Vidya S. Shivatare

Modular synthesis of N -glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120--a glycoprotein found on the surface of the virus envelope--thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

A Neutralizing Antibody Recognizing Primarily N-Linked Glycan Targets the Silent Face of the HIV Envelope

&NA; Virtually the entire surface of the HIV‐1‐envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the “silent face” on the gp120 subunit. From an HIV‐1‐infected donor, #74, we identified antibody VRC‐PG05, which neutralized 27% of HIV‐1 strains. The crystal structure of the antigen‐binding fragment of VRC‐PG05 in complex with gp120 revealed an epitope comprised primarily of N‐linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC‐PG05 in donor #74 involved shifting of glycan‐N448 to N446 or mutation of glycan‐proximal residue E293. HIV‐1 neutralization can thus be achieved at the silent face center by glycan‐recognizing antibody; along with other known epitopes, the VRC‐PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer. Graphical Abstract Figure. No caption available. HighlightsIdentified and defined crystal structure of antibody VRC‐PG05 in complex with gp120VRC‐PG05 epitope is at the center of the glycosylated silent face of HIV‐1 gp120VRC‐PG05 utilizes both glycopeptide and glycan‐cluster mechanisms of recognitionVRC‐PG05 completes neutralizing antibody coverage of the prefusion‐closed Env trimer &NA; The center of the “silent face” on the HIV‐1 envelope is shielded by glycans and has been devoid of antibody recognition. Zhou et al. identify the antibody VRC‐PG05, which binds a glycan‐dominated epitope at the silent face center and completes antibody recognition of all major exposed regions of the envelope trimer.

Identification of four rotamers of m-methoxystyrene by resonant two-photon ionization and mass analyzed threshold ionization spectroscopy

We report the vibronic and cation spectra of four rotamers of m-methoxystyrene, recorded by using the two-color resonant two-photon ionization and mass-analyzed threshold ionization techniques. The excitation energies of the S1← S0 electronic transition are found to be 32 767, 32 907, 33 222, and 33 281 cm(-1), and the corresponding adiabatic ionization energies are 65 391, 64 977, 65 114, and 64 525 cm(-1) for these isomeric species. Most of the observed active vibrations in the electronically excited S1 and cationic ground D0 states involve in-plane ring deformation and substituent-sensitive bending motions. It is found that the relative orientation of the methoxyl with respect to the vinyl group does not influence the vibrational frequencies of the ring-substituent bending modes. The two dimensional potential energy surface calculations support our experimental finding that the isomerization is restricted in the S1 and D0 states.

Influenza A surface glycosylation and vaccine design

Significance Influenza A virus (IAV) is a major threat to global public health, and so understanding the biology of IAV is essential to develop antiflu vaccines and therapeutics. Here, we show the links between viral surface glycosylation and IAV function. The glycosylation of HA modulates virus infectivity, and host immune response; the glycosylation of NA affects its structure, activity, specificity, and thermostability to regulate virus release and virulence. In addition, using live attenuated IAV without the stalk and catalytic domains of NA as vaccine can strongly induce IAV-specific CD8+ T-cell responses to various virus strains. Therefore, our findings have clarified the role of glycosylation in IAV and provided a new direction for the development of universal flu vaccines. We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAV-specific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.

Unprecedented Role of Hybrid N-Glycans as Ligands for HIV-1 Broadly Neutralizing Antibodies

The development of an HIV vaccine has been hampered by the extraordinary mutability and genetic diversity of the virus, particularly the substantial sequence diversity of gp120 and gp 41 envelope glycoproteins existing in more than 2000 HIV variants. The highly diverse glycans on HIV spikes are commonly considered as immunologically silent self-antigens; however, the discovery of highly potent broadly neutralizing antibodies (bNAbs) from HIV patients targeting the viral surface glycans has raised a major question about the origin of their antigens. Recent epitope mapping studies of the bNAb PG9 indicated a requirement of a properly spaced high mannose and a complex type glycan connected by a short peptide spacer. We have recently discovered that a 1:1 mixture of Man5 and sialyl biantennary glycan with well-defined distance and without the peptide spacer is well recognized by PG9 with high avidity and, thus, proposed that a hybrid glycan with oligomannose and complex-type arm could be the proper ligand of PG9. To verify this proposition, we first designed and chemo-enzymatically synthesized a series of unusual hybrid-type N-glycan structures, which may exist on HIV surface glycoproteins through the host-guided N-glycosylation pathway. The synthetic hybrid glycans were then used to prepare glycan arrays for the binding studies of PG9 and several other highly potent bNAbs, including PG16, PGT121, PGT128-3C, 2G12, VRC13, VRC-PG05, VRC26.25, VRC26.09, PGDM1400, 35O22, and 10-1074. Our results demonstrated that PG9 and some other bNAbs bind with strong avidity (subnanomolar Kd) to certain hybrid structures, suggesting that these unusual glycans may serve as epitopes for the design of vaccines against HIV.

Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation

Significance The carbohydrate components of glycoproteins are known to affect the structure and function of glycoproteins, and thus it is important to develop effective tools to manipulate and optimize the glycan components of glycoproteins with therapeutic significance. This study demonstrates the discovery of endoglycosidases for the remodeling of glycans on Herceptin, a monoclonal antibody used in the treatment of breast cancer, to optimize its effector functions, especially the antibody-dependent cellular cytotoxicity (ADCC). In addition, a method has been developed for the expression of antibodies from yeast to enable the large-scale synthesis of antibodies for further manipulation of the glycan moiety, using endoglycosidases and stable transglycosylation donors to prepare a homogeneous glycoform with optimized effector functions. Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycoforms. In this study, we demonstrate that the mAb trastuzumab expressed in glycoengineered P. pastoris can be remodeled through deglycosylation by endoglycosidases identified from the Carbohydrate Active Enzymes database and through transglycosylation using glycans with a stable leaving group to generate a homogeneous antibody designed to optimize the effector functions. The 10 newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS), two of which can even accept sialylated tri- and tetraantennary glycans as substrates.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise α-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial β-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Vibronic and cation spectroscopy of selected rotamers of 4-chloro-3-fluorophenol

We applied the two-colour resonant two-photon ionisation and mass-analysed threshold ionisation techniques to record the vibrationally resolved spectra of the selected rotamers and 35Cl and 37Cl isotopologues of 4-chloro-3-fluorophenol in the electronically excited S1 and cationic ground D0 states. The band origins of the S1 ← S0 electronic transition and the adiabatic ionisation energies of the cis and trans rotamers of 4-chloro-3-fluorophenol are determined to be 35,233 ± 2 and 35,405 ± 2 cm−1, and 69,334 ± 5 and 69,460 ± 5 cm−1, respectively. The electronic transition energies and general spectral features of the two isotopologues are nearly identical. Most of the observed active vibrations result from the in-plane ring deformation and substituent-sensitive motions. The experimental data show that the frequency difference in the observed active vibrations of the rotamers and isotopologues depends on the nature, vibrational pattern, location, and relative orientation of the substituents.