Chi-Hui Liang

Broadly Neutralizing HIV Antibodies Define a Glycan-Dependent Epitope on the Prefusion Conformation of gp41 on Cleaved Envelope Trimers

Broadly neutralizing HIV antibodies are much sought after (a) to guide vaccine design, both as templates and as indicators of the authenticity of vaccine candidates, (b) to assist in structural studies, and (c) to serve as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies has been limited. Here, we describe a set of human monoclonal antibodies that define what is, to the best of our knowledge, a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike.

Hemagglutinin–neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin–neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

Iron Oxide/Gold Core/Shell Nanoparticles for Ultrasensitive Detection of Carbohydrate−Protein Interactions

Changes in the expression of cell surface glycan are often associated with malignant metastasis. The expression level may be dramatically enhanced during tumor progression. A highly sensitive assay that is capable of detecting low levels of cancer-associated carbohydrate antigens can be a powerful tool for early diagnosis. In this work, an ultrasensitive glycans array using iron oxide/gold core/shell nanoparticles conjugated with antibodies or proteins is developed. A magnetic field is applied to quickly bring nanoparticle labeled proteins or antibodies from a solution to an array of carbohydrates immobilized on glass slides and to help them to encounter the carbohydrates at very low concentration. The gold shell provides a well established platform for conjugation of biomolecules. Well-defined recognition systems, namely, mannose derivatives (Man1, Man4, and Man9) with a mannose binding lectin (Concanavalin A) and a stage-specific embryonic antigens-3 (SSEA-3) with a monoclonal antibody (anti-SSEA-3) were chosen to establish this detection tool. Array systems were conducted to determine their surface dissociation constant (K(D,surface)) and their binding specificity for qualitative and quantitative analysis of carbohydrate-protein and carbohydrate-antibody interactions. When coupled with a signal amplification method based on nanoparticle-promoted reduction of silver, the sensitivity of an iron oxide/gold core/shell nanoparticle-based assay reached to subattomole level in carbohydrate detection.

Differential Receptor Binding Affinities of Influenza Hemagglutinins on Glycan Arrays

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.

Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch.

The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.

Effects of Neighboring Glycans on Antibody–Carbohydrate Interaction†

Carbohydrate recognition is a crucial event in many biological processes, including the progression of diseases such as AIDS, influenza, and cancer. Thus, characterization and reconstruction of carbohydrate epitopes to mimic authentic composition and presentation have become one of the goals in glycoscience that may greatly influence the strategy of drugs design. For example, carbohydrate epitopes on virus or cancer cells represent attractive targets for development of carbohydrate-based vaccines. Understanding the presentation of carbohydrate epitopes on cell surface allows us to more closely mimic the natural setting in the context of vaccine design. For instance, the HIV envelope glycoprotein gp120 contains high-mannose clusters on its surface to shield peptides from recognition by the host immune system and facilitate invasion by binding to the C-type lectin DC-SIGN on dendritic cells. Therefore, mimicking the high-mannose clusters on the HIV surface has become a promising approach to develop carbohydrate-based vaccines. However, clustered expression patterns of carbohydrates are complicated. Incomplete mimicry of carbohydrate epitopes on cell surface could lead to a failed vaccine design. Recently, Danishefsky and co-workers have shown that the Man9GlcNAc2-based vaccine elicited a high-titer antibody response that recognizes the Man9GlcNAc2 epitope but fails to neutralize HIV, thereby suggesting that it is not an optimal mimic of the epitope of gp120. To reach an optimal presentation of the carbohydrate epitope, several research groups have attempted to address the question by modulating antigen density and flexibility, but an optimal method has not yet emerged. Furthermore, there is a concern that carbohydrate–protein interactions may be either enhanced by multivalency or suppressed by steric hindrance. In addition, though not well understood, the proximity effect by other molecules may be significant. Herein, we investigated the effects of neighboring glycans on carbohydrate–antibody interaction using glycan microarrays. Interestingly, we found that heterogeneous glycans, which were prepared by mixing two distinct oligosaccharides and spotted onto glass slides, provide the superior binding affinity compared to the individual components in the microarray experiments. These results additionally suggest that heterogeneous-ligand glycans can serve as a novel strategy for the development of carbohydrate-based vaccine design. To understand the effects of neighboring glycans on carbohydrate–antibody recognition, we first employed heterogeneous glycans arrays to study anti-SSEA3 (stage-specific embryonic antigen 3, Gb5) antibody interactions. All glycans were amine-modified and covalently bonded to a glass surface activated by N-hydroxysuccinimide. The slides were spotted with 50 mm of Gb5 (5 10 14 mole/spot), six heterogeneous glycans (SSEA4/Gb5, Globo H/Gb5, Gb4/Gb5, Gb3/Gb5, Gb2/Gb5, Bb2/Gb5; 1:1 mole ratio by mixing the glycan with 50 mm Gb5), and one mixture of 50 mm Gb5 and 50 mm 5amino-1-pentanol (the linker). The binding schemes are each shown in Figure 1a. Each sample was printed with 12 replicates horizontally to form an array of 12 8 spots on each subarray. The heterogeneous glycan subarray was repeated through a series of dilution steps with the density of viable glycan samples decreasing by a factor of two in each step. Slide images obtained from a fluorescence scan after anti-Gb5 antibody incubation are shown in Figure 1b, and their fluorescence intensities have been compared in a bar chart (Figure 1c). Because all these samples contained the same amount of Gb5 for anti-Gb5 antibody binding, they would have had equal fluorescence intensities if the structures of neighboring glycans did not affect the antibody recognition. However, our results clearly show that the relative binding strength, based on fluorescence intensities, was SSEA4/Gb5>Gb5 onlyffilinker/Gb5>Gb3/Gb5> Gb2/Gb5>Bb2/Gb5>Gb4/Gb5>Globo H/Gb5. The highest fluorescence intensity in SSEA4/Gb5 was likely a result of the cross-reactivity and multiligand effects which were caused by one antibody binding with two different structures of glycans simultaneously. Furthermore, the antibody binding avidity in heterogeneous samples seemed to be influenced by the affinity and steric effects of neighboring glycans. As revealed in Figure 1c, a longer length of neighboring glycans [*] Dr. C.-H. Liang, C.-W. Lin, Prof. C.-H. Wong, Prof. C.-Y. Wu The Genomics Research Center, Academia Sinica, Taiwan 128 Academia Road, Section 2, Nankang, Taipei 115 (Taiwan) E-mail: chwong@gate.sinica.edu.tw cyiwu@gate.sinica.edu.tw Dr. S.-K. Wang, Dr. C.-C. Wang, Prof. C.-H. Wong Department of Chemistry, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)

A Prominent Site of Antibody Vulnerability on HIV Envelope Incorporates a Motif Associated with CCR5 Binding and Its Camouflaging Glycans.

The dense patch of high-mannose-type glycans surrounding the N332 glycan on the HIV envelope glycoprotein (Env) is targeted by multiple broadly neutralizing antibodies (bnAbs). This region is relatively conserved, implying functional importance, the origins of which are not well understood. Here we describe the isolation of new bnAbs targeting this region. Examination of these and previously described antibodies to Env revealed that four different bnAb families targeted the (324)GDIR(327) peptide stretch at the base of the gp120 V3 loop and its nearby glycans. We found that this peptide stretch constitutes part of the CCR5 co-receptor binding site, with the high-mannose patch glycans serving to camouflage it from most antibodies. GDIR-glycan bnAbs, in contrast, bound both (324)GDIR(327) peptide residues and high-mannose patch glycans, which enabled broad reactivity against diverse HIV isolates. Thus, as for the CD4 binding site, bnAb effectiveness relies on circumventing the defenses of a critical functional region on Env.

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galβ1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

An HIV-1 antibody from an elite neutralizer implicates the fusion peptide as a site of vulnerability

The induction by vaccination of broadly neutralizing antibodies (bNAbs) capable of neutralizing various HIV-1 viral strains is challenging, but understanding how a subset of HIV-infected individuals develops bNAbs may guide immunization strategies. Here, we describe the isolation and characterization of the bNAb ACS202 from an elite neutralizer that recognizes a new, trimer-specific and cleavage-dependent epitope at the gp120–gp41 interface of the envelope glycoprotein (Env), involving the glycan N88 and the gp41 fusion peptide. In addition, an Env trimer, AMC011 SOSIP.v4.2, based on early virus isolates from the same elite neutralizer, was constructed, and its structure by cryo-electron microscopy at 6.2 Å resolution reveals a closed, pre-fusion conformation similar to that of the BG505 SOSIP.664 trimer. The availability of a native-like Env trimer and a bNAb from the same elite neutralizer provides the opportunity to design vaccination strategies aimed at generating similar bNAbs against a key functional site on HIV-1.

Infection of Differentiated Porcine Airway Epithelial Cells by Influenza Virus: Differential Susceptibility to Infection by Porcine and Avian Viruses

Background Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses. Methodology/Principal Findings To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses. Conclusions/Significance Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.