[show abstract][hide abstract] ABSTRACT: Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.
Biochemical and Biophysical Research Communications 06/2011; 409(2):253-9. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lack of life-long immunity against influenza viruses represents a major global health care problem with profound medical and economic consequences. A greater understanding of the broad-spectrum "heterosubtypic" neutralizing human antibody (BnAb) response to influenza should bring us closer toward a universal influenza vaccine.
Serum samples obtained from 77 volunteers in an H5N1 vaccine study were analyzed for cross-reactive antibodies (Abs) against both subtype hemagglutinins (HAs) and a highly conserved pocket on the HA stem of Group 1 viruses. Cross-reactive Abs in commercial intravenous immunoglobulin were affinity purified using H5-coupled beads followed by step-wise monoclonal antibody competition or acid elution. Enzyme-linked immunosorbent assays were used to quantify cross-binding, and neutralization activity was determined with HA-pseudotyped viruses.
Prevaccination serum samples have detectable levels of heterosubtypic HA binding activity to both Group 1 and 2 influenza A viruses, including subtypes H5 and H7, respectively, to which study subjects had not been vaccinated. Two different populations of Broadly neutralizing Abs (BnAbs) were purified from intravenous immunoglobulin by H5 beads: ~0.01% of total immunoglobulin G can bind to HAs from both Group 1 and 2 and neutralize H1N1 and H5N1 viruses; ~0.001% is F10-like Abs directed against the HA stem pocket on Group 1 viruses.
These data--to our knowledge, for the first time--quantitatively show the presence, albeit at low levels, of two populations of heterosubtypic BnAbs against influenza A in human serum. These observations warrant further investigation to determine their origin, host polymorphism(s) that may affect their expression levels and how to boost these BnAb responses by vaccination to reach sustainable protective levels.
[show abstract][hide abstract] ABSTRACT: The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.
Journal of Experimental Medicine 01/2011; 208(1):181-93. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.
Biochemical and Biophysical Research Communications 11/2010; 403(2):247-51. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.
[show abstract][hide abstract] ABSTRACT: Enveloped viruses use multiple mechanisms to inhibit infection of a target cell by more than one virion. These mechanisms may be of particular importance for the evolution of segmented viruses, because superinfection exclusion may limit the frequency of reassortment of viral genes. Here, we show that cellular expression of influenza A virus neuraminidase (NA), but not hemagglutinin (HA) or the M2 proton pump, inhibits entry of HA-pseudotyped retroviruses. Cells infected with H1N1 or H3N2 influenza A virus were similarly refractory to HA-mediated infection and to superinfection with a second influenza A virus. Both HA-mediated entry and viral superinfection were rescued by the neuraminidase inhibitors oseltamivir carboxylate and zanamivir. These inhibitors also prevented the removal of alpha-2,3- and alpha-2,6-linked sialic acid observed in cells expressing NA or infected with influenza A viruses. Our data indicate that NA alone among viral proteins limits influenza A virus superinfection.
Journal of Virology 06/2008; 82(10):4834-43. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN) lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv) antibody fragments on human cells and lentivirus particles.
Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM) anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6)-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity.
This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.
PLoS ONE 02/2008; 3(9):e3181. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Monoclonal antibodies (mAbs) have long provided powerful research tools for virologists to understand the mechanisms of virus entry into host cells and of antiviral immunity. Even so, commercial development of human (or humanized) mAbs for the prophylaxis, preemptive and acute treatment of viral infections has been slow. This is surprising, as new antibody discovery tools have increased the speed and precision with which potent neutralizing human antiviral mAbs can be identified. As longstanding barriers to antiviral mAb development, such as antigenic variability of circulating viral strains and the ability of viruses to undergo neutralization escape, are being overcome, deeper insight into the mechanisms of mAb action and engineering of effector functions are also improving the efficacy of antiviral mAbs. These successes, in both industrial and academic laboratories, coupled with ongoing changes in the biomedical and regulatory environments, herald an era when the commercial development of human antiviral mAb therapies will likely surge.
[show abstract][hide abstract] ABSTRACT: The cellular receptor for human coronavirus NL63 (HCoV-NL63), a group I coronavirus, is angiotensin-converting enzyme2 (ACE2). ACE2 is also the receptor for the SARS coronavirus (SARS-CoV), a group II coronavirus. Here we describe the ability of HCoV-NL63 to utilize a number of ACE2 variants previously characterized as SARS-CoV receptors. Several ACE2 variants that reduced SARS-CoV S-protein association similarly reduced that of HCoV-NL63, whereas alteration of a number of solvent-exposed ACE2 residues did not interfere with binding by either S protein. One notable exception is ACE2 residue 354, at the boundary of the SARS-CoV binding site, whose alteration markedly inhibited utilization by the HCoV-NL63 but not SARS-CoV S proteins. In addition, the SARS-CoV S-protein receptor-binding domain inhibited entry mediated by the HCoV-NL63 S protein. These studies indicate that HCoV-NL63, like SARS-CoV, associates region of human ACE2 that includes a key loop formed by beta-strands 4 and 5.
[show abstract][hide abstract] ABSTRACT: The chemokine receptor CXCR4 and its ligand stromal-derived factor-1 (SDF-1/CXCL12) are essential for many biological processes and various pathological conditions. However, the relationship between CXCR4 antigenic structure and SDF-1-mediated biological responses is poorly understood. In this report, a panel of human anti-CXCR4 Abs were isolated and used to explore CXCR4 antigenic heterogeneity and function. Multiple fixed CXCR4 antigenic isoforms were detected on the surface of hemopoietic cells. Epitope mapping studies demonstrated the complex nature of the surface-exposed CXCR4 epitopes. Ab-mediated inhibition of chemotaxis correlated strongly with binding affinity, epitope recognition, as well as the level of CXCR4 isoform expression. In addition, detailed genetic analyses of these Abs showed evidence of V(H) replacement. Importantly, structural and biochemical studies demonstrated tyrosine sulfation in novel regions of the V genes that contributed bidirectionally to the binding activity of the Abs. These data provide the first evidence that functional tyrosine sulfation occurs in self-reactive Abs and suggest a potential new mechanism that may contribute to the pathogenesis of Ab-mediated autoimmune disease. These Abs also provide valuable tools to explore the selective in vivo targeting of CXCR4 isoforms that may be preferentially expressed in certain disease states and involved in steady-state CXCR4-SDF-1 homeostasis.
The Journal of Immunology 09/2007; 179(4):2408-18. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 A resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 A resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.
Journal of Biological Chemistry 12/2006; 281(45):34610-6. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rapid elucidation of neutralizing antibody epitopes on emerging viral pathogens like severe acute respiratory syndrome (SARS) coronavirus (CoV) or highly pathogenic avian influenza H5N1 virus is of great importance for rational design of vaccines against these viruses. Here we combined screening of phage display random peptide libraries with a unique computer algorithm "Mapitope" to identify the discontinuous epitope of 80R, a potent neutralizing human anti-SARS monoclonal antibody against the spike protein. Using two different types of random peptide libraries which display cysteine-constrained loops or linear 13-15-mer peptides, independent panels containing 42 and 18 peptides were isolated, respectively. These peptides, which had no apparent homologous motif within or between the peptide pools and spike protein, were deconvoluted into amino acid pairs (AAPs) by Mapitope and the statistically significant pairs (SSPs) were defined. Mapitope analysis of the peptides was first performed on a theoretical model of the spike and later on the genuine crystal structure. Three clusters (A, B and C) were predicted on both structures with remarkable overlap. Cluster A ranked the highest in the algorithm in both models and coincided well with the sites of spike protein that are in contact with the receptor, consistent with the observation that 80R functions as a potent entry inhibitor. This study demonstrates that by using this novel strategy one can rapidly predict and identify a neutralizing antibody epitope, even in the absence of the crystal structure of its target protein.
Journal of Molecular Biology 06/2006; 359(1):190-201. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Meeting abstracts. A s ingle PDF containing all abstracts in this Supplement is available here http://ww w.biomedcentral. com/content/pdf/ 1742-4690-3-S1-i nfo.pdf
[show abstract][hide abstract] ABSTRACT: West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.
Journal of Virology 01/2006; 79(23):14606-13. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding domain. Amino acids critical for 80R binding were identified. In addition, the effects of various 80R-binding domain amino acid substitutions which occur in SARS-like-CoV from civet cats, and which evolved during the 2002/2003 outbreak and in a 2003/2004 Guangdong index patient, were analyzed. The results demonstrated that the vast majority of SARS-CoVs are sensitive to 80R. We propose that by establishing the susceptibility and resistance profiles of newly emerging SARS-CoVs through early S1 genotyping of the core 180-amino-acid neutralizing epitope of 80R, an effective immunoprophylaxis strategy with 80R should be possible in an outbreak setting. Our study also cautions that for any prophylaxis strategy based on neutralizing antibody responses, whether by passive or active immunization, a genotyping monitor will be necessary for effective use.
Journal of Virology 06/2005; 79(10):5900-6. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002-2003 SARS outbreak and during the much less severe 2003-2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002-2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002-2003 SARS epidemic.
The EMBO Journal 05/2005; 24(8):1634-43. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pancreatic cancer is lethal because of its invasiveness, rapid progression, and profound resistance to chemotherapy and radiation therapy. To identify the molecular mechanisms underlying this, we have examined the expression and potency of three major death receptors: tumor necrosis factor receptor (TNF-R), TNF-related apoptosis-inducing ligand receptor (TRAIL-R), and Fas in mediating cytotoxicity in four invasive pancreatic cancer cell lines. We have analyzed the expression of major antiapoptotic factors, cell cycle regulators and death receptor decoys (DcR) in comparison with normal pancreas tissues and five other human malignant tumor cell lines. We have found that different pancreatic cancer cell lines coexpress high-level TRAIL-R, Fas, and TNF-R1 but are strongly resistant to apoptosis triggered by the death receptors. DcR2 and DcR3 overexpression may partly contribute to the resistance of pancreatic cancer cells to TRAIL-R- and Fas-mediated cytotoxicity. Bcl-XL and Bcl-2 are predominantly overexpressed in pancreatic cancer cell lines, respectively. Bcl-XL is also predominantly overexpressed in prostate, colorectal, and intestinal cancer cells. The knockdown of the predominant Bcl-XL overexpression significantly reduces the viability of pancreatic cancer cells to TNFalpha- and TRAIL-mediated apoptosis by sublethal-dose single and combined antitumor drugs, including geldanamycin, PS-341, Trichostatin A, and doxorubicine. Geldanamyin and PS-341 synergistically block NFkappaB activation, suppress Akt/PKB pathway, and down-regulate Bcl-XL, Bcl-2, cIAP-1, and cyclin D1 expression. This combined regimen dramatically enhances TRAIL cytotoxic effects and breaks through chemoresistance. Bcl-XL plays a vital role in pancreatic cancer chemoresistance. Geldanamycin, PS-341, and TRAIL triple combination may be a novel therapeutic strategy for pancreatic cancer.
Cancer Research 04/2005; 65(6):2344-52. · 8.65 Impact Factor