Tag Archives: Molecule

Microbiology

UNIGE researchers identify how the influenza A virus manages to penetrate host cells

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Reviewed by Danielle Ellis, B.Sc.May 31 2023

Influenza epidemics, caused by influenza A or B viruses, result in acute respiratory infection. They kill half a million people worldwide every year. These viruses can also wreak havoc on animals, as in the case of avian flu. A team from the University of Geneva (UNIGE) has identified how the influenza A virus manages to penetrate cells to infect them. By attaching itself to a receptor on the cell surface, it hijacks the iron transport mechanism to start its infection cycle. By blocking the receptor involved, the researchers were also able to significantly reduce its ability to invade cells. These results, published in the journal PNAS, highlight a vulnerability that could be exploited to combat the virus.

Influenza viruses represent a major risk to human and animal health. Their potential for mutation makes them particularly elusive.

‘We already knew that the influenza A virus binds to sugar structures on the cell surface, then rolls along the cell surface until it finds a suitable entry point into the host cell. However, we did not know which proteins on the host cell surface marked this entry point, and how they favored the entry of the virus.”

Mirco Schmolke, Associate Professor, Department of Microbiology and Molecular Medicine and in the Geneva Centre for Inflammation Research (GCIR) at the UNIGE Faculty of Medicine

A receptor as a key to infection

The scientists first identified cell surface proteins present in the vicinity of the viral haemagglutinin, the protein used by the influenza A virus to enter the cell. One of these proteins stood out: transferrin receptor 1. This acts as a revolving door transporting iron molecules into the cell, which are essential for many physiological functions.

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”The influenza virus takes advantage of the continuous recycling of the transferrin receptor 1 to enter the cell and infect it,” explains Béryl Mazel-Sanchez, a former post-doctoral researcher in Mirco Schmolke’s laboratory and first author of this work. ”To confirm our discovery, we genetically engineered human lung cells to remove the transferrin receptor 1, or on the contrary to overexpress it. By deleting it in cells normally susceptible to infection, we prevented influenza A from entering. Conversely, by overexpressing it in cells normally resistant to infection, we made them easier to infect”.

Inhibiting this mechanism

The research team then succeeded in reproducing this mechanism by inhibiting the transferrin receptor 1 using a chemical molecule. ”We tested it successfully on human lung cells, on human lung tissue samples and on mice with several viral strains,” says Béryl Mazel-Sanchez. ”In the presence of this inhibitor, the virus replicated much less. However, in view of its potentially oncogenic characteristics, this product cannot be used to treat humans.” On the other hand, anti-cancer therapies based on the inhibition of the transferrin receptor are under development and could also be interesting in this context.

”Our discovery was made possible thanks to the excellent collaboration within the Faculty of Medicine as well as with the University Hospitals of Geneva (HUG) and the Swiss Institute of Bioinformatics (SIB),” the authors add. In addition to the transferrin receptor 1, scientists have identified some 30 other proteins whose role in the influenza A entry process remains to be deciphered. It is indeed likely that the virus uses a combination involving other receptors. ”Although we are still far from a clinical application, blocking the transferrin receptor 1 could become a promising strategy for treating influenza virus infections in humans and potentially in animals.”

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Source:

University of Geneva

Journal reference:

Mazel-Sanchez, B., et al. (2023) Influenza A virus exploits transferrin receptor recycling to enter host cells. PNAS. doi.org/10.1073/pnas.2214936120.

Microbiology

Experimental decoy provides long-term protection from SARS-Cov-2 infection

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Reviewed by Danielle Ellis, B.Sc.May 30 2023

An experimental “decoy” provided long-term protection from infection by the pandemic virus in mice, a new study finds.

Led by researchers at NYU Grossman School of Medicine, the work is based on how the virus that causes COVID-19, SARS-CoV-2, uses its spike protein to attach to a protein on the surface of the cells that line human lungs. Once attached to this cell surface protein, called angiotensin converting enzyme 2 (ACE2), the virus spike pulls the cell close, enabling the virus to enter the cell and hijack its machinery to make viral copies.

Earlier in the pandemic, pharmaceutical companies designed monoclonal antibodies to glom onto the spike and neutralize the virus. Treatment of patients soon after infection was successful in preventing hospitalization and death. However the virus rapidly evolved through random genetic changes (mutations) that altered the spike’s shape enough to evade even combinations of therapeutic monoclonal antibodies. Thus, such antibodies, which neutralized early variants, became about 300 times less effective against more recent delta and omicron variants.

Published online this week in the Proceedings of the National Academy of Sciences, the study describes an alternative approach from which the virus cannot escape. It employs a version of ACE2, the surface protein to which the virus attaches, which, unlike the natural, cell-bound version, is untethered from the cell surface. The free-floating “decoy” binds to the virus by its spikes so that it can no longer attach to ACE2 on cells in airways. Unlike the monoclonal antibodies, which are shaped to interfere with a certain spike shape, the decoy mimics the spike’s main target, and the virus cannot easily evolve away from binding to ACE2 and still invade cells.

Treatment with the decoy, either by injection or droplets in the nose, protected 100 percent of the study mice when they were infected in the lab with an otherwise lethal dose of SARS-CoV-2. The decoy lowered the virus load in the mice by 100,000-fold, while mice exposed to a non-active control treatment died. Decoy treatment of mice that were already infected with SARS-CoV-2 caused a rapid drop in viral levels and return to health. This suggests that the decoy could be effective as a therapy post-infection, similar to monoclonal antibodies, the researchers say.

What is remarkable about our study is that we delivered the decoy using a harmless, adeno-associated virus or AAV vector, a type of gene therapy that has been found in previous studies to be safe for use in humans. The viral vector instructs cells in the body to produce the decoy so that the mouse or person is protected long-term, without the need for continual treatment.”

Nathanial Landau, PhD, senior study author, professor, Department of Microbiology at NYU Langone Health

Administered with the vector, says Landau, the treatment caused cells, not only to make the decoy, but to continue making it for several months, and potentially for years.

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Importantly, vaccines traditionally include harmless parts of a virus they are meant to protect against, which trigger a protective immune response should a person later be exposed. Vaccines are less effective, however, if a person’s immune system has been compromised, by diseases like cancer or in transplant patients treated with drugs that suppress the immune response to vaccination. Decoy approaches could be very valuable for immunocompromised patients globally, adds Landau.

Future pandemics

For the new study, the research team made key changes to a free ACE2 receptor molecule, and then fused the spike-binding part of it to the tail end of an antibody with the goal of strengthening its antiviral effect. Attaching ACE2 to the antibody fragment to form what the team calls an “ACE2 microbody” increases the time that the molecule persists in tissues (its half-life). The combination also causes the molecules to form dimers, mirror-image molecular pairs that increase the strength with which the decoy attaches to the viral spike.

Whether administered via injection into muscle, or through droplets in the nasal cavity, the study’s AAV vectors provided mice with long-lasting protection COVID infection, including the current Omicron variants.

The approach promises to be effective even if another coronavirus, a type of virus common in birds and bats or apes, were to be transferred to humans in the future, an event termed “zoonosis.” As long as the future virus also uses ACE2 to target cells, the decoy would be ready for “off-the-shelf” soon after an outbreak. If the virus were to somehow switch its receptor a different protein on the surface of lung cells, the decoy could be modified to target the new virus, says Landau.

Along with Landau, the study authors were Takuya Tada and Julia Minnee in the Department of Microbiology at NYU Grossman School of Medicine. The study was supported by a grant from the National Institutes of Health.

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Source:

NYU Langone Health / NYU Grossman School of Medicine

Journal reference:

Tada, T., et al. (2023) Vectored immunoprophylaxis and treatment of SARS-CoV-2 infection in a preclinical model. PNAS. doi.org/10.1073/pnas.2303509120.

Microbiology

Study highlights two strategies used by Salmonella to escape the human body’s defenses

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Reviewed by Danielle Ellis, B.Sc.May 26 2023

Like thieves that constantly look for ways to evade capture, Salmonella enterica, a disease-causing bacterium, uses various tactics to escape the human body’s defense mechanisms. In a new study, researchers from the Department of Microbiology and Cell Biology (MCB), IISc, highlight two such strategies that the bacterium uses to protect itself, both driven by the same protein.

When Salmonella enters the human body, each bacterial cell resides within a bubble-like structure known as Salmonella-containing vacuole (SCV). In response to the bacterial infection, the immune cells in our body produce reactive oxygen species (ROS) and reactive nitrogen species (RNS), along with pathways triggered to break down these SCVs and fuse them with cellular bodies called lysosomes or autophagosomes, which destroy the bacteria. However, these bacteria have developed robust mechanisms to maintain vacuolar integrity, which is crucial for their survival. For example, when a bacterial cell divides, the vacuole surrounding it also divides, enabling every new bacterial cell to be ensconced in a vacuole. This also ensures that more vacuoles are present than the number of lysosomes which can digest them.

In the study published in Microbes and Infection, the IISc team deduced that a critical protein produced by Salmonella, known as SopB, prevents both the fusion of SCV with lysosomes as well as the production of lysosomes, in a two-pronged approach to protect the bacterium. “[This] gives the upper hand to bacteria to survive inside macrophages or other host cells,” explains Ritika Chatterjee, former PhD student in MCB and first author of the study. The experiments were carried out on immune cell lines and immune cells extracted from mice models.

SopB acts as a phosphatase – it aids in removing phosphate groups from phosphoinositide, a type of membrane lipid. SopB helps Salmonella change the dynamics of the vacuole – specifically it alters the type of inositol phosphates in the vacuole membrane – which prevents the vacuole’s fusion with lysosomes.

A previous study from the same team had reported that the number of lysosomes produced by the host cells decreases upon infection with Salmonella. The researchers also found that mutant bacteria that were unable to produce SopB were also unable to reduce host lysosome numbers. Therefore, they decided to look more closely at the role that SopB was playing in the production of lysosomes, using advanced imaging techniques.

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What they found was that SopB prevents the translocation of a critical molecule called Transcription Factor EB (TFEB) from the cytoplasm of the host cell into the nucleus. This translocation is vital because TFEB acts as a master regulator of lysosome production.

This is the first time we deciphered that SopB can work in a dual manner – it changes the phosphoinositide dynamics of SCV and affects TFEB’s translocation into the nucleus. While other groups have already reported the function of SopB in mediating invasion in epithelial cells, the novelty of our study lies in identification of the function of SopB in inhibiting the vacuolar fusion with existing autophagosomes/lysosomes, and the second mechanism, which provides Salmonella with a survival advantage by increasing the ratio of SCV to lysosomes.”

Dipshikha Chakravortty, Professor at MCB and corresponding author of the study

The researchers suggest that using small molecule inhibitors against SopB or activators of TFEB can help counter Salmonella infection.

In subsequent studies, the team plans to explore the role of another host protein called Syntaxin-17 whose levels also reduce during Salmonella infection. “How do the SCVs reduce the levels of Syntaxin-17? Do they exchange it with some other molecules, or do the bacteria degrade it? We [plan to] look into it next,” says Chakravortty.

Source:

Indian Institute of Science (IISc)

Journal reference:

Chatterjee, R., et al. (2023) Deceiving The Big Eaters: Salmonella Typhimurium SopB subverts host cell Xenophagy in macrophages via dual mechanisms. Microbes and Infection. doi.org/10.1016/j.micinf.2023.105128.

Microbiology

Novel antibodies target human receptors to neutralize SARS-CoV-2 variants and future sarbecoviruses

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Neha MathurBy Neha MathurMay 17 2023Reviewed by Lily Ramsey, LLM

In a recent study published in the Nature Microbiology Journal, researchers generated six human monoclonal antibodies (mAbs) that prevented infection by all human angiotensin-converting enzyme 2 (ACE2) binding sarbecoviruses tested, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, Delta and Omicron.

They targeted the hACE2 epitope that binds to the SARS-CoV-2 spike (S) glycoprotein rather than targeting the S protein, which all previous therapeutic mAbs for SARS-CoV-2 targeted.

Study: Pan-sarbecovirus prophylaxis with human anti-ACE2 monoclonal antibodies. Image Credit: paulista/Shutterstock.comStudy: Pan-sarbecovirus prophylaxis with human anti-ACE2 monoclonal antibodies. Image Credit: paulista/Shutterstock.com

Background

The emergence of new variants of SARS-CoV-2, especially Omicron sublineages, made all therapeutic mAbs targeting SARS-CoV-2 S obsolete.

Any new S-targeting mAb therapy will also probably have limited utility because SARS-CoV-2 will continue to adapt to human antibodies. Ideally, mAbs developed in anticipation of future pandemics caused by sarbecoviruses should be resilient to mutations that arise in them.

About the study

In the present study, researchers developed hACE2-binding mAbs that blocked infection by pseudotypes of all tested sarbecoviruses at potencies matching SARS-CoV-2 S targeting therapeutic mAbs. The binding affinity of these mAbs to hACE2 was in the nanomolar to picomolar range.

To develop these mAbs, researchers used the KP and Av AlivaMab mouse strains that generate a human Kappa (κ) light chain and Kappa (κ) and Lambda (λ) light chains carrying antibodies, respectively.

They immunized these mice with monomeric and dimeric recombinant hACE2 extracellular domains. Fusion to the fraction, crystallizable (Fc) portion of human immunoglobulin G1 (IgG1) rendered them dimeric.

Further, the team generated hybridomas from mice using sera that inhibited SARS-CoV-2 pseudotyped viruses. They used enzyme-linked immunosorbent assay (ELISA) to screen hybridoma supernatants for hACE2-binding mAbs.

Furthermore, the researchers tested the ability of the six most potent mAbs to inhibit Wuhan-hu-1 S pseudotyped infection in Huh-7.5 target cells.

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The team purified chimeric mAbs from the hybridoma culture supernatants and used a SARS-CoV-2 pseudotype assay to reconfirm their antiviral activity. They also sequenced the human Fab variable regions, VH and VL.

The team cloned VH and VL domains from the six most potent chimeric human-mouse mAbs into a human IgG1 expression vector to generate fully human anti-hACE2 mAbs.

They used single-particle cryo-electron microscopy (cryo-EM) to delineate the structural basis for broad neutralization of anti-hACE2 mAbs.

Specifically, they determined the structure of soluble hACE2 bound to the antigen-binding fragment (Fab) of 05B04, one of the most potent mAbs unaffected by naturally occurring human ACE2 variations.

Finally, the researchers tested these hACE2 mAbs in an animal model and determined their pharmacokinetic behavior.

Results

The researchers identified 82 hybridomas expressing hACE2-binding mAbs, of which they selected ten based on their potency in inhibiting pseudotyped virus infection of Huh-7.5 cells.

These ten mAbs were 1C9H1, 4A12A4, 05B04, 2C12H3, 2F6A6, 2G7A1, 05D06, 05E10, 05G01 and 05H02. Four of the five mAbs from the KP AlivaMab mice, viz., 05B04, 05E10, 05G01, and 05D06, shared identical complementarity-determining regions (CDRs). Conversely, AV AlivaMab mice-derived mAbs were diverse.

While allosteric inhibition of hACE2 activity by the mAbs was theoretically feasible, such inhibition did not occur.

Also, the anti-hACE2 mAbs did not affect hACE2 internalization or recycling, suggesting that the anti-hACE2 mAbs would unlikely undergo accelerated target-dependent clearance from the circulation during in vivo use.

These two findings confirmed that these mAbs would not have harmful side effects based on their target specificity.

In addition, the anti-hACE2 mAbs showed favorable pharmacokinetics and no ill effects on the hACE2 knock-in mice. When used prophylactically in hACE2 knock-in mice, these mAbs conferred near-sterilizing protection against lung SARS-CoV-2 infection.

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Moreover, they presented a high genetic barrier to the acquisition of resistance by SARS-CoV-2.

The six anti-hACE2 mAbs also inhibited infection by pseudotyped SARS-CoV-2 variants, Delta, and Omicron, with similar potency, i.e., half maximal inhibitory concentration (IC50) values ranging between 8.2 ng ml−1 and 197 ng ml−1.

A cryo-EM structure of the 05B04-hACE2 complex at 3.3 Å resolution revealed a 05B04 Fab bound to the N-terminal helices of hACE2.

05B04-mediated inhibition of ACE2-binding sarbecoviruses through molecular mimicry of SARS-CoV-2 receptor-binding domain (RBD) interactions, providing high binding affinity to hACE2 despite the smaller binding footprint on hACE2.

None of the four most potent mAbs affected hACE2 enzymatic activity or induced the internalization of hACE2 localized on the host cell surface. Thus, based on their target specificity, these mAbs shall not have deleterious side effects.

Though these anti-ACE2 antibodies could effectively inhibit sarbecovirus infection in humans, the fact that the antibodies target a host receptor molecule rather than the SARS-CoV-2 S protein will necessitate their testing in terms of safety, efficacy, and pharmacological behavior in primate models before human clinical trials.

Conclusions

SARS-CoV-2 might evolve and start using receptors other than ACE2, creating another genetic hurdle to overcome for researchers working on the development of SARS-CoV-2 therapeutics.

However, the human anti-hACE2 mAbs engineered in this study showed exceptional breadth and potency in inhibiting infection by hACE2-utilizing sarbecoviruses.

Thus, they represent a long-term, ‘resistance-proof’ prophylaxis and treatment for SARS-CoV-2, even for future outbreaks of SARS-like coronaviruses.

In addition, these mAbs might prove particularly useful for susceptible patients like those with immunodeficiency and in which vaccine-induced protective immunity is unattainable or difficult to attain.

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Journal reference:
Microbiology

New compound with antibacterial activity shows promising results within one hour in laboratory trials

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Reviewed by Emily Henderson, B.Sc.May 15 2023

Resistance to antibiotics is a problem that alarms the medical and scientific community. Bacteria resistant to three different classes of antibiotics, known as multi-drug resistant (MDR) bacteria, are far from rare. Some are even resistant to all currently available treatments and are known as pan-drug resistant (PDR). They are associated with dangerous infections and listed by the World Health Organization (WHO) as priority pathogens for drug development with maximum urgency.

An article published in a special issue of the journal Antibiotics highlights a compound with antibacterial activity that presented promising results within one hour in laboratory trials.

The study was led by Ilana Camargo, last author of the article, and conducted during the doctoral research of first author Gabriela Righetto at the Molecular Epidemiology and Microbiology Laboratory (LEMiMo) of the University of São Paulo’s São Carlos Institute of Physics (IFSC-USP) in Brazil.

The compound we discovered is a new peptide, Pln149-PEP20, with a molecular framework designed to enhance its antimicrobial activity and with low toxicity. The results can be considered promising insofar as the trials involved pathogenic bacteria associated with MDR infections worldwide.”

Adriano Andricopulo, co-author of the article

Although novel antibacterial drugs are urgently needed, the pharmaceutical industry is notoriously uninterested in pursuing them, mainly because research in this field is time-consuming and costly, requiring very long lead times to bring viable active compounds to market.

The Center for Innovation in Biodiversity and Drug Discovery (CIBFar), a Research, Innovation and Dissemination Center (RIDC) set up and funded by FAPESP, looks for molecules that can be used to combat multidrug-resistant bacteria.

Camargo and Andricopulo are researchers at CIBFar, as are two other co-authors who study promising bactericidal compounds: Leila Beltramini and José Luiz Lopes.

For over a decade, the group formed by the collaboration between Beltramini and Lopes has analyzed Plantaricin 149 and its analogs. Plantaricins are substances produced by the bacterium Lactobacillus plantarum to combat other bacteria.

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Lactobacillus plantarum is commonly found in nature, especially in anaerobic plant matter, and in many fermented vegetable, meat and dairy products.

In the case of Plantaricin 149, Japanese researchers were the first to report its bactericidal action (in 1994) and since then scientists have been interested in obtaining more efficient synthetic analogs (molecules with small structural differences). In 2007, one of the first projects completed by the CIBFar team showed that the peptide inhibits pathogenic bacteria such as Listeria spp. and Staphylococcus spp. They then began studying synthetic analogs with stronger bactericidal activity than the original (causing more damage to the membrane of the combated microorganisms).

With the support of a scholarship from FAPESP, Righetto synthesized 20 analogs of Plantaricin 149, finding that Pln149-PEP20 had the best results so far and was also half the size of the original peptide. “The main advances in our research consist of the development of this smaller, more active and less toxic molecule, and the characterization of its action and propensity to develop resistance. It has proven to be highly promising in vitro – active against MDR bacteria and extensively resistant bacteria,” said Camargo, principal investigator for the project.

LEMiMo, the laboratory where the studies were conducted, has experience in characterizing bacterial isolates involved in outbreaks of hospital infections and holds a collection of bacteria selected for these trials in search of novel active compounds. The bacteria have the resistance profiles currently of greatest concern and were isolated during hospital outbreaks.

They are known in the scientific community by the term ESKAPE, an acronym for the scientific names of six highly virulent and antibiotic-resistant bacterial pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

Further research can now be conducted to investigate the molecule’s action mechanism in more depth, to look for formulations, and possibly to develop an application. “In terms of the action mechanism, it’s also possible to use the cell morphology of the bacteria to identify cellular pathways affected by the peptide,” Righetto said. “As for optimization, the molecule can be functionalized by being linked to macrostructures, and the amino acid sequence can be modified.” Research is also needed on its cytotoxicity and on its selectivity (whether it affects healthy cells).

“We’re living in times of major global public health hazards due to a lack of antimicrobials that can be used to treat infections caused by extremely resistant bacteria. Antimicrobial peptides are targets of great interest for the development of novel candidate drugs. This novel molecule has the potential to be used as an innovative antimicrobial therapy, but further modifications and molecular optimizations still need to be investigated,” Andricopulo said.

Publication of the article also involved Harvard Medical School’s Infectious Disease Institute in Boston (USA) via researchers Paulo José Martins Bispo and Camille André.

Source:

São Paulo Research Foundation (FAPESP)

Journal reference:

Righetto, G. M., et al. (2023). Antimicrobial Activity of an Fmoc-Plantaricin 149 Derivative Peptide against Multidrug-Resistant Bacteria. doi.org/10.3390/antibiotics12020391.

Microbiology

Anticoronavirals: the development of COVID-19 therapies and the challenges that remain

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Neha MathurBy Neha MathurMay 8 2023Reviewed by Danielle Ellis, B.Sc.

In a recent article published in Nature Microbiology, researchers highlighted the pace of development of coronavirus disease 2019 (COVID-19) therapies during the pandemic and the challenges that hinder the widespread availability of anticoronavirals.

Study: Therapeutics for COVID-19. Image Credit: Viacheslav Lopatin/Shutterstock.com
Study: Therapeutics for COVID-19. Image Credit: Viacheslav Lopatin/Shutterstock.com

Background

COVID-19 is the third coronavirus disease in the past 20 years after severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). While the two predecessors caused severe mortality, they did not cause a pandemic. On the contrary, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered a pandemic, and by 21 February 2023, it had caused more than 757 million confirmed cases, including >6.8 million deaths worldwide.

Vaccines and monoclonal antibody (mAb) treatments for COVID-19 became available within a year of the pandemic. Yet, there is a substantial need for more effective therapeutics to treat unvaccinated and immunocompromised patients and those whose vaccine immunity waned over time.

About the study

In this study, the authors highlighted four stages of SARS-CoV-2 infection that require different therapeutic interventions as critical for identifying COVID-19 therapeutic targets. At stage 1, when viral replication begins inside the host, oral or intravenous administration of monoclonal antibodies and antiviral therapies are effective. However, an ideal time for prophylactic administration of vaccines is Stage 0 preceding the infection.

Clinical trials have established that mAbs and antivirals effectively combat COVID-19 when administered up to 10 days after symptom onset and within three to five days following the onset of symptoms, respectively. COVID-19 patients in stage 2 develop viral pneumonia, cough and fever, lung inflammation causing shortness of breath, and lung aberrations, such as ground glass opacities.

The most serious is stage 3 characterized by a hyperinflammatory state or acute respiratory distress syndrome (ARDS). Some patients might also develop coagulation disorders or shock or systemic inflammatory response syndrome (SIRS). Thus, at stage 3, a patient needs antiviral drugs and immunomodulatory therapy.

Stage 4 represents post-COVID-19 conditions when patients experience hyperinflammatory illnesses, e.g., multi-system inflammatory syndrome in children (MISC), following acute SARS-CoV-2 infection. Unfortunately, possible preventative measures and treatment for post-acute sequelae of SARS-CoV-2 (PASC) are not fully understood. It is a growing area of unmet medical need; thus, extensive research efforts are ongoing to classify PASC, which might be a conglomeration of several syndromes, and determine its cause(s).

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The National Institutes of Health (NIH) Treatment Guidelines Panel makes recommendations for the treatment and prevention of COVID-19. Early in the pandemic, clinicians used azithromycin and hydroxychloroquine as a possible COVID-19 treatment for hospitalized patients based on in vitro evidence of their synergistic effect on SARS-CoV-2 infection. Later, clinical trials found this combination ineffective. Similarly, the NIH panel did not specify recommendations for empirical antimicrobials.

The NIH rejected giving vitamin/mineral supplements, e.g., zinc, for hospitalized COVID-19 patients. On the contrary, they recommended prompt use of supplemental oxygenation and high-flow nasal cannula in patients with ARDS. In the absence of effective treatments, clinical recommendations by NIH continue to change and evolve.

Early drug repurposing efforts targeted nucleotide prodrugs, e.g., remdesivir (or GS-5734), AT-527, favipiravir, and molnupiravir (or MK-4482). However, only three antivirals received full Emergency Use Authorization (EUA) approval from the United States Food and Drug Administration (US-FDA), remdesivir, molnupiravir, and nirmatrelvir.

Pre-clinical characterization of remdesivir for other coronaviruses, pharmacokinetic and safety evaluation in humans in a failed clinical trial for Ebola virus, all acquired before the beginning of the COVID-19 pandemic, enabled rapid progression of remdesivir.

A phase 3 study conducted among patients in outpatient facilities and nursing facilities showed that remdesevir administration within seven days of symptom onset decreased hospitalization risk by 87%. Thus, its approval extended to high-risk non-hospitalized patients as well. Currently, phase 1b/2a study for inhaled remdesivir, and pre-clinical evaluation of an oral prodrug based on remdesivir is ongoing.

Another randomized phase III trial evaluated ivermectin, metformin, and fluvoxamine, all repurposed drug candidates, for early COVID-19 treatment of overweight or obese adults. Earlier pivotal efficacy and clinical studies found that molnupiravir provided no clinical benefit in hospitalized COVID-19 patients.

Conversely, the MOVe-OUT outpatient study demonstrated that treatment initiated within five days of symptom onset reduced the hospitalization risk or death. Accordingly, molnupiravir attained an EUA in the US on in late 2021 for treatment of mild-to-moderately ill COVID-19 patients at high risk of progression to severe disease. However, an outpatient study suggested that molnupiravir might augment SARS-CoV-2 evolution in immunocompromised individuals.

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In the USA, multiple initiatives have been undertaken to identify candidate agents that may be repurposed as COVID-19 drugs. For instance, the Bill and Melinda Gates Foundation launched the Therapeutics Accelerator in March 2020, wherein they adopted a three-way approach to test approved drugs, screen drug repositories, and evaluate novel small molecules, including mAbs against SARS-CoV-2.

Encouragingly, apilimod, a PIKfyve kinase inhibitor developed for treating autoimmune diseases, is being tested for COVID-19 in clinical studies. Likewise, multiple clinical trials are ongoing for camostat mesilate, an inhibitor of transmembrane protease serine 2 (TMPRSS2), an approved chronic pancreatitis treatment in Japan.

Among anti-inflammatory and immunomodulating drugs, dexamethasone, a corticosteroid, baricitinib, a Janus kinase (JAK) inhibitor, and tocilizumab have received FDA approval. Among mAb therapies, casirivimab with imdevimab and bamlanivimab with etesevimab, Sotrovimab, Bebtelovimab, Tixagevimab–cilgavimab have received FDA approval. However, as SARS-CoV-2 continues to evolve, changes in the spike protein led to EUAs being withdrawn for all mAb therapies due to loss of efficacy.

Conclusions

There is a vast knowledge gap regarding COVID-19 pathogenesis. Despite the absence of a viral reservoir, severe disease persists for weeks or even months after COVID-19 recovery. Another intriguing area of investigation is why autoantibodies increase over time during COVID-19. In February 2022, the government of the United States of America (USA) started a flagship program, RECOVER, to understand, prevent and treat COVID-19-related long-term health effects.

Amid decreasing vaccine uptake and waning efficacy of mAbs as SARS-CoV-2 mutates, there is a need for new, safe, and effective COVID-19 therapies for population-level deployment and the potential to reduce resistance development. Researchers need to accelerate research targeting small molecule candidates that would mechanistically target the conserved region of SARS-CoV-2 and not become ineffective across mutant strains.

To be prepared for another pandemic, a large repository of small molecules that have already progressed through early pre-clinical and clinical evaluation is needed to develop drugs, like remdesivir, developed in a short span of two years.

More importantly, research efforts should continue to advance the development of antivirals for other pathogens, including coronaviruses, in preparation for the next pandemic.

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Journal reference:
Microbiology

Bioengineered drug candidate can counter S. aureus infection in early tests

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Reviewed by Emily Henderson, B.Sc.Apr 24 2023

Researchers at NYU Grossman School of Medicine and Janssen Biotech, Inc. have shown in early tests that a bioengineered drug candidate can counter infection with Staphylococcus aureus – a bacterial species widely resistant to antibiotics and a major cause of death in hospitalized patients.

Experiments demonstrated that SM1B74, an antibacterial biologic agent, was superior to a standard antibiotic drug at treating mice infected with S. aureus, including its treatment-resistant form known as MRSA.

Published online April 24 in Cell Host & Microbe, the new paper describes the early testing of mAbtyrins, a combination molecule based on an engineered version of a human monoclonal antibody (mAb), a protein that clings to and marks S. aureus for uptake and destruction by immune cells. Attached to the mAb are centyrins, small proteins that prevent these bacteria from boring holes into the human immune cells in which they hide. As the invaders multiply, these cells die and burst, eliminating their threat to the bacteria.

Together, the experimental treatment targets ten disease-causing mechanisms employed by S. aureus, but without killing it, say the study authors. This approach promises to address antibiotic resistance, say the researchers, where antibiotics kill vulnerable strains first, only to make more space for others that happen to be less vulnerable until the drugs no longer work.

To our knowledge, this is the first report showing that mAbtyrins can drastically reduce the populations of this pathogen in cell studies, and in live mice infected with drug-resistant strains so common in hospitals. Our goal was to design a biologic that works against S. aureus inside and outside of cells, while also taking away the weapons it uses to evade the immune system.”

Victor Torres, PhD, Lead Study Author, the C.V. Starr Professor of Microbiology and director of the NYU Langone Health Antimicrobial-Resistant Pathogen Program

One-third of the human population are carriers of S. aureus without symptoms, but those with weakened immune systems may develop life-threatening lung, heart, bone, or bloodstream infections, especially among hospitalized patients.

Inside out

The new study is the culmination of a five-year research partnership between scientists at NYU Grossman School of Medicine and Janssen to address the unique nature of S. aureus.

The NYU Langone team together with Janssen researchers, published in 2019 a study that found that centyrins interfere with the action of potent toxins used by S. aureus to bore into immune cells. They used a molecular biology technique to make changes in a single parental centyrin, instantly creating a trillion slightly different versions of it via automation. Out of this “library,” careful screening revealed a small set of centyrins that cling more tightly to the toxins blocking their function.

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Building on this work, the team fused the centyrins to a mAb originally taken from a patient recovering from S. aureus infection. Already primed by its encounter with the bacteria, the mAb could label the bacterial cells such that they are pulled into bacteria-destroying pockets inside of roving immune cells called phagocytes. That is unless the same toxins that enable S. aureus to drill into immune cells from the outside let it drill out of the pockets to invade from the inside.

In a “marvel of bioengineering,” part of the team’s mAbtyrin serves as the passport recognized by immune cells, which then engulf the entire, attached mAbtyrin, along with its centyrins, and fold it into the pockets along with bacteria. Once inside, the centyrins block the bacterial toxins there. This, say the authors, sets their effort apart from antibody combinations that target the toxins only outside of cells.

The team made several additional changes to their mAbtyrin that defeat S. aureus by, for instance, activating chain reactions that amplify the immune response, as well by preventing certain bacterial enzymes from cutting up antibodies and others from gumming up their action.

In terms of experiments, the researchers tracked the growth of S. aureus strains commonly occurring in US communities in the presence of primary human immune cells (phagocytes). Bacterial populations grew almost normally in the presence of the parental antibody, slightly less well in the presence of the team’s engineered mAb, and half as fast when the mAbtyrin was used.

In another test, 98% of mice treated with a control mAb (no centyrins) developed bacteria-filled sores on their kidneys when infected with a deadly strain of S. aureus, while only 38% of mice did so when treated with the mAbtyrin. Further, when these tissues were removed and colonies of bacteria in them counted, the mice treated with the mAbtyrin had one hundred times (two logs) fewer bacterial cells than those treated with a control mAb.

Finally, the combination of small doses of the antibiotic vancomycin with the mAbtyrin in mice significantly improved the efficacy of the mAbtyrin, resulting in maximum reduction of bacterial loads in the kidneys and greater than 70% protection from kidney lesions.

“It is incredibly important,” said Torres, “that we find new ways to boost the action of vancomycin, a last line of defense against MRSA.”

Along with Torres, authors from the Department of Microbiology at NYU Langone were Rita Chan, Ashley DuMont, Keenan Lacey, Aidan O’Malley, and Anna O’keeffe. The study authors included 13 scientists from Janssen Research & Development (for details see the study manuscript).

This work was supported by Janssen Biotech, Inc., one of the Janssen Pharmaceutical Companies of Johnson & Johnson, under the auspices of an exclusive license and research collaboration agreement with NYU. Torres has recently received royalties and consulting compensation from Janssen and related entities. These interests are being managed in accordance with NYU Langone policies and procedures.

Source:

NYU Langone Health / NYU Grossman School of Medicine

Journal reference:

Buckley, P. T., et al. (2023). Multivalent human antibody-centyrin fusion protein to prevent and treat Staphylococcus aureus infections. Cell Host & Microbe. doi.org/10.1016/j.chom.2023.04.004.

Microbiology

Elucidating the function of BRCA2 gene offers insight into cancer development

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Reviewed by Emily Henderson, B.Sc.Apr 3 2023

A new study shows exactly how the gene BRCA2, linked to susceptibility to breast and ovarian cancer, functions to repair damaged DNA. By studying BRCA2 at the level of single molecules, researchers at the University of California, Davis, have generated new insights into the mechanisms of DNA repair and the origins of cancer. The work was published the week of March 27 in the Proceedings of the National Academy of Sciences.

Elucidating the function of BRCA2 is essential for understanding the molecular etiology of cancer development in breast and ovarian cells, as well as many other cell types including prostate.”

Stephen Kowalczykowski, distinguished professor of microbiology and molecular genetics, UC Davis College of Biological Sciences

By visualizing BRCA2 function at a single molecule level, Kowalczykowski’s team discovered that it acts as a molecular chaperone, delivering another protein, RAD51, to single-stranded DNA. It ensures formation of a functional filament of RAD51 and the repair of broken DNA.

“When BRCA2 is defective, broken DNA is not faithfully repaired, the genome loses integrity, and cancer ultimately ensues,” Kowalczykowski said.

Mutations in the BRCA2 gene are linked to an increased risk of cancer, especially breast and ovarian cancer. In 2010, teams led by Kowalczykowski and by Professor Wolf-Dietrich Heyer in the same department at UC Davis succeeded in purifying the BRCA2 protein and showed that it plays a key role in DNA repair.

The new work, using techniques developed in Kowalczykowski’s lab to image single proteins and DNA molecules in real time, gives new insight into the mechanics of this repair process.

Our DNA is under constant assault by both processes inside cells and by outside factors, such as sunlight or chemical exposures. Accumulating damage to DNA can cause cells to become cancerous. Fortunately, our cells have several mechanisms to repair DNA. One of these is homologous recombination to repair double-stranded breaks.

Repairing double-stranded breaks

When a break crosses both strands of the DNA double helix, one strand is trimmed back a little to leave a single exposed strand. This strand then goes hunting for its counterpart in the same gene in the matching paired chromosome. It inserts into the healthy DNA and uses it as a template for repair.

For this insertion to work, the single strand of DNA has to be coated with RAD51. Earlier work from Kowalczykowski’s lab measured how quickly RAD51 could be added onto DNA, like beads on a string.

The function of BRCA2 is to load up with RAD51 (each BRCA2 can carry up to six RAD51s), push another protein called RPA out of the way and put the proteins onto the DNA.

Postdoctoral researcher Jason Bell carried out the experiments observing RAD51 and BRCA2 working their way along the DNA. Bell manipulated pieces of DNA with a single-stranded gap and exposed them to RAD51 with and without BRCA2 under different conditions.

The resulting movies show how BRCA2 chaperones RAD51 onto single-stranded DNA, displacing RPA.

Understanding the role of BRCA2 in DNA repair has two important implications. First, it helps us understand why mutations of BRCA2 lead to an increased risk of cancer. Second, some drugs to treat cancer work by damaging DNA. By understanding how DNA repair works, we can develop new drugs to target it specifically in cancer cells.

Additional co-authors on the paper are Christopher Dombrowski and Jody Plank, both at UC Davis, and Ryan Jensen, formerly at UC Davis and now at the Yale University School of Medicine. The work was supported by grants from the National Institutes of Health.

Source:

University of California – Davis

Journal reference:

Bell, J. C., et al. (2023). BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA. Proceedings of the National Academy of Sciences. doi.org/10.1073/pnas.2221971120.

Microbiology

Researchers report surprising first steps that promote resistance to commonly prescribed antibiotics

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Reviewed by Emily Henderson, B.Sc.Mar 31 2023

Antibiotic resistance is a global health threat. In 2019 alone, an estimated 1.3 million deaths were attributed to antibiotic resistant bacterial infections worldwide. Looking to contribute a solution to this growing problem, researchers at Baylor College of Medicine have been studying the process that drives antibiotic resistance at the molecular level.

They report in the journal Molecular Cell crucial and surprising first steps that promote resistance to ciprofloxacin, or cipro for short, one of the most commonly prescribed antibiotics. The findings point at potential strategies that could prevent bacteria from developing resistance, extending the effectiveness of new and old antibiotics.

Previous work in our lab has shown that when bacteria are exposed to a stressful environment, such as the presence of cipro, they initiate a series of responses to attempt to survive the toxic effect of the antibiotic.”

Dr. Susan M. Rosenberg, co-corresponding author, Ben F. Love Chair in Cancer Research and professor of molecular and human genetics, biochemistry and molecular biology and molecular virology and microbiology at Baylor

She also is program leader in Baylor’s Dan L Duncan Comprehensive Cancer Center (DLDCCC). “We discovered that cipro triggers cellular stress responses that promote mutations. This phenomenon, known as stress-induced mutagenesis, generates mutant bacteria, some of which are resistant to cipro. Cipro-resistant mutants keep on growing, sustaining an infection that can no longer be eliminated with cipro.”

Cipro induces breaks in the DNA molecule, which accumulate inside bacteria and consequently trigger a DNA damage response to repair the breaks. The Rosenberg lab’s discoveries of the steps involved in stress-induced mutagenesis revealed that two stress responses are essential: the general stress response and the DNA-damage response.

Some of the downstream steps of the process that leads to increased mutagenesis have been revealed previously by the Rosenberg lab and her colleagues. In this study, the researchers discovered the molecular mechanisms of the first steps between the antibiotic causing DNA breaks and the bacteria turning on the DNA damage response.

“We were surprised to find an unexpected molecule involved in modulating DNA repair,” said first author Dr. Yin Zhai, postdoctoral associate in the Rosenberg lab. “Usually, cells regulate their activities by producing specific proteins that mediate the desired function. But in this case, the first step to turn on the DNA repair response was not about activating certain genes that produce certain proteins.”

Instead, the first step consisted of disrupting the activity of a protein already present, RNA polymerase. RNA polymerase is key to protein synthesis. This enzyme binds to DNA and transcribes DNA-encoded instructions into an RNA sequence, which is then translated into a protein.

“We discovered that RNA polymerase also plays a major role in regulating DNA repair,” Zhai said. “A small molecule called nucleotide ppGpp, which is present in bacteria exposed to a stressful environment, binds to RNA polymerase through two separate sites that are essential for turning on the repair response and the general stress response. Interfering with one of these sites turns off DNA repair specifically at the DNA sequences occupied by RNA polymerase.”

“ppGpp binds to DNA-bound RNA polymerase, telling it to stop and backtrack along the DNA to repair it,” said co-corresponding author Dr. Christophe Herman, professor of molecular and human genetics, molecular virology and microbiology and member of the DLDCCCC. The Herman lab found the repair-RNA-polymerase connection previously, reported in Nature.

Rosenberg’s lab discovered that DNA repair can be an error-prone process. As repair of the broken DNA strands progresses, errors occur that alter the original DNA sequence producing mutations. Some of these mutations will confer bacteria resistance to cipro. “Interestingly, the mutations also induce resistance to two other antibiotic drugs the bacteria have not seen before,” Zhai said.

“We are excited about these findings,” Rosenberg said. “They open new opportunities to design strategies that would interfere with the development of antibiotic resistance and help turn the tide on this global health threat. Also, cipro breaks bacterial DNA in the same way that the anti-cancer drug etoposide breaks human DNA in tumors. We hope this may additionally lead to new tools to combat cancer chemotherapy resistance.”

Other contributors to this work include P.J. Minnick, John P. Pribis, Libertad Garcia-Villada and P.J. Hastings, all at Baylor College of Medicine.

Source:

Baylor College of Medicine

Journal reference:

Zhai, Y., Minnick, P. J., Pribis, J. P., Garcia-Villada, L., Hastings, P. J., Herman, C., & Rosenberg, S. M. (2023). ppGpp and RNA-polymerase backtracking guide antibiotic-induced mutable gambler cells. Molecular Cell. doi.org/10.1016/j.molcel.2023.03.003.

Microbiology

Study offers a novel therapeutic option to combat antibiotic-resistant pneumonia

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Reviewed by Emily Henderson, B.Sc.Mar 29 2023

Increases in multidrug-resistance in the bacteria Streptococcus pneumoniae have made it the fourth-leading cause of death associated with antibiotic resistance.

In a study in PLOS Biology, researchers report a new target to fight against pneumonia due to infections by this opportunistic lung pathogen -; interference with the bacteria’s fermentation metabolism. This may offer a novel therapeutic option in the urgent need to discover new strategies to combat drug-resistant S. pneumoniae.

In a proof of principle, University of Alabama at Birmingham researchers showed that giving an existing drug -; one already approved by the United States Food and Drug Administration to treat methanol poisoning – in combination with the antibiotic erythromycin significantly reduced disease in mice infected with a virulent, multidrug-resistant S. pneumoniae. The combination therapy reduced bacterial burden in the lungs by 95 percent, and bacterial burdens in the spleen and heart by 100- and 700-fold, respectively. The FDA-approved drug alone, or erythromycin alone, had no effect.

Fomepizole, the FDA-approved drug, disrupts activity of the enzyme alcohol dehydrogenase in the bacteria. The mice were infected intratracheally with the multidrug-resistant clinical isolate S. pneumoniae serotype 35B strain 162–5678, which has high resistance to erythromycin. Notably, the S. pneumoniae 35B serotype has been reported as an emerging multidrug-resistant serotype in clinical settings. Eighteen hours after infection, the mice were given a single injection of erythromycin, with or without fomepizole.

Fomepizole, or other drugs that inhibit bacterial metabolism, have potential to dramatically increase the efficacy of erythromycin and other antibiotics, respectively, in vivo.”

Carlos Orihuela, Ph.D., professor and interim chair of the UAB Department of Microbiology

A broad foundation of basic research preceded this proof-of-principle experiment.

S. pneumoniae relies on fermentation and glycolysis to produce energy. During fermentation, pyruvate is converted to lactate, acetate and ethanol, and NADH is oxidized to regenerate NAD+, which is needed for glycolysis. Accordingly, maintenance of an available NAD+ pool, necessary for redox balance, is vital for sustained energy production, bacterial growth and survival.

Orihuela and UAB colleagues made S. pneumoniae mutants in five enzymes involved in fermentation and NAD+ production, and they found, in general, that the mutants had impaired metabolism. Two of the mutants, one for lactate dehydrogenase and one for alcohol dehydrogenase, had stark decreases in intracellular pool of ATP, the energy molecule of living cells. The other three mutants had significant, but more modest, decreases.

NAD+/NADH redox imbalances in the mutants generally interfered with production of S. pneumoniae virulence factors and colonization in the mouse nasopharynx. Some of the mutations influenced susceptibility to antibiotics, as tested with three antibiotics, including erythromycin, that interfere with protein synthesis, two antibiotics that disrupt cell wall synthesis and one antibiotic that targets DNA transcription.

Researchers found that treating a wildtype S. pneumoniae, which did not have mutations in alcohol dehydrogenase or the other enzymes, with fomepizole alone caused redox imbalances. In vitro tests showed that treatment of S. pneumoniae with fomepizole enhanced the susceptibility to antibiotics, including fourfold decreases in the minimal inhibitory concentrations of the antibiotics erythromycin and gentamicin.

“We also evaluated whether fomepizole treatment impacted the antibiotic susceptibility of other anaerobic gram-positive bacteria, including other streptococcal pathogens, including Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecium, to erythromycin or gentamicin,” Orihuela said. “We observed from twofold to eightfold decreased minimal inhibitory concentration with fomepizole in most cases, including E. faecium.”

“Our results indicate that the blocking of NAD+ regeneration pathways during infection is a way to increase antibiotic susceptibility in drug-resistant gram-positive anaerobic pathogens,” Orihuela said. “This has clinical potential with regard to microbial eradication and treatment of disseminated infection.”

Globally, more than 3 million individuals are hospitalized due to pneumococcal disease annually, and hundreds of thousands die as a result.

Source:

University of Alabama at Birmingham

Journal reference:

Im, H., et al. (2023). Targeting NAD+ regeneration enhances antibiotic susceptibility of Streptococcus pneumoniae during invasive disease. PLOS Biology. doi.org/10.1371/journal.pbio.3002020.