The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to ...global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.
Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development ...focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan density on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organisation of glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the protein surface.
The HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, ...such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length unmodified Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.
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•N-linked glycans of ACE2 have been suggested to play a role in SARS-CoV-2 binding.•Using glycan engineering we generated a panel of glycan modified ACE2 variants.•The binding of ...these variants to spike protein was determined using SPR and LC-MS.•These results suggest a limited role for the glycans of ACE2 in SARS-CoV-2 binding.•SARS binding with ACE2 is slightly influenced by sialylation and mannosylation.
The severity of SARS-CoV-2 infection is highly variable and yet the molecular basis for this effect remains elusive. One potential contribution are differences in the glycosylation of target human cells, particularly as SARS-CoV-2 has the capacity to bind sialic acid which is a common, and highly variable, terminal modification of glycans. The viral spike glycoprotein (S) of SARS-CoV-2 and the human cellular receptor, angiotensin-converting enzyme 2 (ACE2) are both densely glycosylated. We therefore sought to investigate whether the glycosylation state of ACE2 impacts the interaction with SARS-CoV-2 viral spike. We generated a panel of engineered ACE2 glycoforms which were analyzed by mass spectrometry to reveal the site-specific glycan modifications. We then probed the impact of ACE2 glycosylation on S binding and revealed a subtle sensitivity with hypersialylated or oligomannose-type glycans slightly impeding the interaction. In contrast, deglycosylation of ACE2 did not influence SARS-CoV-2 binding. Overall, ACE2 glycosylation does not significantly influence viral spike binding. We suggest that any role of glycosylation in the pathobiology of SARS-CoV-2 will lie beyond its immediate impact of receptor glycosylation on virus binding.
In vaccine design, antigens are often arrayed in a multivalent nanoparticle form, but in vivo mechanisms underlying the enhanced immunity elicited by such vaccines remain poorly understood. We ...compared the fates of two different heavily glycosylated HIV antigens, a gp120-derived mini-protein and a large, stabilized envelope trimer, in protein nanoparticle or "free" forms after primary immunization. Unlike monomeric antigens, nanoparticles were rapidly shuttled to the follicular dendritic cell (FDC) network and then concentrated in germinal centers in a complement-, mannose-binding lectin (MBL)-, and immunogen glycan-dependent manner. Loss of FDC localization in MBL-deficient mice or via immunogen deglycosylation significantly affected antibody responses. These findings identify an innate immune-mediated recognition pathway promoting antibody responses to particulate antigens, with broad implications for humoral immunity and vaccine design.
Lassa virus is an Old World arenavirus endemic to West Africa that causes severe hemorrhagic fever. Vaccine development has focused on the envelope glycoprotein complex (GPC) that extends from the ...virion envelope. The often inadequate antibody immune response elicited by both vaccine and natural infection has been, in part, attributed to the abundance of N-linked glycosylation on the GPC. Here, using a virus-like–particle system that presents Lassa virus GPC in a native-like context, we determine the composite population of each of the N-linked glycosylation sites presented on the trimeric GPC spike. Our analysis reveals the presence of underprocessed oligomannose-type glycans, which form punctuated clusters that obscure the proteinous surface of both the GP1 attachment and GP2 fusion glycoprotein subunits of the Lassa virus GPC. These oligomannose clusters are seemingly derived as a result of sterically reduced accessibility to glycan processing enzymes, and limited amino acid diversification around these sites supports their role protecting against the humoral immune response. Combined, our data provide a structure-based blueprint for understanding how glycans render the glycoprotein spikes of Lassa virus and other Old World arenaviruses immunologically resistant targets.
Incessant antigenic evolution enables the persistence and spread of influenza virus in the human population. As the principal target of the immune response, the hemagglutinin (HA) surface antigen on ...influenza viruses continuously acquires and replaces N-linked glycosylation sites to shield immunogenic protein epitopes using host-derived glycans. Anti-glycan antibodies, such as 2G12, target the HIV-1 envelope protein (Env), which is even more extensively glycosylated and contains under-processed oligomannose-type clusters on its dense glycan shield. Here, we illustrate that 2G12 can also neutralize human seasonal influenza A H3N2 viruses that have evolved to present similar oligomannose-type clusters on their HAs from around 20 years after the 1968 pandemic. Using structural biology and mass spectrometric approaches, we find that two N-glycosylation sites close to the receptor binding site (RBS) on influenza hemagglutinin represent the oligomannose cluster recognized by 2G12. One of these glycan sites is highly conserved in all human H3N2 strains and the other emerged during virus evolution. These two N-glycosylation sites have also become crucial for fitness of recent H3N2 strains. These findings shed light on the evolution of the glycan shield on influenza virus and suggest 2G12-like antibodies can potentially act as broad neutralizers to target human enveloped viruses.
A key aspect of successful viral vaccine design is the elicitation of neutralizing antibodies targeting viral attachment and fusion glycoproteins that embellish viral particles. This observation has ...catalyzed the development of numerous viral glycoprotein mimetics as vaccines. Glycans can dominate the surface of viral glycoproteins and as such, the viral glycome can influence the antigenicity and immunogenicity of a candidate vaccine. In one extreme, glycans can form an integral part of epitopes targeted by neutralizing antibodies and are therefore considered to be an important feature of key immunogens within an immunization regimen. In the other extreme, the existence of peptide and bacterially expressed protein vaccines shows that viral glycosylation can be dispensable in some cases. However, native-like glycosylation can indicate native-like protein folding and the presence of conformational epitopes. Furthermore, going beyond native glycan mimicry, in either occupancy of glycosylation sites or the glycan processing state, may offer opportunities for enhancing the immunogenicity and associated protection elicited by an immunogen. Here, we review key determinants of viral glycosylation and how recombinant immunogens can recapitulate these signatures across a range of enveloped viruses, including HIV-1, Ebola virus, SARS-CoV-2, Influenza and Lassa virus. The emerging understanding of immunogen glycosylation and its control will help guide the development of future vaccines in both recombinant protein- and nucleic acid-based vaccine technologies.
Dried blood spot (DBS) samples can be used for the detection of severe acute respiratory syndrome coronavirus 2 spike antibodies. DBS sampling is comparable to matched serum samples with a relative ...98.1% sensitivity and 100% specificity. Thus, DBS sampling offers an alternative for population-wide serologic testing in the coronavirus pandemic.
Background Hyperkalemia has been associated with higher mortality in long-term hemodialysis (HD) patients. There are few data concerning the relationship between dietary potassium intake and outcome. ...Study Design The mortality predictability of dietary potassium intake from reported food items estimated using the Block Food Frequency Questionnaire (FFQ) at the start of the cohort was examined in a 5-year (2001-2006) cohort of 224 HD patients in Southern California using Cox proportional hazards regression. Setting & Participants 224 long-term HD patients from 8 DaVita dialysis clinics. Predictors Dietary potassium intake ranking using the Block FFQ. Outcomes 5-year survival. Results HD patients with higher potassium intake had greater dietary energy, protein, and phosphorus intakes and higher predialysis serum potassium and phosphorus levels. Greater dietary potassium intake was associated with significantly increased death HRs in unadjusted models and after incremental adjustments for case-mix, nutritional factors (including 3-month averaged predialysis serum creatinine, potassium, and phosphorus levels; body mass index; normalized protein nitrogen appearance; and energy, protein, and phosphorus intake) and inflammatory marker levels. HRs for death across the 3 higher quartiles of dietary potassium intake in the fully adjusted model (compared with the lowest quartile) were 1.4 (95% CI, 0.6-3.0), 2.2 (95% CI, 0.9-5.4), and 2.4 (95% CI, 1.1-7.5), respectively ( P for trend = 0.03). Restricted cubic spline analyses confirmed the incremental mortality predictability of higher potassium intake. Limitations FFQs may underestimate individual potassium intake and should be used to rank dietary intake across the population. Conclusions Higher dietary potassium intake is associated with increased death risk in long-term HD patients, even after adjustments for serum potassium level; dietary protein; energy, and phosphorus intake; and nutritional and inflammatory marker levels. The potential role of dietary potassium in the high mortality rate of HD patients warrants clinical trials.
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