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1.  Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation 
Journal of Virology  2013;87(20):10980-10996.
Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal.
PMCID: PMC3807285  PMID: 23903846
2.  Ebolavirus VP35 Coats the Backbone of Double-Stranded RNA for Interferon Antagonism 
Journal of Virology  2013;87(18):10385-10388.
Recognition of viral double-stranded RNA (dsRNA) activates interferon production and immune signaling in host cells. Crystal structures of ebolavirus VP35 show that it caps dsRNA ends to prevent sensing by pattern recognition receptors such as RIG-I. In contrast, structures of marburgvirus VP35 show that it primarily coats the dsRNA backbone. Here, we demonstrate that ebolavirus VP35 also coats the dsRNA backbone in solution, although binding to the dsRNA ends probably constitutes the initial binding event.
PMCID: PMC3753998  PMID: 23824825
3.  Structural Characterization of Cleaved, Soluble HIV-1 Envelope Glycoprotein Trimers 
Journal of Virology  2013;87(17):9865-9872.
Human immunodeficiency virus type 1 (HIV-1) infection is a significant global public health problem for which development of an effective prophylactic vaccine remains a high scientific priority. Many concepts for a vaccine are focused on induction of appropriate titers of broadly neutralizing antibodies (bNAbs) against the viral envelope (Env) glycoproteins gp120 and gp41, but no immunogen has yet accomplished this goal in animals or humans. One approach to induction of bNAbs is to design soluble, trimeric mimics of the native viral Env trimer. Here, we describe structural studies by negative-stain electron microscopy of several variants of soluble Env trimers based on the KNH1144 subtype A sequence. These Env trimers are fully cleaved between the gp120 and gp41 components and stabilized by specific amino acid substitutions. We also illustrate the structural consequences of deletion of the V1/V2 and V3 variable loops from gp120 and the membrane-proximal external region (MPER) from gp41. All of these variants adopt a trimeric configuration that appropriately mimics native Env spikes, including the CD4 receptor-binding site and the epitope for the VRC PG04 bNAb. These cleaved, soluble trimer designs can be adapted for use with multiple different env genes for both vaccine and structural studies.
PMCID: PMC3754114  PMID: 23824817
4.  Influences on Trimerization and Aggregation of Soluble, Cleaved HIV-1 SOSIP Envelope Glycoprotein 
Journal of Virology  2013;87(17):9873-9885.
We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.
PMCID: PMC3754145  PMID: 23824824
5.  Integrative Structural Biology 
Science (New York, N.Y.)  2013;339(6122):913-915.
PMCID: PMC3633482  PMID: 23430643
6.  Development of a Preclinical Orthotopic Xenograft Model of Ewing Sarcoma and Other Human Malignant Bone Disease Using Advanced In Vivo Imaging 
PLoS ONE  2014;9(1):e85128.
Ewing sarcoma and osteosarcoma represent the two most common primary bone tumours in childhood and adolescence, with bone metastases being the most adverse prognostic factor. In prostate cancer, osseous metastasis poses a major clinical challenge. We developed a preclinical orthotopic model of Ewing sarcoma, reflecting the biology of the tumour-bone interactions in human disease and allowing in vivo monitoring of disease progression, and compared this with models of osteosarcoma and prostate carcinoma. Human tumour cell lines were transplanted into non-obese diabetic/severe combined immunodeficient (NSG) and Rag2−/−/γc−/− mice by intrafemoral injection. For Ewing sarcoma, minimal cell numbers (1000–5000) injected in small volumes were able to induce orthotopic tumour growth. Tumour progression was studied using positron emission tomography, computed tomography, magnetic resonance imaging and bioluminescent imaging. Tumours and their interactions with bones were examined by histology. Each tumour induced bone destruction and outgrowth of extramedullary tumour masses, together with characteristic changes in bone that were well visualised by computed tomography, which correlated with post-mortem histology. Ewing sarcoma and, to a lesser extent, osteosarcoma cells induced prominent reactive new bone formation. Osteosarcoma cells produced osteoid and mineralised “malignant” bone within the tumour mass itself. Injection of prostate carcinoma cells led to osteoclast-driven osteolytic lesions. Bioluminescent imaging of Ewing sarcoma xenografts allowed easy and rapid monitoring of tumour growth and detection of tumour dissemination to lungs, liver and bone. Magnetic resonance imaging proved useful for monitoring soft tissue tumour growth and volume. Positron emission tomography proved to be of limited use in this model. Overall, we have developed an orthotopic in vivo model for Ewing sarcoma and other primary and secondary human bone malignancies, which resemble the human disease. We have shown the utility of small animal bioimaging for tracking disease progression, making this model a useful assay for preclinical drug testing.
PMCID: PMC3883696  PMID: 24409320
7.  Overexpression, crystallization and preliminary X-ray crystallographic analysis of the variable lymphocyte receptor 2913 ectodomain fused with internalin B 
The VLR2913 ectodomain fused with internalin B was crystallized and diffraction data were collected to a maximum resolution of 2.04 Å.
In jawless vertebrates, variable lymphocyte receptors (VLRs) play a crucial role in the recognition of antigens as part of the adaptive immune system. Leucine-rich repeat (LRR) modules and the highly variable insert (HVI) of VLRs contribute to the specificity and diversity of antigen recognition. VLR2913, the antigen of which is not known, contains the same HVI amino-acid sequence as that of VLR RBC36, which recognizes the H-trisaccharide from human blood type O erythrocytes. Since the HVI sequence is rarely identical among all known VLRs, identification of the antigen for VLR2913 and the main contributing factors for antigen recognition based on a comparison of VLR2913 and VLR RBC36 has been attempted. To initiate and facilitate this structural approach, the ectodomain of VLR2913 was fused with the N-terminal domain of internalin B (InlB-VLR2913-ECD). Three amino-acid residues on the concave surface of the LRR modules of InlB-VLR2913-ECD were mutated, considering important residues for hydrogen bonds in the recognition of H-trisaccharide by VLR RBC36. InlB-VLR2913-ECD was overexpressed in Escherichia coli and was crystallized at 295 K using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 2.04 Å resolution and could be indexed in the tetragonal space group P41212 (or P43212), with unit-cell parameters a = 91.12, b = 91.12, c = 62.87 Å. Assuming that one monomer molecule was present in the crystallographic asymmetric unit, the calculated Matthews coefficient (V M) was 2.75 Å3 Da−1 and the solvent content was 55.2%. Structural determination of InlB-VLR2913-ECD by molecular replacement is in progress.
PMCID: PMC3539700  PMID: 23295483
variable lymphocyte receptor; VLR; adaptive immunity; antigen recognition
8.  Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120 
Nature structural & molecular biology  2013;20(7):10.1038/nsmb.2594.
A substantial fraction of broadly neutralizing antibodies (bnAbs) in certain HIV-infected donors recognizes glycan-dependent epitopes on HIV-1 gp120. Here, we elucidate how bnAb PGT 135 recognizes its Asn332 glycan-dependent epitope from its crystal structure with gp120, CD4 and Fab 17b at 3.1 Å resolution. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield to access the gp120 protein surface. Electron microscopy reveals PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. The combined structural studies of PGT 135, PGT 128 and 2G12 show this Asn332-dependent epitope is highly accessible and much more extensive than initially appreciated, allowing for multiple binding modes and varied angles of approach, thereby representing a supersite of vulnerability for antibody neutralization.
PMCID: PMC3823233  PMID: 23708606
10.  Organization of the Influenza Virus Replication Machinery 
Science (New York, N.Y.)  2012;338(6114):1631-1634.
Influenza virus ribonucleoprotein complexes (RNPs) are central to the viral life cycle and in adaptation to new host species. RNPs are composed of the viral genome, viral polymerase and many copies of the viral nucleoprotein. In vitro cell expression of all RNP protein components with four of the eight influenza virus gene segments enabled structural determination of native influenza virus RNPs by cryo-electron microscopy. The cryo-EM structure reveals the architecture and organization of the native RNP, thereby defining the attributes of its largely helical structure and how polymerase interacts with NP and the viral genome. Observations of branched-RNP structures in negative stain EM and their putative identification as replication intermediates suggest a mechanism for viral replication by a second polymerase on the RNP template.
PMCID: PMC3578580  PMID: 23180774
11.  The Enhanced In Vivo Activity of the Combination of a MEK and a PI3K Inhibitor Correlates with [18F]-FLT PET in Human Colorectal Cancer Xenograft Tumour-Bearing Mice 
PLoS ONE  2013;8(12):e81763.
Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3′-deoxy-3′-[18F]-fluorothymidine ([18F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [18F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [18F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [18F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.
PMCID: PMC3858267  PMID: 24339963
12.  Structure of a Classical Broadly Neutralizing Stem Antibody in Complex with a Pandemic H2 Influenza Virus Hemagglutinin 
Journal of Virology  2013;87(12):7149-7154.
We report the structural characterization of the first antibody identified to cross-neutralize multiple subtypes of influenza A viruses. The crystal structure of mouse antibody C179 bound to the pandemic 1957 H2N2 hemagglutinin (HA) reveals that it targets an epitope on the HA stem similar to those targeted by the recently identified human broadly neutralizing antibodies. C179 also inhibits the low-pH conformational change of the HA but uses a different angle of approach and both heavy and light chains.
PMCID: PMC3676097  PMID: 23552413
13.  The Effects of Somatic Hypermutation on Neutralization and Binding in the PGT121 Family of Broadly Neutralizing HIV Antibodies 
PLoS Pathogens  2013;9(11):e1003754.
Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121–134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121–134 but were still capable of neutralizing roughly 40–80% of PGT121–134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121–134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121–134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.
Author Summary
A majority of the over 30 million HIV-1 infected individuals worldwide live in poorly resourced areas where multiple boost strategies, which are likely needed to generate highly mutated antibodies, present formidable logistical challenges. Accordingly, developing new vaccination strategies that are capable of generating highly mutated antibodies should be an active area of research. Another approach, that is not mutually exclusive, is to identify new bnAbs that are both broad and potent in neutralization, but are much less mutated than the bnAbs that currently exist. Here, we have identified bnAbs that are approximately half the mutation frequency of known bnAbs, but maintain high potency and moderate breadth. These less mutated bnAbs offer an important advantage in that they would likely be easier to induce through vaccination than more mutated antibodies. By characterizing these putative intermediates, we can also better estimate how affinity maturation proceeded to result in an antibody with broad and potent neutralization activity and offer more focused strategies for designing immunogens capable of eliciting these less mutated bnAbs.
PMCID: PMC3836729  PMID: 24278016
14.  Recombinant expression of TLR5 proteins by ligand supplementation and a leucine-rich repeat hybrid technique 
Biochemical and biophysical research communications  2012;427(1):10.1016/j.bbrc.2012.09.021.
Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variable lymphocyte receptor (VLR), and determined the crystal structure of its complex with flagellin D1–D2–D3 domains. Although the structure provides valuable information about the interaction, it remains to be revealed how the C-terminal region of TLR5-ECD contributes to the interaction. Here, we present two methods to obtain recombinant TLR5 proteins that contain the C-terminal region in a baculovirus expression system. First, production of biologically active full-length drTLR5-ECD was substantially enhanced by supplementation of expression culture with purified flagellin proteins. Second, we designed TLR5-VLR hybrids using an LRR hybrid technology by single and double LRR fusions and were able to express diverse regions of drTLR5-ECD, allowing us to detect a previously unidentified TLR5/flagellin interaction. The drTLR5-VLR hybrid technique was also successfully applied to human TLR5-ECD whose expression has been highly problematic. These alternative TLR5 expression strategies provide an opportunity to obtain a complete view of the TLR5/flagellin interaction and can be applied to other LRR proteins.
PMCID: PMC3823237  PMID: 22989748
Toll-like receptor 5; Flagellin; Leucine-rich repeat; LRR hybrid; Variable lymphocyte receptor; Innate immunity
15.  Structural insights into key sites of vulnerability on HIV-1 Env and Influenza HA 
Immunological reviews  2012;250(1):180-198.
Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals.
PMCID: PMC3479221  PMID: 23046130
HIV-1; Env; influenza; hemagglutinin; antibody; structure
16.  [18F]-FLT Positron Emission Tomography can be used to image the response of sensitive tumors to PI3-Kinase inhibition with the novel agent GDC-0941 
Molecular cancer therapeutics  2013;12(5):819-828.
The Phosphatidylinositide 3-kinase (PI3-K) pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the PET tracer 3′-Deoxy-3′-[18F]fluorothymidine ([18F]-FLT) is suitable to mark the effect of the novel PI-3K inhibitor GDC-0941 which has entered phase II clinical trial. CBA nude mice bearing U87 glioma and HCT116 colorectal xenografts were imaged at baseline with [18F]-FLT and at acute (18h) and chronic (186h) timepoints after twice-daily administration of GDC-0941 (50mg/kg) or vehicle. Tumor uptake normalized to blood pool was calculated, and tissue was analyzed at sacrifice for PI3-K pathway inhibition and thymidine kinase (TK1) expression. Uptake of [18F]-FLT was also assessed in tumors inducibly overexpressing a dominant-negative form of the PI3-K p85 subunit Δp85α, as well as HCT116 liver metastases after GDC-0941 therapy. GDC-0941 treatment induced tumor stasis in U87 xenografts, whereas inhibition of HCT116 tumors was more variable. Tumor uptake of [18F]-FLT was significantly reduced following GDC-0941 dosing in responsive tumors at the acute timepoint, and correlated with pharmacodynamic markers of PI3-K signaling inhibition and significant reduction in TK1 expression in U87, but not HCT116, tumors. Reduction of PI3-K signaling via expression of Δp85α significantly reduced tumor growth and [18F]-FLT uptake, as did treatment of HCT116 liver metastases with GDC-0941. These results indicate that [18F]-FLT is a strong candidate for the non-invasive measurement of GDC-0941 action.
PMCID: PMC3670082  PMID: 23427298
Positron Emission Tomography; Fluorodeoxythymidine; Imaging Therapy Response
17.  Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo 
mBio  2013;4(5):e00803-13.
Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo.
IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN’s altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.
Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN’s altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.
PMCID: PMC3812707  PMID: 24149514
Cell host & microbe  2012;12(4):396-407.
Despite numerous attempts over many years to develop an HIV vaccine based on classical strategies, none has convincingly succeeded to date. A number of approaches are being pursued in the field, including building upon possible efficacy indicated by the recent RV144 clinical trial, which combined two HIV vaccines. Here, we argue for an approach based, in part, on understanding the HIV envelope spike and its interaction with broadly neutralizing antibodies (bnAbs) at the molecular level and using this understanding to design immunogens as possible vaccines. BnAbs can protect against virus challenge in animal models and many such antibodies have been isolated recently. We further propose that studies focused on how best to provide T cell help to B cells that produce bnAbs are crucial for optimal immunization strategies. The synthesis of rational immunogen design and immunization strategies, together with iterative improvements, offers great promise for advancing toward an HIV vaccine.
PMCID: PMC3513329  PMID: 23084910
19.  Investigation of chronic alcohol consumption in rodents via UHPLC-MS based metabolite profiling 
Journal of chromatography. A  2012;1259:128-137.
Alcohol consumption in man, when seen in its extreme form of alcoholism, is a complex and socially disruptive disorder that can result in significant levels of liver injury. Here the rodent “intragastric feeding model” was used together with UHPLC-TOFMS analysis to determine changes in global metabolite profiles for plasma and urine from alcohol treated rats and mice compared to control animals. Multivariate statistical analysis (using principal components analysis, PCA) revealed robust differences between profiles from control and alcohol-treated animals from both species. A large number of metabolites were seen to differ between control and alcohol-treated animals, for both biofluids.
PMCID: PMC3387526  PMID: 22446076
Alcohol; Metabolite profiling; Ultra Performance Liquid Chromatography (UPLC); Time of Flight mass spectrometry (TOF-MS); metabonomics; metabolomics
20.  New World Bats Harbor Diverse Influenza A Viruses 
PLoS Pathogens  2013;9(10):e1003657.
Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.
Author Summary
Previous studies indicated that a novel influenza A virus (H17N10) was circulating in fruit bats from Guatemala (Central America). Herein, we investigated whether similar viruses are present in bat species from South America. Analysis of rectal swabs from bats sampled in the Amazon rainforest region of Peru identified another new influenza A virus from bats that is phylogenetically distinct from the one identified in Guatemala. The genes that encode the surface proteins of the new virus from the flat-faced fruit bat were designated as new subtype H18N11. Serologic testing of blood samples from several species of Peruvian bats indicated a high prevalence of antibodies to the surface proteins. Phylogenetic analyses demonstrate that bat populations from Central and South America maintain as much influenza virus genetic diversity in some gene segments as all other mammalian and avian species combined. The crystal structures of the hemagglutinin and neuraminidase proteins indicate that sialic acid is not a receptor for virus attachment nor a substrate for release, suggesting a novel mechanism of influenza A virus attachment and activation of membrane fusion for entry into host cells. In summary, our findings indicate that bats constitute a potentially important reservoir for influenza viruses.
PMCID: PMC3794996  PMID: 24130481
21.  Neutralization of influenza A viruses by insertion of a single antibody loop into the receptor binding site 
Nature  2012;489(7417):526-532.
Immune recognition of protein antigens relies upon the combined interaction of multiple antibody loops, which provides a fairly large footprint and constrains the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of antibody C05 that neutralizes strains from multiple subtypes of influenza A viruses, including H1, H2, and H3. Crystal and EM structures show that C5 recognizes conserved elements of the receptor binding site on the hemagglutinin (HA) surface glycoprotein. Recognition of the HA receptor binding site is dominated by a single HCDR3 loop, with minor contacts from HCDR1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved, functional sites on otherwise hypervariable antigens.
PMCID: PMC3538848  PMID: 22982990
22.  A Next-Generation Cleaved, Soluble HIV-1 Env Trimer, BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies 
PLoS Pathogens  2013;9(9):e1003618.
A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.
Author Summary
A protective HIV-1 vaccine is badly needed, but no candidate has yet provided an adequate level of protection against infection. Most existing vaccines provide immune protection by inducing neutralizing antibodies, also a goal of many HIV-1 immunogen design projects. The trimeric envelope protein complex on the HIV-1 surface is the only relevant target for neutralizing antibodies, and is the basis for most strategies aimed at their induction. However, making a soluble, recombinant envelope protein complex that adequately mimics the structure present on the virus has been challenging. Here, we describe a newly designed and engineered Env protein that has the appropriate properties. This protein, termed BG505 SOSIP.664 gp140, binds most of the known neutralizing antibodies but generally does not bind antibodies that lack neutralization activity. Its appearance in negative stain electron micrographs also resembles native envelope complexes.
PMCID: PMC3777863  PMID: 24068931
23.  Highly Conserved Protective Epitopes on Influenza B Viruses 
Science (New York, N.Y.)  2012;337(6100):1343-1348.
Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody-based immunotherapy and ‘universal’ vaccines for influenza. However, a significant part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here we report human monoclonal antibodies, CR8033, CR8071 and CR9114, which protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody-based treatments and a universal flu vaccine for all influenza A and B viruses.
PMCID: PMC3538841  PMID: 22878502
24.  A recurring motif for antibody recognition of the receptor-binding site of influenza hemagglutinin 
Influenza virus hemagglutinin (HA) mediates receptor binding and viral entry during influenza infection. The development of receptor analogs as viral entry blockers has not been successful, suggesting that sialic acid may not be an ideal scaffold to obtain broad and potent HA inhibitors. Here we report crystal structures of Fab fragments from three human antibodies that neutralize the 1957 pandemic H2N2 influenza virus in complex with H2 HA. All three antibodies use an aromatic residue to plug a conserved cavity in the HA receptor-binding site. Each antibody interacts with the absolutely conserved HA1 Trp153 at the cavity base through π-π stacking with the signature Phe54 of two VH1-69 antibodies or a tyrosine from HCDR3 in the other antibody. This remarkably conserved interaction can be used as a starting point to design inhibitors targeting this conserved hydrophobic pocket in influenza viruses.
PMCID: PMC3594569  PMID: 23396351
25.  The CD100 receptor interacts with its plexin B2 ligand to regulate epidermal γδ T cell function 
Immunity  2012;37(2):314-325.
γδ T cells respond rapidly to keratinocyte damage in the skin, providing essential contributions to the wound healing process, but the molecular interactions regulating their response are unknown. Here we identify a role for the interaction of plexin B2 and the CD100 receptor in epithelial repair. In vitro blocking of plexin B2 or CD100 inhibited γδ T cell activation. Furthermore, CD100 deficiency in vivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ T cell response to keratinocyte damage. Direct ligation of CD100 in γδ T cells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providing a mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ T cells and the neighboring cells they support.
PMCID: PMC3430606  PMID: 22902232

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