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Archive for the ‘Mechanisms of infection by SARS-CoV-2’ Category


Glycosylation and its Role in SARS-CoV-2 Viral Pathogenesis

Author: Meg Baker, PhD

 

N-Glycosylation and COVID19

Glycobiology

N-linked glycosylation (NLG) is a complex biosynthetic process that regulates proper folding of proteins through and intracellular transport of proteins to the secretory pathway. This co- and post-translational modification occurs by a series of enzymatic reactions, which results in the transfer of a core glycan from the lipid carrier to a protein substrate and the possibility for further remodeling of the glycan. The enzymes are located in the cytosolic and the luminal side of the ER membrane. The study of NLG and related effects of glycans is called glycobiology.

NLG takes place at sites specified in the protein sequence itself. N-linked oligosaccharides are attached via a GlcNAc linked to the side chain nitrogen of Asn found in the consensus sequence NXT/S (X ≠ P) known as the ‘glycosylation sequon’. Formation of a precursor branched carbohydrate chain, the lipid-linked oligosaccharide (LLO) structure, takes place in the endoplasmic reticulum. The LLO consists of a Glc3Man9GlcNAc2 molecule (three glucose, nine mannose, and two N-acetylglucosamine sugars) linked to a dolichol pyrophosphate. The enzyme oligosaccharyltransferase then moves it to an Asn in the polypeptide.

The removal of the three glucose sugars from the new N-linked glycan signals that the structure is ready for transport to the Golgi where mannose is removed yielding a carbohydrate chain containing five–nine mannose sugars. Further removal of mannose residues can lead to the core structure containing three mannose and two N-acetylglucosamine residues, which may then be elongated with a variety of different monosaccharides including galactose, N-acetylglucosamine (aka NAG or GlcNac), N-acetylgalactosamine, fucose, and sialic acid, many of which can also exist in sulfated form.

The enzymes involved in this essential process are evolutionarily conserved. However, the genes and their specific functions, have evolved uniquely for each selected organism. Therefore, each organism and each individual cell, depending on genetic background and influenced by nutritional and such things as disease status, will decorate secreted proteins in a unique manner.

The advent of biologic medicines (protein based therapeutics) presents the challenge of making sure that the primary protein sequence is specified but also that the manufacture of the protein – typically in a eukaryotic cell host capable of glycosylation – will take place with some degree of reproducibility. The large number of monoclonal antibody therapeutics absolutely require glycosylation for proper structural integrity but are generally made in rodent or other nonhuman cells. Thus, the term “biosimilar” rather than generic is the term being used to connote the variation which will necessarily result due to different manufacturing process even of the same genetic sequence.

 

Viral Glycoproteins

It should be obvious that the viral genome is not large enough to encompass the collection of enzymes required for glycosylation of any type and viral glycoproteins are formed by the host cell in which the virus is replicating. The study of the impact of glycan content and composition on viral infectivity and, more importantly, vaccine development is a subject which has been late to be addressed largely due to the technical difficulty and lack of methods for analyzing protein glycan composition. However, progress is being made. Raska et al. (J Biol Chem 2010 Jul 2; 285(27): 20860–20869. Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition)  was able to perform such an analysis on the HIV-1 virus albeit almost 30 years after its emergence in human populations. The findings of this study may explain, in part, the difficulty in developing a vaccine against HIV.

 

SARS-CoV-2 spike protein (P0DTC2 uniprot.org) – as so popularly depicted – is a trimer poking out of the lipid coat that protects it’s genome. The spike protein, like gp120 in HIV, is the point of contact with the human cell ACE2 receptor it uses to gain entry. The spike protein contains two functional external subunits, designated S1 and S2. S1 separated by a furin cleavage site from S2, forms the apex of the trimeric spike structure, is responsible for attachment to the ACE2 receptor. S2 is responsible for fusion to the cell membrane. (PDB: 6VSB shows a 3D image of the protein structure, including glycan positions). There are 22 glycans per polypeptide or 66 per spike trimer protein (Watanabe et al. 2021 Site-specific glycan analysis of the SARS-CoV-2 spike. Science 17 Jul 2020:Vol. 369, Issue 6501, pp. 330-333 ).

Although shielding of receptor binding sites by glycans is a common feature of viral glycoproteins, Watanabe (ibid) note the low mutation rate of SARS-CoV-2 and that as yet, there have been no observed mutations to N-linked glycosylation sites.

The development of a vaccine or individual antibodies or antibody cocktails with neutralizing (viral entry blocking or virocidal activity) is also influenced by the presence or absence of glycans and how well they target the natural conformation of the spike protein. Papageorgiou et al. The SARS-CoV-2 Spike Glycoprotein as a Drug and Vaccine Target: Structural Insights into Its Complexes with ACE2 and Antibodies. Cells 2020 Oct 22;9(11):2343. doi: 10.3390/cells9112343. SARS-CoV-2 Spike – Stanford Coronavirus Antiviral Research Database It should be noted that the mRNA vaccines (or other nucleic acid formats) may obviate these analysis because the immune response is to a spike protein made and glycosylated in the human host’s own body and therefore will be customized to each individual in some sense.

Glycans may themselves represent drug targets. Casolino et al. suggest an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike’s receptor binding domain (RBD), which is responsible for ACE2 recognition (Casolino et al. 2020. Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein ACS Cent Sci. 2020 Oct 28; 6(10): 1722–1734),

 

COVID19 Variants

SARS-CoV-2 lineage B.1.1.7 likely arose in the United Kingdom in September 2019 and is characterized by 17 mutations, including 8 in the spike protein (Rambaut et al., 2020). Other lineages, including B.1.351, initially detected in South Africa (Tegally et al., 2020), and most recently lineage P.1, first documented in the Amazonia region of Brazil (Faria et al., 2020), carry additional mutations. All three lineages are characterised by a N501Y (Asn to Tyr) mutation in the spike protein, while both B.1.351 and P.1 also carry the spike mutation E484K. In addition, both B.1.1.7 and B.1.351, but not P.1, have acquired short sequence deletions in the spike protein. N501Y is in the receptor-binding domain (RBD) but is not a glycosylation site.

Reference

See the CDC Emerging SARS-CoV-2 Variants | CDC

 

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Comparing COVID-19 Vaccine Schedule Combinations, or “Com-COV” – First-of-its-Kind Study will explore the Impact of using eight different Combinations of Doses and Dosing Intervals for Different COVID-19 Vaccines

Reporter: Aviva Lev-Ari, PhD, RN

 

The UK’s COVID-19 vaccine rollout commenced in December, and requires an individual to receive two doses of the same vaccine, either Pfizer/BioNTech’s BNT162b2 or AstraZeneca/Oxford’s ChAdOx1, with a maximum interval of 12 weeks between doses. As of February 3, 10 million first doses have been administered.

Com-COV has been classified as an “Urgent Public Health” study by the National Institutes for Health and Research (NIHR), and it’s hoped that the data produced may offer greater flexibility for vaccine delivery going forward.

“Given the inevitable challenges of immunizing large numbers of the population against COVID-19 and potential global supply constraints, there are definitely advantages to having data that could support a more flexible immunization program, if ever needed and approved by the medicines regulator,” Jonathan Van-Tam, deputy chief medical officer and senior responsible officer for the study, said in a press release.

The study will run for a 13-month period and will recruit over 800 patients across eight sites in the UK, including London – St George’s and UCL, Oxford, Southampton, Birmingham, Bristol, Nottingham and Liverpool.

Com-COV has eight different arms that will test eight different combinations of doses and dose intervals. This is tentative and subject to change should more COVID-19 vaccines be approved for use in the UK. The eight arms include the following dose combinations:

  • Pfizer/BioNTech and Pfizer/BioNTech – 28 days apart
  • Pfizer/BioNTech and Pfizer/BioNTech – 12 weeks apart – (control group)
  • Oxford/AstraZeneca and Oxford/AstraZeneca – 28 days apart
  • Oxford/AstraZeneca and Oxford/AstraZeneca – 12 weeks apart – (control group)
  • Oxford/AstraZeneca and Pfizer/BioNTech – 28 days apart
  • Oxford/AstraZeneca and Pfizer/BioNTech – 12 weeks apart
  • Pfizer/BioNTech and Oxford/AstraZeneca – 28 days apart
  • Pfizer/BioNTech and Oxford/AstraZeneca – 12 weeks apart

Aside from the logistical benefits of using alternative vaccines, there is scientific value to exploring how different vaccines and doses affect the human immune system.

Dr Peter English, consultant in communicable disease control, pointed out that the antigen used across the currently authorized COVID-19 vaccines is the same Spike protein. Therefore, the immune system can be expected to respond just as well if a different product is used for boosting. “It is also the case that many vaccines work better if a different vaccine is used for boosting – an approach described as heterologous boosting,” English said, referencing previously successful trials using Hepatitis B vaccines.

“It is also even possible that by combining vaccines, the immune response could be enhanced giving even higher antibody levels that last longer; unless this is evaluated in a clinical trial we just won’t know,” added Van-Tam.

If warranted by the study data, the Medicines and Healthcare products Regulatory Agency may consider reviewing and authorizing modifications to the UK’s vaccine regimen approach – but only time will tell.

“We need people from all backgrounds to take part in this trial, so that we can ensure we have vaccine options suitable for all. Signing up to volunteer for vaccine studies is quick and easy via the NHS Vaccine Research Registry,” Professor Andrew Ustianowski, national clinical lead for the NIHR COVID Vaccine Research Program, said

SOURCE

First-of-its-Kind Study Will Test Combination of Different COVID-19 Vaccines | Technology Networks

https://www.technologynetworks.com/biopharma/news/first-of-its-kind-study-will-test-combination-of-different-covid-19-vaccines-345245?utm_campaign=NEWSLETTER_TN_Biopharma

WATCH VIDEO

Different Types of COVID-19 Vaccines With Dr Seth Lederman Video | Technology Networks

https://www.technologynetworks.com/biopharma/videos/different-types-of-covid-19-vaccines-with-dr-seth-lederman-345207

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Rise of a trio of mutated viruses hints at an increase in transmissibility, speeding the virus’ leaps from one host to the next

Reporter: Aviva Lev-Ari, PhD, RN

“We have uncontrolled viral spread in much of the world,” says Adam Lauring, an infectious disease physician and virologist at the University of Michigan. “So the virus has a lot of opportunity to evolve.”

“The variants may be more transmissible, but physics has not changed,” says Müge Çevik, an infectious disease physician at the University of St. Andrews in Scotland.

Many changes don’t affect the virus’ function, and some even harm SARS-CoV-2’s ability to multiply, but they keep happening. “Viruses mutate; that’s what they do,” says Akiko Iwasaki, an immunologist at Yale School of Medicine in Connecticut.

U.K., Brazil, and South Africa. In the United Kingdom, variant B.1.1.7 likely drove the region’s record-setting spike of COVID-19 cases in January. The variant is now circulating in more than 60 countries, including the United States—and projections suggest it will become the most common virus variety in the U.S. by mid-March.

An independently arising lineage called P.1 might also be driving a wave of cases in Manaus, Brazil, where it accounted for nearly half of new COVID-19 infections in December. On January 26, Minnesotan officials reported the first U.S. case of P.1 in a resident who previously traveled to Brazil. And a third lineage raising alarms, known as B.1.351, was first spotted amid a December wave of infections in South Africa. On January 28, the first known U.S. cases of the variant were reported in South Carolina.

One specific mutation, known as N501Y, popped up independently in all three variants, suggesting it could provide an advantage to the virus. “That’s a sign that there is natural selection going on,” Lauring says. The N501Y mutation affects the virus’ spike protein, which is the key it uses to unlock entry into its host’s cells.

Another possibility is that new variants cause people who are infected to harbor more copies of the virus. This results in greater viral “shedding” in airborne droplets spewed when people talk, sing, cough, and breath.

mutations in 501Y.V2 could diminish the effectiveness of antibodies in the blood of people previously infected with the virus. But understanding whether that could lead to more re-infections, or if it could affect vaccine efficacy.

Dramatically scale up production of high-filtration masks for the general public.

Based on:

Why some coronavirus variants are more contagious‹and how we can stop them

https://www.nationalgeographic.com/science/2021/01/why-some-coronavirus-variants-are-more-contagious/?cmpid=org=ngp::mc=crm-email::src=ngp::cmp=editorial::add=SpecialEdition_20210129

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A Platform called VirtualFlow: Discovery of Pan-coronavirus Drugs help prepare the US for the Next Coronavirus Pandemic

Reporter: Aviva Lev-Ari, PhD, RN

 

ARTICLE|ONLINE NOW, 102021

A multi-pronged approach targeting SARS-CoV-2 proteins using ultra-large virtual screening

Open AccessPublished:January 04, 2021DOI:https://doi.org/10.1016/j.isci.2020.102021

 

The work was made possible in large part by about $1 million in cloud computing hours awarded by Google through a COVID-19 research grant program.

The work reported, below was sponsored by

  • a Google Cloud COVID-19 research grant. Funding was also provided by the
  • Fondation Aclon,
  • National Institutes of Health (GM136859),
  • Claudia Adams Barr Program for Innovative Basic Cancer Research,
  • Math+ Berlin Mathematics Research Center,
  • Templeton Religion Trust (TRT 0159),
  • U.S. Army Research Office (W911NF1910302), and
  • Chleck Family Foundation

 

Harvard University, AbbVie form research alliance to address emergent viral diseases

This article is part of Harvard Medical School’s continuing coverage of medicine, biomedical research, medical education and policy related to the SARS-CoV-2 pandemic and the disease COVID-19.

Harvard University and AbbVie today announced a $30 million collaborative research alliance, launching a multi-pronged effort at Harvard Medical School to study and develop therapies against emergent viral infections, with a focus on those caused by coronaviruses and by viruses that lead to hemorrhagic fever.

The collaboration aims to rapidly integrate fundamental biology into the preclinical and clinical development of new therapies for viral diseases that address a variety of therapeutic modalities. HMS has led several large-scale, coordinated research efforts launched at the beginning of the COVID-19 pandemic.

“A key element of having a strong R&D organization is collaboration with top academic institutions, like Harvard Medical School, to develop therapies for patients who need them most,” said Michael Severino, vice chairman and president of AbbVie. “There is much to learn about viral diseases and the best way to treat them. By harnessing the power of collaboration, we can develop new therapeutics sooner to ensure the world is better prepared for future potential outbreaks.”

“The cataclysmic nature of the COVID-19 pandemic reminds us how vital it is to be prepared for the next public health crisis and how critical collaboration is on every level—across disciplines, across institutions and across national boundaries,” said George Q. Daley, dean of Harvard Medical School. “Harvard Medical School, as the nucleus of an ecosystem of fundamental discovery and therapeutic translation, is uniquely positioned to propel this transformative research alongside allies like AbbVie.”

AbbVie will provide $30 million over three years and additional in-kind support leveraging AbbVie’s scientists, expertise and facilities to advance collaborative research and early-stage development efforts across five program areas that address a variety of therapeutic modalities:

  • Immunity and immunopathology—Study of the fundamental processes that impact the body’s critical immune responses to viruses and identification of opportunities for therapeutic intervention.

Led by Ulirich Von Andrian, the Edward Mallinckrodt Jr. Professor of Immunopathology in the Blavatnik Institute at HMS and program leader of basic immunology at the Ragon Institute of MGH, MIT and Harvard, and Jochen Salfeld, vice president of immunology and virology discovery at AbbVie.

  • Host targeting for antiviral therapies—Development of approaches that modulate host proteins in an effort to disrupt the life cycle of emergent viral pathogens.

Led by Pamela Silver, the Elliot T. and Onie H. Adams Professor of Biochemistry and Systems Biology in the Blavatnik Institute at HMS, and Steve Elmore, vice president of drug discovery science and technology at AbbVie.

  • Antibody therapeutics—Rapid development of therapeutic antibodies or biologics against emergent pathogens, including SARS-CoV-2, to a preclinical or early clinical stage.

Led by Jonathan Abraham, assistant professor of microbiology in the Blavatnik Institute at HMS, and by Jochen Salfeld, vice president of immunology and virology discovery at AbbVie.

  • Small molecules—Discovery and early-stage development of small-molecule drugs that would act to prevent replication of known coronaviruses and emergent pathogens.

Led by Mark Namchuk, executive director of therapeutics translation at HMS and senior lecturer on biological chemistry and molecular pharmacology in the Blavatnik Institute at HMS, and Steve Elmore, vice president of drug discovery science and technology at AbbVie.

  • Translational development—Preclinical validation, pharmacological testing, and optimization of leading approaches, in collaboration with Harvard-affiliated hospitals, with program leads to be determined.

SOURCE

https://hms.harvard.edu/news/joining-forces

 

 

A Screen Door Opens

Virtual screen finds compounds that could combat SARS-CoV-2

This article is part of Harvard Medical School’s continuing coverage of medicine, biomedical research, medical education, and policy related to the SARS-CoV-2 pandemic and the disease COVID-19.

Less than a year ago, Harvard Medical School researchers and international colleagues unveiled a platform called VirtualFlow that could swiftly sift through more than 1 billion chemical compounds and identify those with the greatest promise to become disease-specific treatments, providing researchers with invaluable guidance before they embark on expensive and time-consuming lab experiments and clinical trials.

Propelled by the urgent needs of the pandemic, the team has now pushed VirtualFlow even further, conducting 45 screens of more than 1 billion compounds each and ranking the compounds with the greatest potential for fighting COVID-19—including some that are already approved by the FDA for other diseases.

“This was the largest virtual screening effort ever done,” said VirtualFlow co-developer Christoph Gorgulla, research fellow in biological chemistry and molecular pharmacology in the labs of Haribabu Arthanari and Gerhard Wagner in the Blavatnik Institute at HMS.

The results were published in January in the open-access journal iScience.

The team searched for compounds that bind to any of 15 proteins on SARS-CoV-2 or two human proteins, ACE2 and TMPRSS2, known to interact with the virus and enable infection.

Researchers can now explore on an interactive website the 1,000 most promising compounds from each screen and start testing in the lab any ones they choose.

The urgency of the pandemic and the sheer number of candidate compounds inspired the team to release the early results to the scientific community.

“No one group can validate all the compounds as quickly as the pandemic demands,” said Gorgulla, who is also an associate of the Department of Physics at Harvard University. “We hope that our colleagues can collectively use our results to identify potent inhibitors of SARS-CoV-2.

In most cases, it will take years to find out whether a compound is safe and effective in humans. For some of the compounds, however, researchers have a head start.

Hundreds of the most promising compounds that VirtualFlow flagged are already FDA approved or being studied in clinical or preclinical trials for other diseases. If researchers find that one of those compounds proves effective against SARS-CoV-2 in lab experiments, the data their colleagues have already collected could save time establishing safety in humans.

Other compounds among VirtualFlow’s top hits are currently being assessed in clinical trials for COVID-19, including several drugs in the steroid family. In those cases, researchers could build on the software findings to investigate how those drug candidates work at the molecular level—something that’s not always clear even when a drug works well.

It shows what we’re capable of computationally during a pandemic.

Hari Arthanari

SOURCE

https://hms.harvard.edu/news/screen-door-opens?utm_source=Silverpop&utm_medium=email&utm_term=field_news_item_1&utm_content=HMNews02012021

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Inflammation and potential links with the microbiome: Mechanisms of infection by SARS-CoV-2

Reporter: Aviva Lev-Ari, PhD, RN

Mechanisms of infection by SARS-CoV-2, inflammation and potential links with the microbiome

Published Online:https://doi.org/10.2217/fvl-2020-0310

Human coronaviruses (HCoVs) were first isolated from patients with the common cold in the 1960s [1–3]. Seven HCoVs known to cause disease in humans have since been identified: HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, the SARS coronavirus (SARS-CoV), the Middle East respiratory syndrome coronavirus and the novel SARS-CoV-2 [4]. The latter was identified after a spike in cases of pneumonia of unknown etiology in Wuhan, Hubei Province, China during December 2019 and was initially named novel coronavirus (2019-nCoV) [5,6]. The virus was renamed SARS-CoV-2 according to the International Committee on Taxonomy of Viruses classification criteria due to its genomic closeness to SARS-CoV; the disease caused by this virus was named coronavirus disease (COVID-19) according to the WHO criteria for naming emerging diseases [7]. SARS-CoV-2 belongs to the genera Betacoronavirus and shares a different degree of genomic similarity with the other two epidemic coronaviruses: SARS-CoV (∼79%) and Middle East respiratory syndrome coronavirus (∼50%) [8].

COVID-19 has caused considerable morbidity and mortality worldwide and has become the central phenomenon that is shaping our current societies. Human-to-human transmission is the main route of spread of the virus, mainly through direct contact, respiratory droplets and aerosols [9–12]. Management of COVID-19 has been extremely challenging due to its high infectivity, lack of effective therapeutics and potentially small groups of individuals (i.e., asymptomatic or mild disease) rapidly spreading the disease [13–17]. Although research describing COVID-19 and the mechanisms of infection by SARS-CoV-2 and its pathogenesis has expanded rapidly, there is still much to be learnt. Important gaps in knowledge which remain to be elucidated are the dynamic and complex interactions between the virus and the host’s immune system, as well as the potential interspecies communications occurring between ecological niches encompassing distinct microorganisms in both healthy individuals and persons living with chronic diseases, and how these interactions could determine or modulate disease progression and outcomes.

In this review, we describe recent insights into these topics, as well as remaining questions whose answers will allow us to understand how interactions between the virus, the immune system and microbial components could possibly be related to disease states in patients with COVID-19, as well as existing studies of the microbiome in patients with COVID-19.

SOURCE

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