CRISPR screen identifies clinically approved immunosuppressants that could treat coronavirus infections

Researchers in Switzerland and Germany have identified host cell factors required for coronavirus replication that could serve as targets for treatment with clinically-approved drugs.

The team found that several autophagy-related genes were common host defense factors required for the replication of both endemic and emerging coronaviruses.

These coronaviruses include the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic.

Autophagy – cellular response to stressors such as hypoxia or infection – involves the recycling of proteins and organelles to maintain homeostasis. Various trafficking pathways enable the transportation of cytoplasmic material to the lysosome, where it is destroyed.

Among the autophagy-related genes were three immunophilins – high affinity-receptor proteins that specifically bind to certain immunosuppressive agents.

Furthermore, inhibition of the immunophilins with the clinically-approved drugs Cyclosporin A and Alisporivir resulted in dose-dependent reduction of coronavirus replication in primary human nasal epithelial cells.

The study was conducted by a team from the Institute of Virology and Immunology in Bern and Mittelhäusern, Switzerland and Ruhr-Universität Bochum in Germany

“Overall, we identified host factors that are crucial for coronavirus replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs,” writes Volker Thiel and the team.

A pre-print version of the paper is available on the bioRxiv* server, while the article undergoes peer review.

Study: A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets. Image Credit: Meletios Verras / Shutterstock

Three highly pathogenic coronaviruses have emerged over the last two decades

The last two decades have seen the emergence of three highly pathogenic coronaviruses, including the SARS-CoV virus responsible for the 2002-2004 SARS outbreaks, the Middle Eastern respiratory syndrome coronavirus (MERS-CoV) that emerged in 2012 and, most recently, the SARS-CoV-2 virus that causes COVID-19.

The severe risk these outbreaks have posed to human health over a relatively short period has highlighted the importance of developing effective approaches to treating both current coronavirus infections and those that could emerge in the future.

Coronaviruses rely on host dependency factors

Coronaviruses rely on cellular host factors – termed host dependency factors (HDFs) – for viral entry, replication and survival.

“The identification of HDFs is therefore important for understanding essential host-virus interactions required for successful viral replication and providing a framework to guide the development of new pharmacological strategies for the treatment of coronavirus infections,” says Thiel and colleagues.

One hallmark process that occurs during coronavirus replication is extensive virus-induced remodeling of host endomembranes to form double-membrane vesicles (DMVs) that are targeted by viral replication and transcription complexes.

“However, the host factors that are required for the formation of these structures remain elusive,” says the team.

What did the researchers do?

The researchers conducted two independent genome-wide loss-of-function CRISPR screens to identify HDFs required for the replication of both endemic and emerging coronaviruses.

The knockout screens were performed in Huh7 cells infected with the highly pathogenic MERS-CoV and with human coronavirus 229E (HCoV-229E) – a less pathogenic endemic coronavirus that generally only causes mild respiratory symptoms.

Enrichment analysis uncovers host biological networks crucial for CoV replication. (A) Enrichment map summarizing major host biological networks co-opted by CoVs during infection. Gene Ontology (GO) enrichment analysis was performed using hits from both MERS-CoV and HCoV-229E CRISPR screens and filtered to contain conserved representative GO terms and genes. Each node represents an individual GO term and nodes that are functionally related cluster together into a larger network. Node size reflects number of significantly enriched genes in the node and color indicates the CoV screen for which the node was significant.

What did the study find?

The team identified multiple virus-specific and conserved HDFs, including several that are required for replication of SARS-CoV-2.

The study revealed that several autophagy-related genes, including the immunophilins FK506 binding protein 8 (FKBP8), transmembrane protein 41B (TMEM41B), and membrane integral NOTCH2-associated receptor 1 (MINAR1) were common HDFs.

The researchers say that the interaction between autophagy components and coronaviruses in the context of replication has been considered for some time because parts of the autophagy process share similarities with the process of DMV formation.

However, “studies investigating the possible involvement of the early autophagy machinery in the conversion of host membranes into DMVs reached conflicting conclusions,” says Thiel and colleagues.

“Another possibility is that single components of the autophagic machinery may be hijacked by coronaviruses independently of their activity in autophagic processing,” they add.

The team says that irrespective of the precise underlying mechanism, the results suggest that FKBP8, TMEM41B, and MINAR1 represent potential therapeutic targets.

CoV HDFs are interactors of the autophagy pathway but do not depend on autophagy for replication. (A) Upon starvation, the mTORC1 complex is blocked and activation of the PI3K complex, as well as the ULK1 complex leads to the initiation of phagophore formation, as an initial step in the autophagy pathway. MERS-CoV and HCoV503 229E top scoring CRISPR knockout screen hits FKBP8, MINAR1, TMEM41B and VMP1 are involved in this early pathway. Furthermore, the ATG8 system containing among others LC3, which is recruited by VPM1 or FBKP8 is necessary for targeting cellular cargo to the autophagosome. PPP3R1 is upregulated and initiates TFEB translocalization to the nucleus, where it catalyzes transcription of ATGs. MERS-CoV or conserved host dependency factors (HDFs) are indicated in respective colors. Inhibitor intervention in this pathway is shown in red.

Targeting the immunophilins with clinically-approved drugs

Next, the researchers showed that inhibition of the immunophilin family with the clinically-approved and well-tolerated drugs Tacrolimus, Cyclosporin A and Alisporivir reduced the replication of MERS-CoV, SARS-CoV, and SARS-CoV-2 in a dose-dependent manner.  

However, the team noted that while Huh7 cells are valuable for studying coronaviruses, they are likely less effective at capturing important aspects of infection than primary human airway epithelial cells.

To address this limitation, the researchers also tested the drugs in primary human nasal epithelial cell cultures.

This revealed that Cyclosporin A and Alisporivir potently inhibited SARS-CoV-2 replication at concentrations known to be achievable and efficacious in patients.

“Overall, the genes and pathways identified in our coronavirus screens expand the current repertoire of essential HDFs required for replication that can be exploited to identify novel therapeutic targets for host-directed therapies against both existing and future emerging CoVs,” writes Thiel and colleagues.

“Together these findings depict a promising path towards the repurposing of Cyclosporin A and Alisporivir as COVID-19 treatment options,” concludes the team.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Thiel V, et al. A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets. bioRxiv, 2021. doi: https://doi.org/10.1101/2021.02.24.432634, https://www.biorxiv.org/content/10.1101/2021.02.24.432634v1

Posted in: Device / Technology News | Medical Research News | Disease/Infection News

Tags: Autophagy, Cell, Coronavirus, Coronavirus Disease COVID-19, CRISPR, Drugs, Gene, Genes, Genome, Hypoxia, Immunology, Knockout, MERS-CoV, Pandemic, Protein, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Syndrome, Tacrolimus, Transcription, Virology, Virus

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Written by

Sally Robertson

Sally first developed an interest in medical communications when she took on the role of Journal Development Editor for BioMed Central (BMC), after having graduated with a degree in biomedical science from Greenwich University.

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Lab study of South African SARS-CoV-2 variant and Moderna vaccine: reduced neutralization, but still protective

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic rages on, several virus variants have been emerging with mutations in the structural and non-structural proteins. The SARS-CoV-2 spike protein binds to the host angiotensin-converting enzyme 2 (ACE2) receptor, facilitating viral entry into the host cell. Studies have shown many different mutations in the spike protein over the last twelve months.

The first significant spike protein variant emerged with a mutation from aspartic acid (D) to glycine (G) at position 614, leading to increased viral fitness, replication, and binding to ACE2 and conformational changes within the protein. Several other variants have emerged over the past few months, raising concerns about changes to transmission, nature of the disease, and viral fitness.

When SARS-CoV-2 infects humans, our immune system rapidly responds against the viral spike protein. The receptor-binding motif in the spike protein interacts with the ACE2 receptor and is a key target of neutralization for antibodies. Longitudinal studies have found that the antibodies to the spike protein can remain in the body for at least a year following infection.

The mRNA-1273 vaccine encodes the SARS-CoV-2 spike protein and triggers a potent neutralizing antibody response to the virus that lasts for several months. The B.1.351 variant originated in South Africa has three mutations in the receptor-binding domain and many other mutations in the spike protein, all of which may influence viral binding to the ACE2 receptor and viral resistance to neutralization by antibodies.

Comparing antibody binding and viral neutralization against two different SARS-CoV-2 variants

Researchers from the US recently compared antibody binding and viral neutralization against 2 SARS-CoV-2 variants that emerged in different parts of the world. The researchers used sera from spike mRNA vaccinated and naturally infected individuals against a circulating B.1 variant and the emerging B.1.351 variant. The study is published on the preprint server bioRxiv*.

Study: Reduced binding and neutralization of infection- and vaccine-induced antibodies to the B.1.351 (South African) SARS-CoV-2 variant. Image Credit: NIAID

EHC-083E (the B.1 variant) belongs to the B.1 PANGO lineage and was isolated in March 2020 from a nasopharyngeal swab of a patient in Atlanta, GA. This variant has the D614G mutation in the viral spike protein. The B.1.351 variant was isolated in November 2020 from an oropharyngeal swab of a patient in KwaZulu-Natal, South Africa. This variant of the virus contains amino acid mutations (L18F, D80A, D215G) within the viral spike protein and deletion at positions 242-244 (L242del, A243del, and L244del), K417N, E484K, N501Y, and D614G.

Neutralizing antibodies for B.1.351 variant are produced early in the infection phase

The researchers observed decreased antibody binding to the B.1.351-derived receptor binding domain of the SARS-CoV-2 spike protein and neutralization power against the B.1.351 variant in sera from both infected and vaccinated individuals. Their longitudinal convalescent COVID-19 cohort assessed the impact on antibody binding to the receptor-binding domain and neutralization across the SARS-CoV-2 variants. Interestingly, most convalescent COVID-19 individuals showed less impact on neutralization against the B.1.351 variant at longer durations post-infection. This showed that neutralizing antibodies for the B.1.351 variant is produced early during infection and last for several months.

Most SARS-CoV-2-infected individuals showed binding and neutralizing titers against the B.1.351 variant in both acute and convalescent sera

According to the observations, most sera samples from acute and convalescent COVID-19 individuals showed antibody binding to the B.1.351-dervied receptor binding domain.  Most samples also showed a neutralizing capacity for the B.1.351 variant, and the effector functions of these neutralizing antibodies might contribute to SARS-CoV-2 infection control.

To summarize, although decreased by a few folds, most SARS-CoV-2 infected individuals showed binding and neutralizing titers against the B.1.351 variant in acute as well as convalescent sera. Moreover, all mRNA-1273 vaccinated individuals still maintained viral neutralization. These findings agree with previous notions that natural infection- and vaccine-induced immunity can offer protection against COVID-19 in the context of the SARS-CoV-2 B.1.351 variant.

“Our results show that despite few fold decrease, most infected individuals showed binding and neutralizing titers against the B.1.351 variant in acute and convalescent sera, and further, all mRNA-1273 vaccinated individuals still maintained neutralization.”

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Reduced binding and neutralization of infection- and vaccine-induced antibodies to the B.1.351 (South African) SARS-CoV-2 variant, Venkata Viswanadh Edara, Carson Norwood, Katharine Floyd, Lilin Lai, Meredith E. Davis-Gardner, William H. Hudson, Grace Mantus, Lindsay E. Nyhoff, Max W. Adelman, Rebecca Fineman, Shivan Patel, Rebecca Byram, Dumingu Nipuni Gomes, Garett Michael, Hayatu Abdullahi, Nour Beydoun, Bernadine Panganiban, Nina McNair, Kieffer Hellmeister, Jamila Pitts, Joy Winters, Jennifer Kleinhenz, Jacob Usher, James B. O’Keefe, Anne Piantadosi, Jesse J. Waggoner, Ahmed Babiker, David S. Stephens, Evan J. Anderson, Srilatha Edupuganti, Nadine Rouphael, Rafi Ahmed, Jens Wrammert, Mehul S. Suthar, bioRxiv, 2021.02.20.432046; doi: https://doi.org/10.1101/2021.02.20.432046, https://www.biorxiv.org/content/10.1101/2021.02.20.432046v1

Posted in: Medical Research News | Disease/Infection News

Tags: ACE2, Amino Acid, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Aspartic Acid, Cell, Coronavirus, Coronavirus Disease COVID-19, Enzyme, Glycine, Immune System, Infection Control, Mutation, Pandemic, Protein, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Vaccine, Virus

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Written by

Susha Cheriyedath

Susha has a Bachelor of Science (B.Sc.) degree in Chemistry and Master of Science (M.Sc) degree in Biochemistry from the University of Calicut, India. She always had a keen interest in medical and health science. As part of her masters degree, she specialized in Biochemistry, with an emphasis on Microbiology, Physiology, Biotechnology, and Nutrition. In her spare time, she loves to cook up a storm in the kitchen with her super-messy baking experiments.

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COVID-19 vaccine candidate shows potential against SARS-CoV-2 and potential future zoonotic coronaviruses

Over the last two decades, three major outbreaks of highly pathogenic coronaviruses have occurred. The third is the ongoing coronavirus disease 2019 (COVID-19) pandemic that has claimed well over 2.46 million human lives so far, in a little over a year from its onset. Without any targeted, safe and effective antivirals to prevent or treat the infection by the causative pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), population immunity via mass vaccination seems to be the only way out – as complex and expensive as the process is likely to be.

Study: SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys. Image Credit: Numstocker / Shutterstock

Pan-group 2b CoV vaccine

A new study, released on the bioRxiv* preprint server, sheds light on the threat posed by future zoonotic coronaviruses to make similar leaps across species barriers to infect human beings and cause other pandemics. The goal would appear to be a vaccine capable of inducing not limited immunity against SARS-CoV-2 alone, but one that can elicit broadly neutralizing antibody and cellular immune responses against a range of other betaCoVs.

This includes existing SARS-related coronaviruses (SARSr-CoVs) in humans, as well as those that are now circulating in animals.

The first evidence that this could be so came from the observation that SARS-CoV caused the production of cross-neutralizing antibodies against many betacoronaviruses (betaCoVs). This proof-of-concept drove the search for a vaccine that would induce neutralizing antibodies against multiple group 2b Sarbecoviruses.

Cross-neutralizing antibodies

Cross-neutralizing antibodies always target the viral receptor-binding domain (RBD) via a specific epitope. The RBD can be rendered more immunogenic by using a multimeric form. One way to achieve this is by using nanoparticles to mount arrays of RBD proteins, creating a virus-like particle (VLP).

Vaccines have been shown to successfully induce cross-neutralizing antibodies against pseudoviruses expressing CoV antigens in mouse studies. The current study describes a non-human primate (NHP) study that explores the cross-neutralizing ability of a SARS-CoV-2 vaccine based on multimeric SARS-CoV-2 RBD-bearing nanoparticles.

RBD-conjugated nanoparticle vaccine

The RBD-conjugated nanoparticle vaccine comprises 24 RBD protomers on a sortase-ferritin platform for the sake of versatility. This bound not only to the human host cell receptor, the angiotensin-converting enzyme 2 (ACE2), which is thought to be the SARS-CoV-2 entry receptor, but also to potent anti-RBD neutralizing antibodies. These include DH1041, DH1042, DH1043, DH1044, and DH1045.

All these antibodies bind to epitopes within the receptor-binding motif, within the RBD. However, antibodies that bound to epitopes outside the RBD were not able to bind the RBD-bearing nanoparticle. In contrast, it did show binding to the cross-neutralizing antibody DH1047.

This vaccine was assessed by a three-dose regimen, administered at four-week intervals, in a non-human primate (NHP) study. The vaccine was found to result in high plasma levels of antibodies to the SARS-CoV-2 RBD and to the stabilized spike protein.

The antibodies completely blocked the ACE2 binding site on the spike protein after two doses of vaccine and partially blocked the binding of the RBD antibody DH104.

SARS-CoV-2 receptor binding domain (RBD) sortase conjugated nanoparticles (scNPs) elicits extremely high titers of SARS-CoV-2 pseudovirus neutralizing antibodies. a. SARS-CoV-2 RBD nanoparticles were constructed by expressing RBD with a C-terminal sortase A donor sequence (blue and red) and a Helicobacter pylori ferritin nanoparticle with N737 terminal sortase A acceptor sequences (gray) on each subunit (top left). The RBD is shown in blue with the ACE2 binding site in red. The RBD was conjugated to nanoparticles by a sortase A (SrtA) enzyme conjugation reaction (top right). The resultant nanoparticle is modeled on the bottom left. Nine amino acid sortase linker is shown in orange. Two dimensional class averages of negative stain electron microscopy images of actual RBD nanoparticles are shown on the bottom right. b. Antigenicity of RBD nanoparticles determined by biolayer interferometry against a panel of SARS-CoV-2 antibodies and the ACE2 receptor. Antibodies are color-coded based on epitope and function. N-terminal domain (NTD), nonAbs IE, infection enhancing non-neutralizing antibody; nAb, neutralizing antibody; nonAb, non-neutralizing antibody. Mean and standard error from 3 independent experiments are shown. c. Cynomolgus macaque challenge study scheme. Blue arrows indicate 748 RBD-NP immunization timepoints. Intranasal/intratracheal SARS-CoV-2 challenge is indicated at week 10. d. Macaque serum IgG binding determined by ELISA to recombinant SARS-CoV-2 stabilized Spike ectodomain (S-2P), RBD, NTD, and Fusion peptide (FP). Binding titer is shown as area752under-the curve of the log10-transformed curve. Arrows indicate immunization timepoints. e. Plasma antibody blocking of SARS-CoV-2 S-2P binding to ACE2-Fc and RBD neutralizing antibody DH1041. Group mean and standard error are shown. f. Dose-dependent serum neutralization of SARS-COV-2 pseudotyped virus infection of ACE2- expressing 293T cells. Serum was collected after two immunizations. The SARS-CoV-2 pseudovirus spike has an aspartic acid to glycine change at position 614 (D614G). Each curve represents a single macaque. g. Heat map of serum neutralization ID50 and ID80 titers for SARS-COV-2 D614G pseudotyped virus after two immunizations. h. SARS-COV-2 D614G pseudotyped virus serum neutralization kinetics. Each curve represents a single macaque. i. Comparison of serum neutralization ID50 titers from cynomolgus macaques immunized with recombinant protein RBD nanoparticles (blue) or nucleoside-modified mRNA-LNP expressing S- 2P (burgundy) (**P<0.01, Two-tailed Exact Wilcoxon test n = 5). j. Comparison of serum neutralization titers obtained from RBD-scNP-vaccinated macaques (blue) and SARS-CoV-2 infected humans (shades of green). Human samples were stratified based on disease severity as asymptomatic (N=34), symptomatic (n=71), and hospitalized (N=60) (**P<0.01, Two-tailed Wilcoxon test n = 5).

Competitive with the Moderna/Pfizer vaccine for neutralizing antibody titer

When tested against the currently dominant D614G strain of SARS-CoV-2, the RBD-conjugated nanoparticle vaccine induced higher neutralizing antibody titers than another vaccine similar to the Moderna and Pfizer lipid-encapsulated nucleoside-modified mRNA (mRNA-LNP) vaccines that are now being used in the vaccination campaigns against COVID-19.

The measure of antibody titer used here showed an eight-fold increase with the former compared to the latter. The antibody response was also higher with the RBD-nanoparticle vaccine than with natural infection of all grades of severity.

Unaffected by emerging variants

It also showed potent neutralizing activity against the new SARS-CoV-2 variant B.1.1.7, which is rapidly spreading worldwide. This is not only more infective but may be resistant to many RBD-targeting antibodies, as well as more virulent.

While changes in binding affinity of anti-RBD antibody DH1041 to the ACE2 receptor and to the spike protein were observed with different mutations, such as those acquired during mink infection, or those found in the South African or Brazil or UK strains, the cross-neutralizing antibody DH1047 showed unchanged binding to the SARS-CoV-2.

“RBD-scNP (RBD sortase A conjugated nanoparticle) and mRNA-LNP-induced RBD binding antibodies were not sensitive to spike mutations present in neutralization-resistant UK, South Africa or Brazil SARS-CoV-2 variants.”

SARS-CoV-2 spike induces cross-neutralizing antibodies to pre-emergent betaCoVs

SARSr-CoVs still pose a danger of future pandemics to human beings. The researchers, therefore, explored the ability of this vaccine to neutralize other viruses. Similar to the LNP-mRNA vaccines based on the prefusion stabilized spike or the RBD, the RBD-scNP also elicited potent cross-neutralizing antibodies against SARS-CoV and SARSr-bat CoVs (batCoV-WIV-1, and batCoV-SHC014).

The neutralization was most potent against SARS-CoV-2, however. The highest neutralizing antibody titers were observed with RBD-scNP and the least with the RBD-expressing LNP-mRNA vaccine. The high titers may indicate that durable immunity is achieved.  

The RBD-scNP vaccine showed cross-neutralizing activity against batCoV-RaTG13 and pangolin CoV GXP4L spike antigens, in addition to SARS-CoV and SARS-CoV-2. Notably, sera obtained following vaccination with this formulation failed to neutralize the seasonal human CoVs or MERS-CoV, probably because of the difference in RBD among these CoVs, which belong to different groups.

The similarity between the RBD-scNP and DH1047 in terms of cross-neutralizing profile shows that not only do antibodies induced by the former bind near the epitope bound by the latter, but they are not specific to SARS-CoV-2 RBD. In fact, they also block batCoV-SHC01.

Notably, only a third of COVID-19 patients produce antibodies that block DH1047, indicating it is a sub-immunodominant epitope. As such, the RBD-scNP vaccine targets this epitope rather than the immunodominant ACE2 blocking epitope.

Protection against productive infection

The RBD-scNP vaccine was also protective for vaccinated monkeys when challenged with the SARS-CoV-2 virus via the respiratory tract. In all but one of the vaccinated macaques, “RBD-scNP-induced immunity prevented virus replication, and likely provided sterilizing immunity, in the upper and lower respiratory tract.”

What are the implications?

The RBD-scNP platform induced the highest cross-neutralizing antibody titer for group 2b CoVs, and as such, may serve as the basis for a reasonably effective initial broadly neutralizing vaccine against this group – both now, and in the future, if the further zoonotic transmission should occur.

The study also showed that the use of both RBD-scNP and the LNP-spike mRNA vaccines, the latter resembling those which have been recently rolled out, is capable of inducing cross-neutralizing antibodies to the dominant D614G variant and the newer variants of SARS-CoV-2, but at lower titers.

The findings indicate the ability of the SARS-CoV-2 Spike to be included in an RBD-scNP or LNP-mRNA formulation to induce cross-neutralizing antibodies against several SARSr-CoVs. Thus, even the currently used vaccines are likely to prevent future pandemics if immunization is successfully achieved.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Saunders, K. O. et al. (2021). SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys. bioRxiv preprint. doi: https://doi.org/10.1101/2021.02.17.431492. https://www.biorxiv.org/content/10.1101/2021.02.17.431492v1

Posted in: Medical Science News | Medical Research News | Disease/Infection News | Healthcare News

Tags: ACE2, Amino Acid, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Aspartic Acid, binding affinity, Cell, Conjugation, Coronavirus, Coronavirus Disease COVID-19, Electron, Electron Microscopy, Enzyme, Glycine, heat, Helicobacter pylori, Immunization, MERS-CoV, Microscopy, Nanoparticle, Nanoparticles, Nucleoside, Pandemic, Pathogen, Protein, Pseudovirus, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Vaccine, Virus

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Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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Elevated Activin A and FLRG levels correlate with worst outcomes in severe COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes a "cytokine storm" in hospitalized and ICU patients. Studies have shown that the blockade of cytokine signaling and steroid treatment helps achieve positive outcomes in patients. However, a better understanding of the downstream signaling pathways contributing to the massive immune response is crucial to treat patients suffering from severe COVID-19 symptoms.

Study: Activin A correlates with the worst outcomes in COVID-19 patients, and can be induced by cytokines via the IKK/NF-kappa B pathway. Image Credit: Optimarc / Shutterstock

Analyzing the response of COVID-19 relevant cell types to inflammatory cytokines released during the cytokine storm

Recently researchers from the US studied serum from COVID-19 patients to see if they had elevated Activin A levels. They also evaluated PAI-1, another marker found to be associated with ARDS, and one of the parameters said to be associated with ARDS-related mortality. They further determined if the levels of Activin A, follistatin-like related gene (FLRG) and PAI correlated to key COVID-19 disease markers such as the supplemental oxygen requirement, ARDS symptoms, and mortality. The study is published on the preprint server bioRxiv*.

The researchers had previously analyzed IL-1 and TNFα in the setting of skeletal muscle cachexia, where the cytokines induce skeletal muscle atrophy. They learned from their previous studies that IL-1 and TNFα could induce Activin A production in skeletal muscles, which induces skeletal muscle atrophy. They felt that this was relevant in the context of COVID-19, and it had been previously reported. Earlier, it has been reported that high levels of Activin A were found in the bronchial alveolar lavage fluid of patients who had acute respiratory disease syndrome (ARDS).

"The impression, therefore, is that these severely affected patients are not suffering directly from the viral load, but instead from an over-reaction of the immune system – the later occurring cytokine response, which in some patients over-induces Activin A."

Elevated levels of Activin A and FLRG at baseline were predictors of the severe COVID-19 outcomes

In the present study, the researchers wanted to see if other COVID-19 relevant cell types such as bronchial and pulmonary smooth muscles responded in a similar manner to inflammatory cytokines released during the cytokine storm to produce Activin A. They had performed a trial with COVID-19 patients using a Regeneron anti-IL-6R antibody in which they evaluated the serum of these patients post-randomization and before therapy to measure baseline Activin A, PAI-1, and FLRG levels. They then correlated these values to baseline clinical and lab variables and key disease outcomes.

The study results demonstrated that cytokines responsible for activating the NF-kappaβ pathway could also induce Activin A and its downstream marker, FLRG. In hospitalized COVID-19 patients, high levels of Activin A / FLRG at baseline were predictors of the severe COVID-19 outcomes, such as the need for ventilation and all-cause mortality. Patients with higher than median levels of Activin A / FLRG were nearly three times more likely to die compared to patients who had Activin A / FLRG levels below the sample median.

Activin A, FLRG, and PAI-1 levels vs disease severity in COVID-19 patients and in non-Covid 19 controls. A. Activin A (pg/mL) levels plotted for control, severe COVID-19, and critical COVID-19 subjects. Significant differences between control and critical COVID-19, and severe and critical COVID-19 were found. B. FLRG (pg/mL) levels plotted as in A. All groups were significantly different from each other, with FLRG levels increasing with disease severity. C. PAI-1 (ng/mL) levels plotted as in A. Significant differences were found between control and severe COVID-19, and control and critical COVID-19. Number of subjects tested in each group (n) is indicated under respective plots. **** p < 0.0001.

Inhibiting Activin A can be beneficial in the treatment of COVID-19 patients experiencing ARDS

The findings demonstrate that both Activin A and FLRG were significantly upregulated in COVID-19 patients in the ICU. Some patients experienced an over 2-fold increase in Activin A. Interestingly, Activin A levels were not significantly increased in severe COVID-19 patients who did not require invasive mechanical ventilation showing that the marker can clearly distinguish the patient populations.

While FLRG levels increased in patients with severe disease who were not in the ICU, it was even more elevated in ICU patients, especially those who had ARDS and needed invasive mechanical ventilation. Additionally, subjects with extremely high increases of FLRG were also found to be more likely to need invasive ventilation. Thus, high levels of Activin A and FLRG increases the risk of ARDS in COVID-19 patients, and the blockade of Activin A can be beneficial in the treatment of COVID-19 patients experiencing ARDS.

"We, therefore, suggest it's reasonable to try inhibiting Activin A or its induced pathway to treat COVID-19 patients who are experiencing ARDS."

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Activin A correlates with the worst outcomes in COVID-19 patients, and can be induced by cytokines via the IKK/NF-kappa B pathway Megan McAleavy, Qian Zhang, Jianing Xu, Li Pan, Matthew Wakai, Peter J. Ehmann, Matthew F. Wipperman, Tea Shavlakadze, Sara C. Hamon, Anita Boyapati, Lori G. Morton, Christos A. Kyratsous, David J. Glass, bioRxiv, 2021.02.04.429815; doi: https://doi.org/10.1101/2021.02.04.429815, https://www.biorxiv.org/content/10.1101/2021.02.04.429815v1

Posted in: Medical Research News | Disease/Infection News

Tags: Antibody, Cachexia, Cell, Coronavirus, Coronavirus Disease COVID-19, Cytokine, Cytokines, Gene, Immune Response, Immune System, Mortality, Muscle, Muscle Atrophy, Oxygen, Respiratory, Respiratory Disease, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Steroid, Syndrome

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Written by

Susha Cheriyedath

Susha has a Bachelor of Science (B.Sc.) degree in Chemistry and Master of Science (M.Sc) degree in Biochemistry from the University of Calicut, India. She always had a keen interest in medical and health science. As part of her masters degree, she specialized in Biochemistry, with an emphasis on Microbiology, Physiology, Biotechnology, and Nutrition. In her spare time, she loves to cook up a storm in the kitchen with her super-messy baking experiments.

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Substrain of SARS-CoV-2 variant in UK may resist antibody neutralization

Researchers at the Polish Academy of Sciences in Warsaw have identified a substrain of the recently emerged B.1.1.7 variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may confer resistance to antibody neutralization.

The SARS-CoV-2 virus is the agent responsible for the coronavirus disease 2019 (COVID-19) pandemic that has now claimed the lives of more than 2.35 million people.

The substrain of the B.1.1.7 variant of concern (VOC) contains mutations that have previously been shown to compromise the binding of neutralizing antibodies.

Tomasz Lipniacki and colleagues say mutations in the receptor-binding domain (RBD) of the viral spike protein are of particular concern, especially those identified in the receptor-binding motif (RBM).

The spike protein is the surface structure the virus uses to bind to and infect cells by attaching to the host cell receptor angiotensin-converting enzyme 2 (ACE2).

The researchers say the mutations could eventually lead to “immune escape” strains that can reinfect convalescent individuals and reduce the efficacy of the vaccines currently being used in mass immunization efforts.

“Such mutants may hinder the efficiency of existing vaccines and expand in response to the increasing after‐infection or vaccine‐induced seroprevalence,” writes the team.

A pre-print version of the research paper is available on the medRxiv* server, while the article undergoes peer review.

Study: L18F substrain of SARS-CoV-2 VOC-202012/01 is rapidly spreading in England. Image Credit: NIAID

The B.1.1.7 variant has spread rapidly since mid-October 2020

The B.1.1.7 variant has rapidly spread since mid-October 2020, and by January 2021, it constituted about 80% of all SARS-CoV-2 genomes sequenced in England.

The high transmissibility of this VOC may be expressed in terms of its replicative advantage – defined as the ratio of the VOC reproduction number to that of non-VOC strains.

To date, a number of studies have estimated the replicative advantage as lying somewhere between 1.47 and 2.24.

As is the case with all viral strains, the B.1.1.7 variant will continue to mutate, and given its significant replicative advantage, any mutations acquired are likely to spread globally.

“As this strain will likely spread globally towards fixation, it is important to monitor its molecular evolution,” say the researchers.

What did the current study involve?

Using the Global Initiative on Sharing Avian Influenza Data (GISAID) database, Lipniacki and colleagues estimated growth rates of the mutations that B.1.1.7 has acquired.

This revealed a substrain with an L18F substitution in the spike protein that is rapidly growing in the UK.

This leucine‐to‐phenylalanine substitution in residue 18 was first reported to have occurred in a VOC strain genome collected on December 4th, 2020.

As of February 5th, 2021, as many as 850 spikes L18F VOC genomes had been reported in England.

Based on data collected between December 7th, 2020 and January 17th, 2021, the researchers showed that the L18F substrain had spread exponentially in England. They estimated a replicative advantage of 1.70 relative to the remaining B.1.1.7 substrains.

RBM mutations are particularly concerning

Lipniacki and colleagues say that mutations in the RBD of the spike protein are particularly concerning, especially substitutions E484K and S494P found in the RBM.

Importantly, the LI8F mutation has expanded in the South African variant 501Y.V2 that contains the spike mutations E484K and N501Y. Studies have suggested that E484K may compromise the binding of class 2 neutralizing antibodies, while the A501V mutation compromises the binding of class 1 antibodies.

Furthermore, in a 2021 study published in Science, the S494P substitution was characterized as an escape mutation, along with six other escape residues in the RBM that included F490.

In the current study, Lipniacki and colleagues also identified F490S as a potential escape mutation.

What do the authors advise?

“These mutations may potentially lead to immune escape mutants, resulting in reinfection of convalescent individuals and lowering efficacy of current vaccines,” warn the researchers.

“Propagation of such mutations is facilitated by high replicative advantage of the VOC strain and potential selection due to the increasing number of convalescent or immunized individuals,” they add.

Correspondingly, a study published in 2021 showed that L18F substitution compromises the binding of neutralizing antibodies, suggesting that the replicative advantage of L18F mutants may be partly associated with the ability to infect seroprevalent individuals (who already have anti-SARS-CoV-2 antibodies).

“In turn, propagation of mutations in escape residues (L18, E484, F490S, or S494) on the VOC strain suggests an increasing selection pressure resulting from the growth of the seroprevalent fraction of the population of England,” says Lipniacki and colleagues.

“This trend can be enhanced by the ongoing English vaccination program, in which the relatively large time span between the first and second dose can be a contributing factor,” concludes the team.

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Lipniacki T, et al. L18F substrain of SARS-CoV-2 VOC-202012/01 is rapidly spreading in England. medRxiv, 2021. doi: https://doi.org/10.1101/2021.02.07.21251262, https://www.medrxiv.org/content/10.1101/2021.02.07.21251262v1

Posted in: Medical Research News | Disease/Infection News

Tags: ACE2, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Avian Influenza, Cell, Coronavirus, Coronavirus Disease COVID-19, Efficacy, Enzyme, Evolution, Genome, Immunization, Influenza, Leucine, Mutation, Pandemic, Phenylalanine, Propagation, Protein, Receptor, Reproduction, Research, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Vaccine, Virus

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Sally Robertson

Sally has a Bachelor's Degree in Biomedical Sciences (B.Sc.). She is a specialist in reviewing and summarising the latest findings across all areas of medicine covered in major, high-impact, world-leading international medical journals, international press conferences and bulletins from governmental agencies and regulatory bodies. At News-Medical, Sally generates daily news features, life science articles and interview coverage.

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