A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence.

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.

1-deoxysphingolipids bind to COUP-TF to modulate lymphatic and cardiac cell development.

Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differentiation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2-mediated transcription.

Variable posttranslational modifications of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.

Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2.

The emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race and pre-existing conditions of COVID-19 patients.

Vaccines and Therapeutics for COVID-19 - How Can Understanding SARS-CoV-2 Glycosylation Lead to Pharmaceutical Advances?

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 160 million people worldwide. Researchers have targeted the SARS-CoV-2 structural proteins to better combat the pandemic. Of the four structural proteins, spike (S), membrane (M), envelope (E) and nucleocapsid (N), the S, M and E proteins are glycosylated whereas the N protein is phosphorylated. The glycosylation of the S protein has been reported previously by multiple research groups, and this knowledge has assisted the pharmaceutical industry in developing vaccines and treatment options. In the United States, there are currently three approved COVID-19 vaccines. All three of these vaccines use the S protein to teach host cells how to react when SARS-CoV-2 particles are present. Treatment options utilizing antivirals and immunosuppressants are being developed in addition to vaccines. Different treatment approaches are needed based on the severity of COVID-19 infection. The therapeutic options currently available are not derived through the direct knowledge on SARS-CoV-2 glycosylation. However, more research on the glycosylation of the structural proteins and how this effects SARS-CoV-2 and host cell binding could lead to new and more effective therapeutics. Herein we outline the current vaccine and therapeutic options against COVID-19 available to the public, as well as those still in development.

Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2.

The current emergence of the novel coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high-resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor-binding domain of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation are strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research toward the development of a suitable vaccine candidate.

Manipulation of cytokine secretion in human dendritic cells using glycopolymers with picomolar affinity for DC-SIGN.

The human C-type lectin DC-SIGN (CD209) is a significant receptor on the surface of dendritic cells (DCs) - crucial components of host defense that bridge the innate and adaptive immune systems. A range of linear glycopolymers, constructed via controlled radical polymerization techniques have been shown to interact with DC-SIGN with affinities in the physiologically active range. However, these first generation glycopolymers possess limited structural definition and their effects on DCs were not known. Here we report the development of star-shaped mannose glycopolymers with the aim of targeting the clustered domain arrangement of DC-SIGN and these were shown to bind with picomolar affinity. Increased secretion of IL-10 with simultaneous decrease in secreted IL-12p70 occurred in activated DCs incubated with star-shaped glycopolymers - a cytokine secretion pattern characteristic of wound-healing tissue environments. Incorporating stellar architecture into glycopolymer design could be key to developing selective and very high-affinity therapeutic materials with distinct immunomodulatory and tissue repair potential.

Specific and Differential Binding of N-Acetylgalactosamine Glycopolymers to the Human Macrophage Galactose Lectin and Asialoglycoprotein Receptor.

A range of glycopolymers composed of N-acetylgalactosamine were prepared via sequential Cu(I)-mediated polymerization and alkyne-azide click (CuAAC). The resulting polymers were shown, via multichannel surface plasmon resonance, to interact specifically with human macrophage galactose lectin (MGL; CD301) with high affinity (KD = 1.11 µM), but they did not bind to the mannose/fucose-selective human lectin dendritic-cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN; CD209). The effect of sugar ligand valency on the binding (so-called "glycoside cluster effect") of poly(N-acetylgalactosamine) to MGL was investigated by varying first the polymer chain length (DP: 100, 64, 40, 23, 12) and then the architecture (4- and 8-arm star glycopolymers). The chain length did not have a significant effect on the binding to MGL (KD = 0.17-0.52 µM); however, when compared to a hepatic C-type lectin of a similar monosaccharide specificity, the asialoglycoprotein receptor (ASGPR), the binding affinity was more noticeably affected (KD = 0.37- 6.65 µM). These data suggest that known differences in the specific configuration/orientation of the carbohydrate recognition domains of MGL and ASGPR are responsible for the differences in binding observed between the different polymers of varied chain length and architecture. In the future, this model has the potential to be employed for the development of tissue-selective delivery systems.