Gene Editing/Gene Therapies: CHALLENGE TO DEVELOP GMP COMPLIANT MANUFACTURING PROCESS FOR COXSACKIE VIRUS TYPE B3

Background & Aim: Gene therapies has become recognized for its remarkable clinical benefits in a variety of medical applications, in particular recent approval of an Ad vector-based COVID-19 vaccines have attracted recent global attention. Here, we present key considerations for GMP compliant process development for Coxsackie virus type B3 (CVB3), an oncolytic virus designed for clinical trial in triple-negative breast cancer. Methods, Results & Conclusion(s): CVB3 is a non-enveloped, linear single-strand RNA virus with a size of approximately 27-33 um in diameter. From the initial type using the zonal rotor centrifuge to the advanced type using the tangential flow filtration system and ion chromatograph, we considered the points of the design concept in constructing the manufacturing process. The final design system is constructed as a closed and single-use manufacturing system in which all processes from upstream large-scale cell culture to downstream target purification and concentration steps. In brief, HEK293 cell suspension extended in 3L serum-free medium infected with CVB3, up to 3.6 times 10 to 7 of TCID50 /mL before going to downstream steps, made total 150 mL of final products as 8.43 times 10 to 7 of TCID50/mL concentration. Although further quality control challenges remain that is removal of product-related impurities such as human cellular proteins and residual DNA/RNA to increase virus purity, this concept is effectively applicable even for other types of viruses as GMP manufacturing processes, and would be also important for technology transfer to future commercial production.Copyright © 2023 International Society for Cell & Gene Therapy

Virus-Specific T Cells From Cryopreserved Blood During an Emergent Virus Outbreak for a Potential Off-the-Shelf Therapy.

During the first outbreak of an emergent virus, methods need to be developed to rapidly establish suitable therapies for patients with high risk of severe disease caused by the pathogen. Considering the importance of the T-cell response in controlling viral infections, adoptive cell therapy with virus-specific T cells has been used as a safe and effective antiviral prophylaxis and treatment for immunocompromised patients. The main objective of this study was to establish an effective and safe method to cryostore whole blood as starting material and to adapt a T-cell activation and expansion protocol to generate an off-the-shelf antiviral therapeutic option. Additionally, we studied how memory T-cell phenotype, clonality based on T-cell receptor, and antigen specificity could condition characteristics of the final expanded T-cell product. Twenty-nine healthy blood donors were selected from a database of convalescent plasma donors with a confirmed history of SARS-CoV-2 infection. Blood was processed using a fully automated, clinical-grade, and 2-step closed system. Eight cryopreserved bags were advanced to the second phase of the protocol to obtain purified mononucleated cells. We adapted the T-cell activation and expansion protocol, without specialized antigen-presenting cells or presenting molecular structures, in a G-Rex culture system with IL-2, IL-7, and IL-15 cytokine stimulation. The adapted protocol successfully activated and expanded virus-specific T cells to generate a T-cell therapeutic product. We observed no major impact of post-symptom onset time of donation on the initial memory T-cell phenotype or clonotypes resulting in minor differences in the final expanded T-cell product. We showed that antigen competition in the expansion of T-cell clones affected the T-cell clonality based on the T-cell receptor ß repertoire. We demonstrated that good manufacturing practice of blood preprocessing and cryopreserving is a successful procedure to obtain an initial cell source able to activate and expand without a specialized antigen-presenting agent. Our 2-step blood processing allowed recruitment of the cell donors independently of the expansion cell protocol timing, facilitating donor, staff, and facility needs. Moreover, the resulting virus-specific T cells could be also banked for further use, notably maintaining viability and antigen specificity after cryopreservation.

Cell Therapy: cGMP Facilities and Manufacturing: Second Edition

This new edition presents a fully-updated and expanded look at current Good Manufacturing Practice (cGMP) for cell therapy products. It provides a complete discussion of facility design and operation including details specific to cord blood banking, cell processing, vector production and qualification of a new facility. Several chapters cover facility infrastructure including cleaning and maintenance, vendor qualification, writing a Standard Operating Procedure, staff training, and process validation. The detailed and invaluable product information covers topics like labelling, release and administration, transportation and shipment, et al. Further chapters cover relevant topics like writing and maintaining investigational new drug applications, support opportunities in North America and the European Union, commercial cell processing and quality testing services, and financial considerations for academic GMP facilities. A chapter on future directions rounds out Cell Therapy: cGMP Facilities and Manufacturing making it essential reading for any cell therapy professional involved in the development, use, or management of this type of facility. © Springer Nature Switzerland AG 2009, 2022, Corrected Publication 2022.

QGIS Application for Spatial and Temporal Visualisation of Data for Food Safety Audits in Malaysia

Introduction: Advancement in digital technology opens new doors for food safety auditors when it comes to performing food safety audits. Surge of Covid cases since year 2020 has seen an unprecedented switch to remote auditing by the Food Safety and Quality Programme under the arm of Ministry of Health in Malaysia. Methods: This paper presents the use of QGIS, an open-source cross-platform for geographic information system (GIS) to store, manage and visualise 2 types of data, i.e. real time data collected via a mobile device using QField, an open-source mobile application and also fixed data retrieved from existing database. New data from obtained from field sampling and surveillance presents updated information for food safety auditing and enforcement purposes. A total of 4972 datasets were obtained from the Ministry of Health's Food Safety and Quality Division database on food factories from all 13 states and 3 federal territories in Malaysia. These datasets were transformed and stored into QGIS point layer for performing data classification analysis on clustering of HACCP, GMP and MeSTI certifications. Results: The Penang state has the most HACCP certified companies in fish and fish product category, Selangor is the highest for confectionery industry and Sabah for food services. The general output of mobile GIS provides a big picture of distribution of food safety certifications in Malaysia while more specific adoption of QField can assist in effective field work planning for enforcement officers and auditors leading to cost calculation via information on location, distance and time. Conclusion: QGIS application for spatial and temporal visualisation of data benefits the food safety auditing in Malaysia. © 2023 UPM Press. All rights reserved.

Development and Validation of an Innovative Analytical Approach for the Quantitation of Tris(Hydroxymethyl)Aminomethane (TRIS) in Pharmaceutical Formulations by Liquid Chromatography Tandem Mass Spectrometry.

A novel COVID-19 vaccine (BriLife®) has been developed by the Israel Institute for Biological Research (IIBR) to prevent the spread of the SARS-CoV-2 virus throughout the population in Israel. One of the components in the vaccine formulation is tris(hydroxymethyl)aminomethane (tromethamine, TRIS), a buffering agent. TRIS is a commonly used excipient in various approved parenteral medicinal products, including the mRNA COVID-19 vaccines produced by Pfizer/BioNtech and Moderna. TRIS is a hydrophilic basic compound that does not contain any chromophores/fluorophores and hence cannot be retained and detected by reverse-phase liquid chromatography (RPLC)-ultraviolet (UV)/fluorescence methods. Among the few extant methods for TRIS determination, all exhibit a lack of selectivity and/or sensitivity and require laborious sample treatment. In this study, LC−mass spectrometry (MS) with its inherent selectivity and sensitivity in the multiple reaction monitoring (MRM) mode was utilized, for the first time, as an alternative method for TRIS quantitation. Extensive validation of the developed method demonstrated suitable specificity, linearity, precision, accuracy and robustness over the investigated concentration range (1.2−4.8 mg/mL). Specifically, the R2 of the standard curve was >0.999, the recovery was >92%, and the coefficient of variance (%CV) was <12% and <6% for repeatability and intermediate precision, respectively. Moreover, the method was validated in accordance with strict Good Manufacturing Practice (GMP) guidelines. The developed method provides valuable tools that pharmaceutical companies can use for TRIS quantitation in vaccines and other pharmaceutical products.

THE FUTURE OF REMOTE PHARMA GMP AUDITING

The COVID-19 pandemic saw pharma industry auditing move increasingly online and in place of on-site visits, we became familiar with virtual checks and inspections. Some auditors now say that virtual auditing could become a permanent solution, but deeper analysis shows that clients are not in favour of this option, says Alasdair Leckie, Operations Manager at Rephine Ltd. © 2022 TeknoScienze. All rights reserved.

Propagation of SARS-CoV-2 in a Closed Cell Culture Device: Potential GMP Compatible Production Platform for Live-Attenuated Vaccine Candidates under BSL-3 Conditions?

Live-attenuated SARS-CoV-2 vaccines present themselves as a promising approach for the induction of broad mucosal immunity. However, for initial safety assessment in clinical trials, virus production requires conditions meeting Good Manufacturing Practice (GMP) standards while maintaining biosafety level 3 (BSL-3) requirements. Since facilities providing the necessary complex ventilation systems to meet both requirements are rare, we here describe a possibility to reproducibly propagate SARS-CoV-2 in the automated, closed cell culture device CliniMACS Prodigy® in a common BSL-3 laboratory. In this proof-of-concept study, we observed an approximately 300-fold amplification of SARS-CoV-2 under serum-free conditions with high lot-to-lot consistency in the infectious titers obtained. With the possibility to increase production capacity to up to 3000 doses per run, this study outlines a potential fast-track approach for the production of live-attenuated vaccine candidates based on highly pathogenic viruses under GMP-like conditions that may contribute to pandemic preparedness.

QGIS Application for Spatial and Temporal Visualisation of Data for Food Safety Audits in Malaysia

Introduction: Advancement in digital technology opens new doors for food safety auditors when it comes to performing food safety audits. Surge of Covid cases since year 2020 has seen an unprecedented switch to remote auditing by the Food Safety and Quality Programme under the arm of Ministry of Health in Malaysia. Methods: This paper presents the use of QGIS, an open-source cross-platform for geographic information system (GIS) to store, manage and visualise 2 types of data, i.e. real time data collected via a mobile device using QField, an open-source mobile application and also fixed data retrieved from existing database. New data from obtained from field sampling and surveillance presents updated information for food safety auditing and enforcement purposes. A total of 4972 datasets were obtained from the Ministry of Health's Food Safety and Quality Division database on food factories from all 13 states and 3 federal territories in Malaysia. These datasets were transformed and stored into QGIS point layer for performing data classification analysis on clustering of HACCP, GMP and MeSTI certifications. Results: The Penang state has the most HACCP certified companies in fish and fish product category, Selangor is the highest for confectionery industry and Sabah for food services. The general output of mobile GIS provides a big picture of distribution of food safety certifications in Malaysia while more specific adoption of QField can assist in effective field work planning for enforcement officers and auditors leading to cost calculation via information on location, distance and time. Conclusion: QGIS application for spatial and temporal visualisation of data benefits the food safety auditing in Malaysia. [ FROM AUTHOR]

Basic good manufacturing practices (GMP), special conditions and inspection processes in the Covid19 (SARS-CoV-2) pandemic

The COVID-19 pandemic has severely impacted the world economy, businesses and global health. Exceptional situations are evident in the food, cosmetics and pharmaceutical production in the supply chain, where many businesses have been shut down completely following government restrictions. In the pandemic process, it has become much more important to ensure hygienic products and employee safety and to deliver quality and healthy products to the consumer. In extraordinary conditions, it is necessary to provide additional contributions to the principles of good manufacturing practices. With the additional measures to be taken during the epidemic period, a healthy and safe operation can be brought to production. In addition, the assurance system can continue to function despite restrictions and failing audits. In addition to the updated guides, the development of roadmaps specific to the epidemic period for production may provide the advantage of being ready for problems. Copyright © 2022 Marmara University Press.

Current GMP standards for the production of vaccines and antibodies: An overview.

The manufacture of pharmaceutical products made under good manufacturing practices (GMP) must comply with the guidelines of national regulatory bodies based on international or regional compendia. The existence of this type of regulation allows pharmaceutical laboratories to count on the standardization of high-quality production processes, obtaining a safe product for human use, with a positive impact on public health. In addition, the COVID-19 pandemic highlights the importance of having more and better-distributed manufacturing plants, emphasizing regions such as Latin America. This review shows the most important GMP standards in the world and, in particular, their relevance in the production of vaccines and antibodies.

COVID-19 Molecular Pathophysiology: Acetylation of Repurposing Drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces immune-mediated type 1 interferon (IFN-1) production, the pathophysiology of which involves sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) tetramerization and the cytosolic DNA sensor cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. As a result, type I interferonopathies are exacerbated. Aspirin inhibits cGAS-mediated signaling through cGAS acetylation. Acetylation contributes to cGAS activity control and activates IFN-1 production and nuclear factor-κB (NF-κB) signaling via STING. Aspirin and dapsone inhibit the activation of both IFN-1 and NF-κB by targeting cGAS. We define these as anticatalytic mechanisms. It is necessary to alleviate the pathologic course and take the lag time of the odds of achieving viral clearance by day 7 to coordinate innate or adaptive immune cell reactions.

Chemistry Manufacturing and Controls Development, Industry Reflections on Manufacture, and Supply of Pandemic Therapies and Vaccines.

This publication provides some industry reflections on experiences from the Chemistry, Manufacturing, and Controls (CMC) development and manufacture and supply of vaccines and therapies in response to the COVID-19 pandemic. It integrates these experiences with the outcomes from the collaborative work between industry and regulators in recent years on innovative science- and risk-based CMC strategies to the development of new, high-quality products for unmet medical needs. The challenges for rapid development are discussed and various approaches to facilitate accelerated development and global supply are collated for consideration. Relevant regulatory aspects are reviewed, including the role of Emergency Use/Conditional Marketing Authorizations, the dialogue between sponsors and agencies to facilitate early decision-making and alignment, and the value of improving reliance/collaborative assessment and increased collaboration between regulatory authorities to reduce differences in global regulatory requirements. Five areas are highlighted for particular consideration in the implementation of strategies for the quality-related aspects of accelerated development and supply: (1) the substantial need to advance reliance or collaborative assessment; (2) the need for early decision making and streamlined engagement between industry and regulatory authorities on CMC matters; (3) the need to further facilitate 'post-approval' changes; (4) fully exploiting prior and platform knowledge; and (5) review and potential revision of legal frameworks. The recommendations in this publication are intended to contribute to the discussion on approaches that can result in earlier and greater access to high-quality pandemic vaccines and therapies for patients worldwide but could also be useful in general for innovative medicines addressing unmet medical needs.

Recent Status of Development of Hb-based Blood Substitute With Pharmacologic Properties of ATP, Adenosine and Reduced Glutathione

Background: SARS-CoV-2 pandemic brought new attention to blood substitutes as a potential remedy for treatment of COVID-19 associated hypoxemia and more importantly to provide relief for sagging blood banks. In fact the American Red Cross declared a national blood crisis, the worst in decades. Although commercialization attempts of 1st generation blood substitutes have been unsuccessful due to toxicity and/or poor efficacy problems, the artificial oxygen carriers' field is rapidly expanding again creating more innovative products. To attenuate the adverse effects of blood substitutes caused by intrinsic toxicity of Hb we implemented a novel concept of 'pharmacologic cross-linking' and formulated a safe and effective blood substitute - HemoTech. Methods: HemoTech consists of pure bovine Hb cross-linked intramolecularly with o-ATP and intermolecularly with oadenosine and conjugated with reduced glutathione (GSH). HemoTech cGMP manufacturing includes a novel, validated orthogonal technology platform for effective clearance of endotoxin, prions and non-enveloped and enveloped viruses. Regulatory mandated requirements have been met including CMC and GLP non-clinical pharmacology, toxicology, genotoxicity and efficacy studies. HemoTech's clinical 'proof-of-concept' was performed in children with sickle cell anemia. Results: HemoTech is formulated as a 6.5 g/dL modified Hb solution in oxy- or carbon monoxy-form, enriched with electrolytes and mannitol. The 'pharmacologic cross-linking' does not interfere with Hb respiratory function, but eradicates Hb's intrinsic toxicity and provides Hb molecules with new medicinal properties of ATP, adenosine and GSH. The results of preclinical and clinical studies indicate that HemoTech is non-toxic and works as a physiological oxygen carrier with prolonged intravascular persistence, is vasodilatory and can reduce vasoconstriction that follows hemorrhage, has antioxidant and anti-inflammatory activities, and erythropoietic properties. Conclusion: HemoTech promises to be an effective blood substitute for various clinical indications.

Public Cord Blood Banks as a source of starting material for clinical grade HLA-homozygous induced pluripotent stem cells.

BACKGROUND: The increasing number of clinical trials for induced pluripotent stem cell (iPSC)-derived cell therapy products makes the production on clinical grade iPSC more and more relevant and necessary. Cord blood banks are an ideal source of young, HLA-typed and virus screened starting material to produce HLA-homozygous iPSC lines for wide immune-compatibility allogenic cell therapy approaches. The production of such clinical grade iPSC lines (haplolines) involves particular attention to all steps since donor informed consent, cell procurement and a GMP-compliant cell isolation process. METHODS: Homozygous cord blood units were identified and quality verified before recontacting donors for informed consent. CD34+ cells were purified from the mononuclear fraction isolated in a cell processor, by magnetic microbeads labelling and separation columns. RESULTS: We obtained a median recovery of 20.0% of the collected pre-freezing CD34+, with a final product median viability of 99.1% and median purity of 83.5% of the post-thawed purified CD34+ population. CONCLUSIONS: Here we describe our own experience, from unit selection and donor reconsenting, in generating a CD34+ cell product as a starting material to produce HLA-homozygous iPSC following a cost-effective and clinical grade-compliant procedure. These CD34+ cells are the basis for the Spanish bank of haplolines envisioned to serve as a source of cell products for clinical research and therapy.

Quantitative PBMC ADCC bioassay and ADCC reporter bioassays for immunotherapy and SARS-CoV-2 monoclonal antibody development

Fc effector function is one of the main mechanisms of action (MoA) for therapeutic monoclonal antibodies (mAbs). Quantitative measurement of antibody-dependent cellular cytotoxicity (ADCC) is critically required for understanding the Fc function in mAb drug development. Despite the increasing interest and clinical success of the mAb therapeutic, it has been highly challenging to measure their ADCC activity in a reproducible and quantitative manner due to the lack of consistency in current methods that are based on primary PBMCs or NK cells and use tedious assay procedures. To improve ADCC assay precision so they can be validated as potency assay in cGMP laboratories, we developed reporter based ADCC bioassays using engineered effector cell line stably expressing a luciferase reporter and FcγRIIIa (V or F variant) to replace primary PBMC to overcome the assay variation. The ADCC reporter bioassays have been validated according to ICH guidelines by many laboratories and are demonstrated to be suitable for product release and stability studies in a quality-controlled environment. For early research and antibody characterization, we developed an improved PBMC ADCC assay using ADCC-prequalified PBMCs and engineered HiBiT target cells so they can measure the target specific lysis in ADCC. The PBMCs used in the study are isolated from prescreened blood donors and QC tested in ADCC assay. When HiBiT target cells are incubated with an antibody and PBMCs, HiBiT are released to the culture medium where it binds to LgBiT in the detection reagent to form a functional NanoBiT luciferase to generate luminescence signal. This new PBMC ADCC bioassay is simple, homogenous, highly sensitive, and gives a robust assay window. We demonstrate that it can quantitatively measure the potency for mAb drugs in cancer immunotherapy (e.g., rituximab, trastuzumab), and for anti-SARS-CoV-2 spike antibodies in antiviral drug development. Additionally, it shows antibody potency comparable with the ADCC reporter bioassay. In summary, the new PBMC ADCC bioassay using HiBiT target cells can be a valuable tool for early antibody discovery and characterization and also for method bridging study with ADCC reporter bioassay.

IMPAIRED BETA-2ADRENERGIC ENDOTHELIUM-DEPENDENT VASODILATION IS REVERSED BY PHOSPHODIESTERASE INHIBITION IN PATIENTS PREVIOUSLY HOSPITALIZED WITH COVID-19

Objective: Endothelial dysfunction is thought to underlie many of the complications of COVID-19 but to what degree this persists after recovery is unknown. Here we examine endothelial function in subjects previously hospitalized with COVID- 19, those with mild symptoms who were not hospitalized and negative controls (absence of SARS-CoV-2-antibodies). Endothelial function was measured as pulse wave response to the β2 adrenergic agonist salbutamol (PWRS) which is mediated through the nitric oxide - cyclic guanosine monophosphate pathway (NO-cGMP). Design and method: Echocardiography was used to exclude subjects with cardiac abnormalities. Tonometry of the radial artery (SphygmoCor, AtCor Medical, Sydney, Australia) was performed in duplicate by a single operator before and after inhalation of 200 mcg of salbutamol using a spacer device. The PWRS was taken as the change from baseline in augmentation index (Aix) as calculated by the SphygmoCor system. In a sub-sample, PWRS was assessed in the presence and absence of the phosphodiesterase type 5 inhibitor sildenafil which inhibits the breakdown of cGMP. Results: We recruited 88 subjects (49 men) aged 47.9 ± 14.3 (mean ± SD) years of whom 32 were previously hospitalized with COVID-19 (~6 months). Subjects previously hospitalized with COVID-19 were all previously assessed in a dedicated pulmonary clinic. Age, gender, BMI, smoking status, diabetes and estimated 10-year cardiovascular risk (Q-RISKâ3) were similar between the groups. Administration of salbutamol reduced AIx in controls and those with mild COVID-19 but produced an increase in AIx in previously hospitalized COVID-19 cases (mean [95% CI]): -2.85 [-5.52, -0.188] %, -2.32 [-5.17,0.54] %, and 3.03 [0.06, 6.00] % respectively, P = 0.017 between the groups. In a sub-sample (11 hospitalized and 11 non-hospitalized) the PWRS was measured again 30 minutes after oral administration of sildenafil 25 mg. This produced a greater reduction in AIx: -5.28 [-9.00, -1.54] % in non-hospitalized and a reduction: -3.90 [-7.60, -0.21] % in hospitalized patients, and an overall improvement in the PWRS (P = 0.006). Conclusions: In subjects previously hospitalized with severe COVID-19, endothelial function is impaired for many months after hospital discharge and the impaired NO-cGMP mediated vasodilation may be reversed by sildenafil.

GMP Manufacturing and IND-Enabling Studies of a Recombinant Hyperimmune Globulin Targeting SARS-CoV-2.

Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus.

Acute prolonged motor aura resembling ischemic stroke after COVID - 19 vaccination (CoronaVac): the firstcase report

Objective: NA Background: Due to the COVID-19 pandemic, Thailand has started its vaccination program since February 2021. After the launch of the mass vaccination with CoronaVac, there were reports of patients who suffer unusual hemiparesis across the country. We report the first case of a patient who suffered transient focal neurological deficit mimicking stroke following CoronaVac vaccination. However, instead of an ischemic stroke, motor aura was suspected. Design/Methods: A 24 year-old Thai female presented with left hemiparesis fifteen minutes after receiving CoronaVac. She also had numbness of her left arm and legs, flashing lights, and headaches. On physical examination, her BMI was 32.8. Her vital signs were normal. She had moderate left hemiparesis (MRC grade III), numbness on her left face, arms, and legs. Her weakness continued for 5 days. Results: A brain CT scan was done showing no evidence of acute infarction. Acute treatment with aspirin was given. MRI in conjunction with MRA was performed in which no restricted diffusion was seen. A SPECT was performed to evaluate the function of the brain showing significant hypoperfusion of the right hemisphere. The patient gradually improved and was discharged. Conclusions: In this study, we present the first case of stroke mimic after CoronaVac vaccination. After negative imaging studies, stroke is unlikely to be the cause. Asymmetrical abnormal functional imaging study showing multifocal hypoperfusion on the right could represent the ongoing neurological deficits. Therefore, we believed that it might be due to cortical spreading depression, in which motor aura could be responsible. The uniqueness in our case is the prolongation of weakness that we think might be due reverberating spreading depression wave. The cause is unknown, but we proposed that aluminum found to enhance the vaccine that could disrupt the Glutamate - Nitric oxide - cGMP pathway leading to the prolongation of motor aura.

Human infection studies: Key considerations for challenge agent development and production.

Human infection (or challenge) studies involve the intentional administration of a pathogen (challenge agent) to volunteers. The selection, isolation, development and production of the challenge agent is one of the first steps in developing a challenge study and critical for minimising the risk to volunteers. Regulatory oversight for this production differs globally. Manufacturing agents within a Good Manufacturing Practice (GMP) facility reduces the risk of the manufacturing process by including processes such as confirming the identity of the challenge agent and ascertaining that it's pure and free from impurities. However, in some cases it's not possible or feasible to manufacture to GMP standards, for example where the challenge agent requires an intermediate vector for growth. There is lack of clear guidance on what the minimum requirements for high-quality safe manufacture outside of GMP facilities should be and here we describe the development of a considerations document for the selection and production of challenge agents to meet this need.

Safety and efficacy study of allogeneic human menstrual blood stromal cells secretome to treat severe COVID-19 patients: clinical trial phase I & II.

BACKGROUND: Cell-free Mesenchymal stromal cells (MSCs) have been considered due to their capacity to modulate the immune system and suppress cytokine storms caused by SARS-CoV-2. This prospective randomized double-blind placebo-controlled clinical trial aimed to assess the safety and efficacy of secretome derived from allogeneic menstrual blood stromal cells (MenSCs) as a treatment in patients with severe COVID-19. METHODS: Patients with severe COVID-19 were randomized (1:1) to either MenSC-derived secretome treatment or the control group. Subjects received five intravenous infusions of 5 mL secretome or the same volume of placebo for five days and were monitored for safety and efficacy for 28 days after treatment. Adverse events, laboratory parameters, duration of hospitalization, clinical symptom improvement, dynamic of O2 saturation, lymphocyte number, and serial chest imaging were analyzed. RESULTS: All safety endpoints were observed without adverse events after 72 h of secretome injection. Within 28 days after enrollment, 7 patients (50%) were intubated in the treated group versus 12 patients (80%) in the control group. Overall, 64% of patients had improved oxygen levels within 5 days of starting treatment (P < 0.0001) and there was a survival rate of 57% in the treatment group compared to 28% in the control group was (P < 0.0001). Laboratory values revealed that significant acute phase reactants declined, with mean C-reactive protein, ferritin, and D-dimer reduction of 77% (P < 0.001), 43% (P < 0.001), and 42% (P < 0.05), respectively. Significant improvement in lymphopenia was associated with an increase in mean CD4+ and CD8+ lymphocyte counts of 20% (P = 0.06) and 15% (P < 0.05), respectively. Following treatment, percentage of pulmonary involvement showed a significant improvement in the secretome group (P < 0.0001). This improvement differed significantly between survivors and those who were dying (P < 0.005). CONCLUSIONS: For the first time, this study demonstrated that in hospitalized patients with severe COVID-19, therapy with MenSCs-derived secretome leads to reversal of hypoxia, immune reconstitution, and downregulation of cytokine storm, with no adverse effects attributable to the treatment. Given these outcomes, it may be possible to use this type of treatment for serious inflammatory lung disease with a mechanism similar to COVID-19 in the future. However, it is necessary to evaluate the safety and efficacy of MenSCs-derived secretome therapy in clinical trials on a larger population of patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05019287. Registered 24AGUEST 2021, retrospectively registered, https://clinicaltrials.gov/ct2/show/record/NCT05019287 . IRCT, IRCT20180619040147N6. Registered 04/01/2021.