patient survival.Conclusions. NT-proBNP have a significant predictive value for survival of CAPD patients and may be of value in guiding risk stratification and potentially targeted therapeutic interventions.Plasma levels of cardiac natriuretic peptides are elevated in patients with chronic kidney disease, owing to impairment of renal function, hypertension, hypervolemia, and/or concomitant heart disease (1–7). Atrial natriuretic peptide (ANP) and particularly brain natriuretic peptide (BNP) levels are linked independently to left ventricular mass (3–5,8–16) and function (3,6–17) and predict total and cardiovascular mortality (1,3,8,10,12,18) as well as cardiac events (12,19). ANP and BNP decrease significantly during hemodialysis treatment but increase again during the interdialytic interval (1,2,4,6,7,14,17,20–23). Levels in patients on peritoneal dialysis (PD) have been found to be lower than in patients on hemodialysis (11,24–26), but the correlations with left ventricular function and structure are maintained in both types of dialysis modalities (11,15,27,28).The high mortality of patients on peritoneal dialysis and the failure of dialytic interventions to alter this mortality (29,30) necessitate renewed attention into novel methods of stratification and identification of patients at highest risk to be targeted for specific interventions. Cardiac natriuretic peptides are increasingly considered to fulfill this role in nonrenal patients. Evaluations of cardiac natriuretic peptides in patients on PD have been limited by small numbers (3,9,11,12,15,24–26) and only one study examined correlations between natriuretic peptide levels and outcomes (12). The PD population enrolled in the ADEMEX trial offered us the opportunity to evaluate cardiac natriuretic peptides and their value in predicting outcomes in the largest clinical trial ever performed on PD (29,30). It is hoped that such an evaluation would identify patients at risk even in the absence of overt clinical disease and hence facilitate or encourage interventions with salutary outcomes.