Abstract
The COVID-19 Pandemic
MRNA Vaccination Boosts Cross-Variant Neutralizing Antibodies Elicited by SARS-CoV-2 Infection
The protective effects of immunization largely arise from the generation of neutralizing antibodies (nAbs). Studies suggest that transferring nAbs limits respiratory infection and protects infection in animal models and is suggested to protect against infection in humans. A SARS-CoV-2 infection gives rise to nAbs that decline but is detectable in the subsequent months. Most nAbs are directed against the receptor binding domain (RBD), which block interactions against the ACE2 motif as well as other regions of the viral spike. While vaccines have been produced by Pfizer/BioNTech and Moderna, the high global rates of SARS-CoV-2 have resulted in viral variants with increased transmission, such as the UK (B.1.1.7), South African (B.1.351), and Brazilian (P.1) variants. B.1.1.7, B.1.351, and P.1 increase affinity for the ACE2 receptor, while the D614G mutation increases virion spike density, infectivity, and its transmission. Overall, there is growing concern that these variants of concern (VoC) can evade nAbs generated from prior infections and even result in a loss of efficacy of vaccines raised against the original Wuhan strain.
To assess neutralization susceptibility, sera from previously infected donors (PIDs) were collected before or after one or two immunizations or from uninfected (naive) donors (NDs) that received two doses as well as the infected but unvaccinated patients. nAbs obtained from Wuhan strain infections were found to be ~10-fold weaker toward B.1.351 variants. Similarly, other nAbs were approximately three- to fourfold weaker. Certain nAbs—anti-NTD CV1 mAb (monoclonal antibody)—could not neutralize B.1.351 variants. This demonstrates how the B.1.351 variants are more resistant to mAb neutralization generated from variants early during the pandemic. From PIDs, a single dose of either variant boosted RBD-specific IgG Ab titers by ~500-fold. Vaccination also increased RBD-specific IgA titers by 200-fold. When PIDs were compared to NDs, a single dose increased IgG by 4.5-fold, while IgA increased by 7.7-fold compared to double-dosed NDs. Of the 15 PIDs in this study, only 12 generated sera that neutralized the original Wuhan variant. The prevaccine sera from NDs was found to be nonneutralizing. In these PIDs, the median half-maximal neutralizing titers (ID50) were boosted 1000-fold after the first dose. In contrast, two doses given to NDs gave rise to ID50 titers 5- to 10-fold lower than the vaccinated PIDs.
These were then evaluated on B.1.351 mutant pseudoviruses (model infectious organisms). Before vaccination, only 5 of 15 sera from PIDs neutralized the B.1.351 mutant pseudovirus. The median ID50 of the prevaccine sera against the Wuhan strain was significantly higher compared to the B.1.351 variants. The nAb titers from a single immunization in PIDs were significantly greater than a double immunization in NDs against all the tested pseudoviruses. Only 8 of 13 vaccinated NDs could achieve 80% neutralization of one of the B.1.351 variants. RBD-Ab depletion studies showed that this subdomain was key to serum neutralization of Wuhan strains. The results collectively suggest that the mRNA vaccines (based on the Wuhan variant) can generate and/or boost nAb responses, but lose their potency against divergent variants. This study also shows that previously infected individuals strongly benefit from a single-dose immunization against vaccine-matched and emerging VoC. Furthermore, a second dose was not beneficial in boosting neutralizing titers, suggesting that their administration could be delayed. These findings help to determine the necessity or optimal timing of the second dose in previously infected individuals. (Stamatatos, L.; et al. Science
SARS-CoV-2 Infection of Human iPSC–Derived Cardiac Cells Reflects Cytopathic Features in Hearts of Patients with COVID-19
SARS-CoV-2 has been shown to affect a number of different organs, including the cardiovascular system. Reports of acute COVID-19-associated myopathy without prior disease suggests the infection is directly causing cardiac damage. A number of signs include increased troponin-I and natriuretic peptides (typical biomarkers for cardiac damage), impaired cardiac function (by MRI), and abnormal echocardiograms. To develop therapeutic strategies against COVID-19-induced myocardial injury, the mechanism of SARS-CoV-2-induced damage needs to be identified. Furthermore, cardiomyocytes (CMs) express the main receptor that facilitates viral entry—angiotensin-converting enzyme 2 (ACE2), suggesting its susceptibility to SARS-CoV-2. Autopsies also detect SARS-CoV-2 presence in cardiac tissue from COVID-19 patients. Ex vivo studies using human cell models are one prospective and clinically relevant method for studying the effects of cardiac viral infection. Organoid models developed using stem cells have shown hepatocyte, intestinal epithelium, and lung susceptibility.
This study tested the relative susceptibility of SARS-CoV-2 infection of the three main cardiac cells: CMs, cardiac fibroblasts (CFs), and endothelial cells (ECs). These in vitro models facilitate the observed phenotypic biomarkers of infection to serve as a discovery platform to develop cardioprotective therapeutics. Single-cell RNA sequencing (scRNA-seq) and immunofluorescence staining suggest that ACE2 transcripts were only detectable in CMs rather than CFs and ECs.
During infection, CFs and ECs show minimal viral RNA while CMs express >104-fold higher RNA after 48 h. These viral RNA levels were similar in CM and mixed cell cultures, while undifferentiated human induced pluripotent stem cells (hiPSCs) were uninfected. These findings were further affirmed by plaque assays. While CMs support viral replication, all three types exhibited marked cytopathic effects after 48 h of exposure, evidenced by fragmented cells, dissociation from neighboring cells, and significant cell death. Interestingly, cytopathy was most prevalent in CFs and nuclear loss greatest in ECs despite insignificant levels of viral replication. This cell loss was alleviated when the viruses were inactivated. A higher infection dosage resulted in a uniform appearance of virus-positive cells, while lower dosages tended to give rise to local infection foci. The results suggest that SARS-CoV-2 is capable of infecting and rapidly propagating in human CMs.
Several clinically relevant methods prevented SARS-CoV-2 infection of CMs—ACE2 blocking antibodies (Abs), inhibitor drugs, or antivirals (i.e., remdesivir) significantly curtailed viral infection. While the reduction of infection reduced cytopathic effects, remdesivir was toxic to CMs at 10 µM. Further experimentation with various inhibitor molecules suggests that SARS-CoV-2 binds to the ACE2 receptor and exploits cathepsin L (CTSL) for cell entry. Treatment with interferon (IFN-beta) decreased infection, while applying JAK/STAT inhibitors reversed these protective effects.
RNA-seq analysis also inferred an inflammatory cytokine production genetic signature. The CMs reduced their expression of cardiac muscle tissue organization and cellular respiration genes. Dysregulation of contractile machinery, proteasomal subunits, and ubiquitination genes was also observed postinfection. Genes involved in contractile and structural activity were further distorted. Infection also cleaved myofibrils into individual sarcomeric units devoid of alignment. Myofibrillar fragmentation was also observed as early as 24 h after infection until 48 h postexposure. Signs of cellular cytotoxic stress were also observed, including fragmented myofibrils with extended I-bands and the absence of A-bands. This sarcomeric fragmentation phenotype was not observed following infection using other coronaviruses. Proteasome drug inhibition also gave rise to less prevalent and severe characteristics. Cardiotoxicity from doxorubicin did not generate myofibril fragmentation. Samples obtained from autopsy also identified disrupted myocytes with observable loss of DNA staining. Immunostaining revealed severe myofibrillar anomalies such as diffuse or absent cTnT and alpha-actinin 2.
These studies demonstrate how the in vitro models identify the cytopathic effects wrought by SARS-CoV-2 without any diagnosis of cardiac tissue damage. This will eventually lead to efficacious antiviral and cardioprotective strategies to manage and prevent cardiac damage in COVID-19 patients. (Perez-Bermejo, J. A.; et al. Sci. Transl. Med.
A Human-Airway-on-a-Chip for the Rapid Identification of Candidate Antiviral Therapeutics and Prophylactics
Repurposing existing drugs against SARS-CoV-2 is a comparatively rapid method to identify novel therapeutics and strategies to combat the growing incidence against COVID-19. Yet, current methods involving traditional in vitro screening methods have yielded results with poor fidelity, such as hydroxychloroquine and its related toxicity risks. Human organ chip microfluidic technology has demonstrated good mimicry of human lung physiology, making it a good tool in drug repurposing efforts against infectious diseases such as influenza and COVID-19. Apart from conventional cell culture which fails to recapitulate matured lung tissue characteristics, lung tissue explants and primary tissue-derived organoids are alternatives currently being trialed. Whereas explants can be limited in their supply as well as being less uniform, organoid technology cannot readily reproduce the air–liquid interface and immune cell interactions. Furthermore, static culture lung tissue models cannot predict human responses to clinically relevant drug exposure profiles that give rise to complex pharmacokinetics (PK), whereas microfluidic culture may facilitate these.
The human airway chip consists of parallel microfluidic channels separated by a porous membrane—airway and vascular channels—giving rise to many human lung features. This led to human lung epithelium generating high serine protease expression necessary for viral infections. High expressions of the angiotensin-converting enzyme 2 (ACE2) necessary for SARS-CoV-2 infection were also observed. To mimic an airborne infection, fluorescent-tagged H1N1 viruses were introduced into the airway microfluidic channel. This gave rise to damage to the lung epithelium and loss of adherens junctions resembling vascular leakage. While a number of different influenza strains (H1N1, H3N2, H5N1) were tested, they mostly propagated efficiently in the human airway chip model. Interestingly, the H3N2 strain demonstrated ~10-fold higher replication rates compared to H1N1 strains, giving rise to more severe barrier disruption. This corresponded with the higher clinical severity of the H3N2 infections. The human airway chip was also capable of modeling immune cell interactions. Primary neutrophils perfused through influenza-infected epithelium were efficiently recruited, whereas minimal neutrophils adhered in uninfected chips.
Imaging of the chips demonstrated transmigration of neutrophils to infected cells before clearing viruses identified by the loss of fluorescence signal by 2 days. H3N2 virus stimulated more neutrophil recruitment compared to H1N1, consistent with the greater inflammation in patients. Correspondingly, viral titers decreased with neutrophil recruitment and inflammatory cytokines (IL6, RANTES, etc.) were generated. The neutrophils further caused damage to the tissue barrier. The antiviral therapeutic Tamiflu was then tested on the airway chip. Its metabolite oseltamivir inhibited influenza A replication at a concentration of 1 µM. Its administration inhibited viral replication, preventing the disruption of the epithelium barrier, the tight junctions, as well as decreasing cytokines and chemokines. The airway chip was then applied for repurposing existing drugs. The authors explored approved serine protease inhibitors delivering them via the airway channel (mimicking intratracheal aerosol delivery). Nafamostat and Trasylol significantly reduced H1N1 and H3N2 titers, protecting airway barrier function and tight junction integrity, with concurrent reduction in inflammatory response. A significant limitation of oseltamivir is its short window of therapeutic administration—it is recommended for usage within 48 h of influenza infection. When combined with nafamostat, the treatment time window doubled to 96 h.
In view of the COVID-19 pandemic, the human airway chip was also utilized to repurpose existing drugs. Pseudotyped viruses—SARS-CoV-2pp—were inoculated into the airway channel, mimicking airborne infection. Neutralizing antibodies successfully blocked its entry via ACE2 receptors. Experimentation showed that three broad antiviral drugs—amodiaquine, toremiphene, and clomiphene—significantly reduced viral entry with preventive administration. Amodiaquine and its metabolite—desethylamodiaquine—inhibited infection in a dose-dependent manner. To validate its effects on animal subjects, amodiaquine was administered on hamsters before infection with SARS-CoV-2. Amodiaquine resulted in 70% reduced levels of SARS-CoV-2 3 days after the viral challenge. Immunohistochemistry of animal lungs confirmed reduction in SARS-CoV-2 N protein. This was further explored to prevent the spread of COVID-19 within populations. Compared to untreated subjects, amodiaquine reduced disease transmission by 90%. In contrast, hydroxychloroquine at similar concentrations did not generate significant inhibitory activity. When utilized as a drug treatment, it reduced infection by 70% (from N transcripts) with complete clearance by day 7. Given the threat of the COVID-19 pandemic and future ones, drug repurposing using biologically relevant models as the human airway chip can facilitate a rapid drug discovery pipeline against future biothreats. (Si, L.; et al. Nat. Biomed. Eng.
Tissue Engineering and Regeneration
In Situ Bioprinting of Autologous Skin Cells Accelerates Wound Healing of Extensive Excisional Full-Thickness Wounds
Poorly healing wounds—diabetic, venous, pressure ulcers, etc.—represent a costly burden. In the USA alone, 7 million patients cost US$25 billion annually. Patients respond best by closing and protecting the wounds, while early treatment helps to prevent the wound from worsening, minimizing chances of hypertrophic scarring. Extensive scarring may even cause disfigurement and loss of range of limb motion. While autografts are the “gold standard” for severe wounds, they are limited by supply. Allografts are an option but risk graft immune rejection. Biomaterial-based dermal substitutes have been developed, yet these are costly with relatively poor cosmetic outcomes. Cell therapy is a promising approach, involving seeding of keratinocytes and fibroblasts from a small skin tissue biopsy. These cells are deposited via either manual seeding or cell spraying. On the other hand, bioprinting offers greater control to deliver cells to precise locations. Inkjet printers deposit various cells and controlled volumes of liquids.
This study details the design and proof-of-concept validation of a novel, mobile skin bioprinting system. The device combines imaging technology with in situ printing of cells tailored to individual patients. The bioprinting system was designed with several criteria: portability, to identify and measure a wide range of wound sizes, delivering multiple cell types with precise orientation, easy sterilization, easy operation, and cost-effective maintenance. The main components of the system consist of a handheld 3D scanner, a print head with XYZ movement with eight nozzles (260 µm diameter), each driven by dispensing motors. The XYZ axes enable precision delivery at a resolution of 100 µm.
The skin bioprinting system is based on a cartridge delivery system similar to traditional inkjet printing. It comprises three components: a delivery mechanism, material reservoirs, and a pressure source. While the contents of each cartridge are in a fibrinogen and collagen matrix, separate atomizing nozzles deposit thrombin to generate fibrin. The system is pressurized with 6.89 kPa of compressed air. The bioprinting system was sterilized using 70% ethanol, which is flushed every 3 min, followed by water flushing. The laser scanner can be used to first create a wound map for printing. Alternatively, the user can also define areas corresponding to cell deposition and the pixel color corresponding to a given cell type. A laser sight guided deposition of fibrin/collagen hydrogels as well as different cell types to enable accurate deposition to form multicellular, multilayered skin constructs. In athymic mouse models, full-thickness wounds were created in 36 subjects. Epithelial wound coverage occurred by 1 week with fully formed skin observed 10–14 days after bioprinting. Whereas control wounds remained 95% of their original size after 1 week, the bioprinted group recovered to 66%. By 2 weeks, the wounds were now 15% with bioprinting therapy, while control wounds were 40%. At both time points, bioprinting improved wound closure of full-thickness wounds.
Closer analysis of retrieved skin constructs revealed high cellularity of 1-week postprinting, forming an immature epidermal barrier. By weeks 3 and 6 after cell printing, defined epidermis was observed as well as an organized dermis consisting of aligned collagen fibers. In contrast, matrix-printed wounds had poor cellularity. Only after week 6 were more defined epidermis and dermis features observed. All groups closed wounds by the end of week 6. This was further repeated in porcine full-thickness wound models. All wounds generated granulation tissue by 1 week, with the greatest levels of granulation observed in allogeneic and autologous cell-treated wounds by week 2. Importantly, bioprinted autologous cells led to the fastest reduction in wound size, smallest amount of contraction, and most rapid re-epithelialization. Compared to the other groups, bioprinted autologous cells improved wound re-epithelialization by 4–5 weeks. Rete peg epithelial projections are a mature feature of epithelial regeneration. While bioprinted autologous cells showed Rete peg generation by 4 weeks, allogeneic and matrix-treated wounds did not show any mature epithelialization features until week 6. Untreated wounds only presented immature epithelium at week 6.
A number of mature skin tissue features were observed, such as early formation of papillary and reticular dermis. Woven dermal collagen and regularly distributed vasculature were similarly observed, with the tissue structure appearing fully formed at 8 weeks. Longer periods of inflammation often result in greater scarring. Even as early as 2 weeks, autologous bioprinted wounds had suppressed inflammation, with only mild signs of acute inflammation. On the other hand, the other groups experienced prominent inflammation. By the eighth week, acute inflammation was mostly absent or very mild. Further histology analysis revealed a higher density of immature CD31+ blood vessels from bioprinted autologous cells by week 4. By week 8, smaller vessels decreased and were replaced by larger, more mature vessels found throughout the dermis tissue. The other groups (allogeneic, matrix-treated) were comparatively delayed, showing reduced numbers of mature blood vessels. In untreated and matrix-treated groups, the dermis was shown to have densely packed nuclei (inflammatory cells) with disrupted collagen fibers. Allogeneic and autologous cell groups had more diffuse nuclei and better organized collagen fibers. At week 8, cell nuclei numbers were significantly reduced, although patches of concentrated cells were seen more prominently in all groups except for autologous cell treatment. The lower levels of immune cells in autologous cell treatment corresponded with larger and better organized collagen fibers. To gauge the extent of hypertrophic scar formation, the presence of myofibroblasts was analyzed by staining for α-smooth muscle actin (αSMA). The trend suggested that autologous and allogeneic cells generated moderate amounts of staining at 4 weeks, whereas this was mostly absent in untreated and matrix-treated wounds.
At week 8, high numbers of staining were seen in the untreated and matrix groups, whereas these appeared to migrate to the proximity of the blood vessels in autologous and allogeneic cell-treated wounds. Ki67—representing proliferating cells—was observed to a greater extent at week 8 for autologous cell-treated wounds compared to the other groups. Compared to the commercially available Fibrijet based on cell spraying, granulation tissue was generated at similar time points as well as wound closure, suggesting it was at least equivalent in regard to wound healing performance. Histology analysis suggests that cell-sprayed wounds did not give rise to epidermis formation, whereas bioprinted wounds had signs of tissue maturity, including the appearance of papillary dermis. These were less apparent in cell-sprayed wounds at earlier stages, suggesting that bioprinting improved early tissue maturity. This study demonstrates the great utility of the bioprinting system for customized delivery of cells and biomaterials to accelerate severe wound healing. In addition, it is a highly flexible system that enables inclusion of novel biomaterials, melanocytes, adipose cells, hair follicles, etc. Following such developments, bioprinting may be expanded to applications such as burns, ulcers, and other deep tissue injury. (Albanna, M.; et al. Sci. Rep.
Anti–USAG-1 Therapy for Tooth Regeneration through Enhanced BMP Signaling
Tooth morphogenesis is regulated by signal transduction involving epithelium and mesenchyme interactions. Although the number of teeth in a given species is usually controlled, it may vary in <1% of individuals. In mouse genetic models, loss of Usag-1 gives rise to the growth of supernumerary teeth (increased number of teeth). Usag-1 has been shown to antagonize BMP and Wnt signaling—two critical pathways for tooth development. Supplementing BMP7 to heterozygous USAG1 mice gave rise to supernumerary teeth. An alternative approach involves rescuing arrested teeth. It has been established that Runx2-deficient mice were rescued by depleting USAG1. This suggests that local inhibition of USAG1 may assist tooth development. In an animal model that impacts tooth generation (knocking out Msx1 and EDA (ectodysplasin A) genes), further knock out of USAG1 promoted growth of the whole tooth. To investigate if a similar function was observed using soluble neutralizing antibodies, the authors obtained five monoclonal antibody candidates. These antibody candidates are directly bound to BMP and the Wnt coreceptor LRP5/6. These USAG1 neutralizing antibodies were then systematically administered to EDA1 pregnant mice prone to agenesis.
Compared to control mice, USAG1 neutralizing antibodies reversed hypodontia to a high extent in a dose-dependent manner. USAG-1 antibodies generated supernumerary teeth in the maxillary incisor, mandibular incisor, or molars of wild-type mice. These antibodies neutralized BMP signaling, suggesting its crucial role in tooth morphogenesis. Importantly, a single systemic administration gives rise to a whole new tooth. Using antibody binding studies, it was identified that stoichiometric binding of USAG1 was observed with the E1-E2 domain of the Wnt coreceptor LRP6. With number 16 USAG1 antibody (Ab), near complete inhibition was observed, further demonstrating the Wnt-modulating ability of USAG1. Through these observations, the inhibitory effects of USAG1 on BMP rather than Wnt were more effective in tooth morphogenesis. Finally, in a nonrodent ferret model, the effects of USAG1 neutralization on BMP signaling were attempted. This led to supernumerary tooth formation in the maxillary incisor position. This tooth had a similar shape to a typical permanent incisor.
Currently, tissue engineering methods have been applied for tooth regeneration, although costs and safety issues are among existing bottlenecks. This is the first demonstration of a molecular therapy approach targeting USAG1, which may help with a range of congenital tooth agenesis disorders as well as the induction of supernumerary teeth. (Murashima-Suginami, A.; et al. Sci. Adv.
Formation of Contractile 3D Bovine Muscle Tissue for Construction of Millimeter-Thick Cultured Steak
Global population increase and increase in meat consumption are creating sustainability issues in regard to ethics and environmental pollution. Thus, sustainable meat production has been proposed to circumvent these issues. Cultured meat is one promising approach produced using tissue culture of animal cells. Crucially, small quantities of cells are obtained without the slaughter of livestock, even using less land and water resources. Using methods based on 3D tissue engineering methods, hydrogels have been cultured with myocyte cells to create tissues suitable for use as minced meat. To obtain realistic cultured steak meat, porous gelatin or porous soy proteins have been used as scaffold biomaterials. Unfortunately, these result in low myotube density and loss of contractility. The authors develop a method to construct 3D bovine muscle tissue comprising unidirectionally aligned myotubes. A method is introduced to fabricate millimeter-thick bovine muscle tissues with highly aligned myotubes using myoblast–hydrogel modules with striped structures.
To obtain contractile bovine muscle tissue, bovine myocyte-laden hydrogels are cultured on a device with pillars. Bovine myocytes were used for tissue construction by culturing cells obtained from commercial fresh beef. Of these, >85% were myocytes (myoblasts and satellite cells). After 14 days, fiber-shaped bovine muscle tissue of ~295 um was generated and immobilized, giving rise to gaps between anchors of 7 mm. To compare cultured bovine muscle tissue, the hydrogels collagen and fibrin+Matrigel were used for fabrication. Electrical stimulation was also applied to both tissue types. For fibrin–Matrigel with electrical stimulation, 100% of muscle tissue contracted (N = 9), whereas without electrical stimulation, 56% muscle tissue contracted. In contrast, collagen-cultured myoblasts, only 33% of electrically stimulated tissue contracted and no muscle tissue without stimulation contracted. The contraction in the fibrin–Matrigel muscle tissue was comparatively larger than the collagen muscle tissue.
To gauge the maturity of the muscle tissue, α-actinin immunostaining was performed. Of all the different groups, the fibrin–Matrigel group gave rise to the highest rate of myotubes with stripe-patterned α-actinin (50%). These fibers were also aligned along the long axis of the tissue. To construct highly aligned, millimeter-thick bovine muscle tissue, 40 myocyte-laden collagen modules were immobilized to pillars arranged parallel at 0.3 mm intervals. These fused into one tissue after 7 days of culture, giving rise to a tissue of 8 mm × 10 mm × 7 mm. Hematoxylin and eosin (H&E) staining showed that the myotubes were uniformly distributed and well integrated into a single tissue. In previous studies where intervals of 1 mm were used on rat myoblasts, integrated muscle tissue was not formed. Further analysis also suggests that the cultured muscle tissue was highly aligned without enucleated cells—indicating the absence of necrosis within the tissue. After releasing the constructed tissue from the immobilizing pillars, the striped structures were still maintained. The addition of red food coloring agents gave rise to realistic-looking meat tissue. The cultured meat was then cooked (70 °C warm bath, 1 h) and compared with beef tenderloin meat. The beef tenderloin weight was reduced by 40%, while the cultured meat lost 90% of its weight. Myocyte-free collagen structures completely melted, suggesting that myocyte–collagen interactions altered thermal responsiveness properties.
The breaking force of the cooked tissue also increased with time, where day 14 samples were closer to commercial tenderloin compared to less mature samples at day 4. A further advantage was the sterility of the cultured meat that had <5 CFU/g, under the detection limit. By contrast, the commercial beef tenderloin had 1.7 × 105 CFU/g, demonstrating sterility as a further advantage. The cultured meat technology demonstrated in this study is promising to generate sustainable meat substitutes. Food characteristics like taste and nutrient composition can further be adjusted through culture conditions or controlled by additives such as heme proteins and vitamins. Expansion into a cultured meat product from existing meat products (as opposed to biopsies from living subjects) demonstrates its great sustainability, as it does not interfere with commercial meat supply chains. (Furuhashi, M.; et al. NPJ Sci. Food
Therapeutics and Nanotechnology
SARS-CoV-2 MPro Inhibitors with Antiviral Activity in a Transgenic Mouse Model
In addition to vaccines for SARS-CoV-2, antiviral drugs may also contribute in combating the disease. For SARS-CoV-2 to replicate, the main protease (MPro) is required. It also acts in a unique manner, unlike any known human protease. This makes it an attractive biological target for drug treatment. While there has been some interest in MPro inhibitors, such as by Pfizer in phase I trials, no animal data have been reported. Antiviral drugs were developed based on approved antivirals against hepatitis C virus infection, boceprevir and telaprevir. The active site of MPro consists of four sites that accommodate four fragments. The fragment P1 was fixed as the optimal fragment P2 from either boceprevir or telaprevir while allowing P3 to change. A total of 32 candidates with various P3 fragments were generated. Using a fluorescence resonance energy transfer (FRET) assay, all 32 compounds showed potent inhibitory activity (IC50 7.6–748.5 nM). Differential scanning fluorimetry (DSF) validated binding between the compounds and the SARS-CoV-2 MPro target. The 20 compounds with IC50 <50 nM examined cytotoxicity and antiviral activity. None of the compounds showed cytotoxicity (Cell Counting Kit-8 [CCK8] assay) (C50 >500 µM) in Vero E6, HPAEpiC, LO2, BEAS-2B, A549, and Huh7 cells.
The compounds were then tested for their ability to retain cell viability during SARS-CoV-2 infection. All compounds had a dose-dependent effect on cells, with 50% effective concentration (EC50) values ranging from 0.53 to 30.49 µM. PCR assays reveal that the compounds inhibited SARS-CoV-2 replication in HPAEpiC cells, with EC50 values between 0.3 and 7.3 nM. These EC50 values were corroborated using CCK8 and PCR assays. To gauge suitability, PK (pharmacokinetics) experiments were performed in Sprague–Dawley (SD) rats. Compounds MI-09 and MI-30 demonstrated good PK properties with oral bioavailabilities of 11.2% and 14.6%, respectively—suggesting their good potential. In toxicity evaluations, no subjects died after high amounts were administered (intravenously, intraperitoneally, or orally) twice daily for 7 consecutive days. The compounds were then tested in human angiotensin-converting enzyme 2 (hACE2) transgenic mouse models susceptible to SARS-CoV-2. In a moderate virus challenge, the compounds administered intraperitoneally and orally reduced viral loads compared to control groups at 5 days after infection. Histopathology showed moderate alveolar septal thickening and inflammatory cell infiltration 3 days after infection, whereas the compounds reduced alveolar septal thickening and mild cell infiltration. MI-09 and MI-30 further reduced IFN-β and CXCL10 levels. Fewer neutrophils and macrophages were also observed following compound treatment, suggesting immune cell infiltration was inhibited. Thus, the antiviral compounds administered intraperitoneally or orally reduced SARS-CoV-2 replication, ameliorating the induced lesions. This suggests a critical step toward developing oral drugs against SARS-CoV-2. (Qiao, J.; et al. Science
Erythrocyte Leveraged Chemotherapy (ELeCt): Nanoparticle Assembly on Erythrocyte Surface to Combat Lung Metastasis
Despite improvements to cancer therapy and diagnosis, lungs are still the most common metastasis site due to their high vascular density. Systemic chemotherapy is the standard of care for lung metastasis. Unfortunately, its efficacy suffers from ineffective targeting and poor drug accumulation at the disease site. Traditional nanoparticles are often impeded from accumulating at the desired site, while the use of tissue-specific ligands has only resulted in modest improvements. To achieve effective therapy, the authors propose erythrocyte leveraged chemotherapy-biodegradable drug NPs assembled on erythrocyte surfaces. The erythrocytes dislodge NP particles that carry large amounts of chemotherapeutic doxorubicin (DOX) in response to the high shear stress in narrow lung capillaries. DOX was encapsulated in biodegradable polymeric (poly[lactic-co-glycolic acid] [PLGA]) NPs. These had an approximate diameter of ~136 nm, with a loading capacity of 197 mg/g. These PLGA particles exhibit a burst release followed by a sustained release profile, with most of the drugs released in the first 6 h. In B16F10-Luc melanoma cells, DOX-loaded PLGA NPs were internalized quickly and efficiently after 20 min. Concurrently, DOX fluorescence signal within the cell nucleus suggested effective intracellular delivery and release of drug. Their median inhibitory concentration (IC50) suggests similar cell killing efficacy to the free drug. Mouse erythrocytes were incubated with NPs at different ratios (50:1 to 800:1). Between 81.6% and >96% of cells were bound with NPs. The cells were able to carry dosages as high as 294.1 μg per 3 × 108 erythrocytes.
Upon further analysis, the murine erythrocytes maintained their characteristic biconconcave shape after binding without loss of integrity. Human erythrocytes also successfully assembled with NPs at high doses (209.1 μg per 1.5 × 108 erythrocytes). Compared to free drug and nanoparticles, the cell-bound NPs extended the circulation time of DOX. The shear-dependent phenomena of release PLGA NPs were then tested in vitro. Mouse lung capillaries are as small as 5 µm, narrowing down as small as 1 µm. With shear stress going from as low as 1 Pa to 6 Pa, NP detachment was shear dependent for both mice and human erythrocytes. At 6 Pa, 76% of drug-loaded NPs were found to be sheared off. In a lung metastasis model bearing B16F10-Luc melanoma cells, erythrocyte hitchhiking drug delivery delivered 16.6-fold higher drug content compared to free NPs 20 min after administration. Six hours later, 8.7-fold higher drug concentrations were observed. Compared to free drug injection, erythrocyte hitchhiking was 6.9-fold higher. Further investigation also showed greater amounts of drug NPs in lung sections as well as NPs found deep within tumor metastasis nodules. This demonstrated how the ELeCt technology precisely delivered chemotherapy to the desired site.
In the lung metastasis model, luciferase-tagged cell burden was measured by the change in bioluminescence signal. One day after injecting the melanoma cells, four doses were administered every other day. ELeCt gave rise to improved inhibition (lower bioluminescence signal) compared to free drug or NPs alone. Whereas free drug and drug NPs led to 17.2- and 1.8-fold lower signal compared to control subjects, ELeCt reduced this by 204.8-fold. Kaplan–Meier survival analysis further showed that free drug and NPs only improved survival slightly (29–32 days’ median survival), while ELeCt-treated mice extended this to 61 days. Moreover, free drug treatment was toxic, resulting in a decline in the subject’s body weight during treatment. This was avoided using ELeCt and NP treatment. In a further study where therapy was administered 7 days after cell inoculation (to mimic late-stage cancer), drug NPs did not inhibit lung metastasis meaningfully. ELeCt, however, managed to slow down metastasis. On day 16 postinoculation, ELeCt performed 2.4-fold better efficacy-inhibiting metastasis. Histology also showed a reduced number (2.3-fold) of surface nodules. Once more, only ELeCt technology extended the median survival time from 28.5 to 37 days, whereas drug NPs were not beneficial.
Finally, the authors showed six other common therapeutics and multicombinations—camptothecin, paclitaxel, docetaxel, 5-fluorouracil, gemcitabine, methotrexate. This demonstrated how ELeCt is a versatile platform to deliver drugs for metastatic cancer. In summary, the ELeCt platform is highly versatile, enabling potential treatment of later-stage cancer metastasis with good translational potential. (Zhao, Z., et al.; Sci. Adv.
Elexacaftor–Tezacaftor–Ivacaftor for Cystic Fibrosis with a Single Phe508del Allele
Cystic fibrosis is a lethal, inherited, autosomal recessive disorder affecting ~80,000 people globally, caused by mutations that reduce cystic fibrosis transmembrane conductance function. The gene is involved in transporting ions across epithelial surfaces, while most (90%) who suffer from it suffer from the Phe508del CFTR mutation. Phe508del causes defective intracellular processing, trafficking, and reduced stability, reducing the availability of CTFR on cell surfaces. To restore full CTFR function, such molecular mutations need to be addressed. Small-molecule drugs have been used to increase cell surface expression of CTFR by improving its trafficking and processing. Combinations of corrector drugs (e.g., lumacaftor or tezacaftor) with the potentiator drug ivacaftor improve lung function. In certain mutations, these drug combinations are ineffective due to the complete absence of protein production or lack of responsiveness to the drugs. A triple-drug combination consisting of the next-generation corrector elexacaftor, the corrector tezacaftor, and the potentiator ivacaftor was proposed to restore the function of nonresponsive cases.
To validate the efficacy in the target population, a randomized, placebo-controlled, phase 3 trial of elexacaftor–tezacaftor–ivacaftor was conducted in patients with the minimal-function mutations. The trial was conducted at 115 sites in 13 countries within a year, involving 405 patients, with 98% of them adhering. The forced expiratory volume in 1 s (FEV1) had a 13.8-point difference relative to placebo (p < 0.001). A sustained improvement was observed through 24 weeks with a separation observed between the two groups. The triple-drug combination also reduced the rate of pulmonary exacerbations compared to the placebo group. Sweat chloride concentrations also improved by ~41.8 mmol/L compared to placebo, sustaining the improvement over 24 weeks. The CFQ-R domain score also similarly improved.
In regard to safety, adverse events in 10% of patients in the respective groups were consistent with cystic fibrosis complications. Serious adverse events occurred in 13.9% of the triple-drug group, while in 20.9% of the placebo group. No deaths occurred in either group, with 1% of patients who discontinued due to adverse events like rash and preexisting organ conditions. Elevated aminotransferase levels were also observed in 10.9% of the triple-drug group compared to 4% in the placebo group. These levels did not exceed three times the upper limit of the normal range. Rash also occurred to a higher extent in female than male patients. Thus, the trial showed that the triple-drug combination for CTFR modulator therapy was suitable for patients 12 years or older heterozygous for the Phe508del CFTR mutation and a minimal-function mutation. The biomarkers observed did not constitute a safety problem. This suggests that the triple-drug combination may address the underlying issue for the majority of cystic fibrosis patients. (Middleton, P. G.; et al. N. Engl. J. Med.
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