Enabling mRNA medicine for brain tumors

mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.

Optimizing mRNA vaccines and virusmediated CAR T-cell therapy for immuno-oncology: Synthetic tools for gene modification

With the success of mRNA vaccines during the COVID-19 pandemic and CAR T-cell therapies in clinical trials, there is growing opportunity for immunotherapies in the treatment of many types of cancers. Lentiviral vectors have proven effective at delivery of genetic material or gene editing technology for ex vivo processing, but the benefits and promise of Adeno-associated virus (AAV) and mRNA tools for in vivo immunotherapy have garnered recent interest. Here we describe complete synthetic solutions for immuno-oncology research programs using either mRNA-vaccines or virus-mediated cell and gene engineering. These solutions optimize workflows to minimize screening time while maximizing successful research results through: (1) Efficiency in lentiviral packaging with versatility in titer options for high-quality particles. (2) A highthroughput viral packaging process to enable rapid downstream screening. (3) Proprietary plasmid synthesis and preparation techniques to maintain ITR integrity through AAV packaging and improve gene delivery. (4) Rapid synthesis, in vitro transcription, and novel sequencing of mRNA constructs for complete characterization of critical components such as the polyA tail. The reported research demonstrates a streamlined approach that improves data quality through innovative synthesis and sequencing methodologies as compared to current standard practices.

Biodegradable Polyester Nanoparticle Vaccines Deliver Self-Amplifying mRNA in Mice at Low Doses

Delivery of self-amplifying mRNA (SAM) has high potential for infectious disease vaccination due to its self-adjuvanting and dose-sparing properties. Yet a challenge is the susceptibility of SAM to degradation and the need for SAM to reach the cytosol fully intact to enable self-amplification. Lipid nanoparticles are successfully deployed at incredible speed for mRNA vaccination, but aspects such as cold storage, manufacturing, efficiency of delivery, and the therapeutic window can benefit from further improvement. To investigate alternatives to lipid nanoparticles, a class of >200 biodegradable end-capped lipophilic poly(beta-amino ester)s (PBAEs) that enable efficient delivery of SAM in vitro and in vivo as assessed by measuring expression of SAM encoding reporter proteins is developed. The ability of these polymers to deliver SAM intramuscularly in mice is evaluated, and a polymer-based formulation that yields up to 37-fold higher intramuscular (IM) expression of SAM compared to injected naked SAM is identified. Using the same nanoparticle formulation to deliver a SAM encoding rabies virus glycoprotein, the vaccine elicits superior immunogenicity compared to naked SAM delivery, leading to seroconversion in mice at low RNA injection doses. These biodegradable nanomaterials may be useful in the development of next-generation RNA vaccines for infectious diseases.Copyright © 2023 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH.

Fulminant myocarditis following SARS-CoV-2 mRNA vaccination rescued with venoarterial ECMO: A report of two cases

Objectives: Cases of fulminant myocarditis after mRNA COVID-19 vaccination have been reported. The most severe may need venoarterial extracorporeal membrane oxygenation (V-A ECMO) support. Here we report two cases successfully rescued with V-A ECMO. Method(s): We included all the cases supported with V-A ECMO for refractory cardiogenic shock due to myocarditis secondary to a mRNA SARS-COV2 vaccine in the high-volume adult ECMO Program in Vall Hebron University Hospital since January 2020. Result(s): We identified two cases (table). One of them was admitted for out-of-hospital cardiac arrest. In both, a peripheral V-A ECMO was implanted in the cath lab. An intra-aortic balloon pump was needed in one case for left ventricle unloading. Support could be successfully withdrawn in a mean of five days. No major bleeding or thrombosis complications occurred. Definite microscopic diagnosis could be reached in one case (Image, 3). Treatment was the same, using 1000mg of methylprednisolone/day for 3 days. A cardiac magnetic resonance 10 days after admission showed a significant improvement in systolic function and diffuse oedema and subepicardial contrast intake in different segments (Image, 1-2). Both patients were discharged fully recovered. Conclusion(s): V-A ECMO should be established in cases of COVID-19 vaccine-associated myocarditis with refractory cardiogenic shock during the acute phase. (Table Presented).

Lipid nanoparticle library towards development next generation genomic medicines

Ionizable amino lipids are a major constituent of the lipid nanoparticles for delivering nucleic acid therapeutics (e.g., DLin-MC3-DMA in ONPATTRO , ALC-0315 in Comirnaty , SM-102 in Spikevax ). Scarcity of lipids that are suitable for cell therapy, vaccination, and gene therapies continue to be a problem in advancing many potential diagnostic/therapeutic/vaccine candidates to the clinic. Herein, we describe the development of novel ionizable lipids to be used as functional excipients for designing vehicles for nucleic acid therapeutics/vaccines in vivo or ex vivo use in cell therapy applications. We first studied the transfection efficiency (TE) of LNP-based mRNA formulations of these ionizable lipid candidates in primary human T cells and established a workflow for engineering of primary immune T cells. We then adapted this workflow towards bioengineering of CAR constructs to T cells towards non-viral CAR T therapy. Lipids were also tested in rodents for vaccine applications using self-amplifying RNA (saRNA) encoding various antigens. We have then evaluated various ionizable lipid candidates and their biodistribution along with the mRNA/DNA translation exploration using various LNP compositions. Further, using ionizable lipids from the library, we have shown gene editing of various targets in rodents. We believe that these studies will pave the path to the advancement in nucleic acid based therapeutics and vaccines, or cell gene therapy agents for early diagnosis and detection of cancer, and for targeted genomic medicines towards cancer treatment and diagnosis.

Chemical synthesis of super messenger RNA medicine.

Messenger RNAmRNAmedicine was urgently approved in 2020 as a vaccine for COVID-19 . However, current mRNA therapeutics are not fully established, with challenges remaining in translation efficiency and drug delivery system. Therefore, further research is needed to adapt mRNA therapeutics to other diseases. Furthermore, the preparation of mRNA drugs is time-consuming and costly because of the biological methods used. Our laboratory has been working on chemical methods to solve these issues. In this paper, we introduce chemical modifications and novel capping reactions as a method to improve the translation efficiency of mRNA and the introduction of disulfide modification to oligonucleotide therapeutics as an effort on the drug delivery system.Copyright © 2023, Japan Society of Drug Delivery System. All rights reserved.

Haematuria and proteinuria associated with COVID-19-mRNA-vaccination: a case-report

Objective: A new generation of vaccine technology platform has been developed to combat the COVID- 19 pandemic, the mRNA vaccine. The EMA granted the Pfizer- BioNTech COVID-19 vaccine an emergency use authorization in December 2020 with limited clinical experience, especially in the pediatric population. Method(s): Here, we present a case-report of a 17-yearold girl, who was vaccinated with the mRNA-COVID vaccine in October 2021, and developed a gross hematuria and proteinuria the day after the vaccination. Result(s): The patient presented at our outpatient clinic three days after the vaccination with new-onset hematuria and proteinuria. Up to this date, she had no former known medical conditions and the family history was negative regarding kidney diseases. We excluded nephrolithiasis, autoimmune glomerulonephritis and urinary tract infection as causes. The laboratory chemistry of the kidney was within normal range. The proteinuria dissolved spontaneously, and a microhematuria persisted. One day after the second dose of Cominarty in November 2021, the gross hematuria with proteinuria relapsed. A treatment with an ACE-inhibitor did not have any effect on the proteinuria. At this point, only a few casereports of patients with a comparable clinical course, especially from Japan, were published. In suspicion of a vaccine-triggered nephritis we started a prednisolon therapy which dissolved the proteinuria and induced a regression of the haematuria to a minimal stage. Conclusion(s): Within the last year, the medical community has gained more insights concerning mRNA vaccines. There is growing evidence, that mRNA vaccines can trigger de novo or relapse IgA nephropathy. But more systematic research and long-term evaluation is desirable to elucidate the underling pathophysiology as well as the influence on kidney survival of affected patients in the future. Furthermore, patient education should incorporate the risk of hematuria and proteinuria in children when applying mRNA vaccines.

Managing severe warm autoimmune haemolytic anaemia after the SARS-Cov- 2 vaccine with emergency splenectomy and eculizumab

Autoimmune haemolytic anaemia (AIHA) is rare but described after the SARS-CoV- 2 Pfizer-BioNTech vaccine. We present a case of severe refractory warm AIHA after this vaccine, managed with emergency splenectomy and complement inhibition with eculizumab. A male in his teens with a history of liver transplant for biliary atresia (aged 2 years) and AIHA (aged 6 years) presented to his district general hospital with jaundice, dark urine, fatigue and chest discomfort 48 h after the first dose of SARS-CoV- 2 Pfizer-BioNTech vaccine (BNT162b2 mRNA). Investigations revealed haemoglobin (Hb) of 70 g/L and bilirubin of 98 mumol/L, which was treated as AIHA. The patient initially responded to prednisolone (1 mg/kg, 60 mg) but subsequently deteriorated and failed to respond to second-line rituximab (375 mg/m2) and two units of packed red blood cells (PRBC). By day 29 the patient had developed life-threatening anaemia culminating in a Hb of 35 g/L (after transfusion), lactate dehydrogenase (LD) of 1293 units/L and bilirubin of 228 mumol/L. This necessitated an immediate transfer to our tertiary centre for specialist support. Further investigations revealed a haptoglobin <0.1 g/L and direct antiglobulin test (DAT) strongly positive for IgG (4+) and negative for C3d. The peripheral blood film showed severe anaemia, nucleated red cells, anisocytosis and spherocytes with no autoagglutination, schistocytes or platelet clumps. Thrombocytopaenia (platelets 49 +/- 109/L) was present. Differentials were ruled out, such as paroxysmal nocturnal haemoglobinuria and heparin-induced thrombocytopaenia. HIV and hepatitis serology were negative, as were adenovirus, cytomegalovirus and Epstein-Barr virus PCR assays. A CT showed splenomegaly of 15.5 cm. Urinalysis found urobilinogen and bilirubin at high concentrations and negative urinary haemosiderin. Together, the investigations were consistent with warm AIHA. On day 29, four units of PRBC were transfused alongside 100 mg methylprednisolone and 1 g/kg IVIG. On day 30 the patient deteriorated despite the escalated treatment: Hb had only increased to 54 g/L, bilirubin was 200 mumol/L and LD was rising. Considering this life-threatening fulminant haemolysis, an emergency splenectomy was performed. This slowed haemolysis but did not completely ameliorate it: by day 33 the patient had received 15 units of PRBC. Thus, eculizumab, a terminal complement pathway inhibitor, was trialled to arrest intravascular haemolysis, alongside rituximab, repeat IVIG 1 g/kg, prednisolone 40 mg and tacrolimus 2 mg. This showed a favourable response, requiring less frequent transfusions and settling haemolysis. This case highlights the rare complication of warm AIHA with the SARS-CoV- 2 Pfizer-BioNTech vaccine, the use of emergency splenectomy for disease control, and the potential of eculizumab for refractory cases.

Challenges in the manufacture, storage, distribution and regulation of traditional and novel vaccines

Since the onset of the COVID-19 pandemic, there has been a significant surge in interest of COVID-19 vaccines in particular, and other traditional vaccines in general. This strong interest is expected to continue as the industry strives to manufacture safer and more effi cacious vaccines against COVID-19 and other infectious diseases. Vaccines are a unique class of products, being biologicals that are administered to healthy individuals to prevent diseases. The equitable distribution and availability of safe, efficacious and good quality vaccines are of utmost importance in preventing and controlling infections and safeguarding public health. The continued existence of poor- quality vaccines suggests a lack of control of manufacturing, storage, distribution, and possibly, their associated regulation. Nonetheless, all these situations - whether positive or negative, present opportunities for improvements. As regulatory authorities step up efforts in regulating existing traditional vaccines, advancements in vaccine research and development churn out novel vaccines that pose further manufacturing and regulatory challenges. This manuscript provides an overview of vaccines, both traditional and novel, and strives to identify challenges in the manufacture, storage, distribution, handling and their associated regulation. It also evaluates whether current regulatory frameworks are adequate, and where applicable, recommends areas for improvements. International harmonization and convergence of national regulatory framework with the view to facilitate quicker approval of safe, efficacious and good quality vaccines, that are accessible and affordable to patients worldwide, are also explored.

Perinatal SARS-CoV-2 infection and vaccination: new insight

Cumulative data regardingCOVID-19 infection during pregnancy have demonstrated the ability of SARS-CoV-2 to infect the placenta. However, the mechanisms of SARS-CoV-2 placental viral entry are yet to be defined. SARS-CoV-2 infects cells by binding to the ACE2 receptor. However, SARS-CoV-2 cell entry also requires co-localization of spike protein cleavage by the serine protease TMPRSS2. However, the co-expression of ACE2 and TMPRSS2 in placental cells is debated, raising the question of whether potential non-canonical molecular mechanismsmay be involved in SARS-CoV-2 placental cells' viral entry. Although published data regarding the ability of the SARS-CoV- 2 to infect the fetus are contradicting, the placenta appears to be an immunological barrier to active SARS-CoV-2 infection and vertical transmission;however, the mechanism is unclear. Our experiments demonstrated the ability of the SARS-CoV-2 virus to directly infect the placenta and induce transcriptomic responses in COVID-positive mothers. These transcriptomic responses were characterized by differential expression of specific mRNAs and miRNAs associated with SARS-CoV-2 infection, with induction of specific placental miRNAs that can inhibit viral replication. Failure in such mechanisms may be associated with vertical transmission. Since the start of the COVID-19 pandemic, the COVID-19 mRNA vaccines have been widely used to reduce the morbidity and mortality of SARS-CoV-2 infection. Historically, non-live vaccines have not caused any harm to pregnant mothers;however, it is unclear whether our current understanding of the effects of non-live vaccines serves as a reliable precedent owing to the novel technology used to create these mRNA vaccines. Since there are no definitive data on the possible biodistribution of mRNA vaccines to the placenta, the likelihood of vaccine mRNA reaching the fetus remains uncertain. Little has been reported on the tissue localization of the lipid nanoparticles (LNPs) after intramuscular (IM) administration of the mRNA vaccine. The biodistribution of LNPs containing the mRNA vaccine has been investigated in animal models but not humans. In the murine model, the vaccine LNPs were rapidly disseminated to several organs, including the heart, liver, kidney, lung, and spleen, following IM administration. However, no traditional pharmacokinetic or biodistribution studies have been performed with the mRNA vaccines, including possible biodistribution to breast milk or the placenta.

Modeling DNA Oligonucleotide Binding to DNA-mimetic Stem-Loop 1 of the SARS-CoV-2 RNA

SARS-CoV-2 is a positive-sense RNA virus that contains open reading frame 1ab (ORF1ab) to produce 16 nonstructural proteins (nsps). Five stem-loops (SL) are found in the 5' UTR of the RNA that are involved in myriad viral functions and are labeled SL1 through SL5. SL1 is crucial to viral replication. Upon viral infection, nsp1 binds the ribosomal 40S subunit to inhibit all host mRNA translation. Upon SL1 binding to nsp1, viral mRNA can be processed by the ribosome, allowing viral proteins to be produced. In this study, we are examining small DNA oligonucleotides that bind to SL1-mimetic DNA in order to block SL1-nsp1 interactions. We designed a DNA analog of the SL1 hairpin and two small DNA oligonucleotides that are complementary to either the helical stem or the loop region of SL1. The binding of these oligonucleotides to the SL1 hairpin should allow the formation of either an alternate duplex or a triplex structure. Isothermal titration calorimetry (ITC) and circular dichroism (CD) techniques were performed in 1 MKCl and 10 mM MgCl2 at two different pH (5.5 and 7.0) to examine structural and thermodynamics of binding. ITC of the two oligonucleotides showed modest binding. Results from DNA binding experiments, thermal denaturation, and CD show the hairpin structure is thermodynamically more favored and mostly remains intact under the conditions examined.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Advances in approved nucleic acid drugs and lipid nanoparticle system.

Nucleic acids, as a next generation of biotechnology drugs, not only can fundamentally treat diseases, but also own significant platform characteristics in view of technology and production. Therefore, nucleic acid-based drugs have broad clinical applications in biomedical fields. However, nucleic acids are degradable and unstable, and have very low intracellular delivery efficiency in vitro and in vivo, which greatly limits their applications. In recent years, ionizable lipid-based lipid nanoparticles have shown promising application potentials and have been successfully applied to COVID-19 (Coronavirus Disease 2019) vaccines in clinic. Lipid nanoparticles demonstrate high in vivo delivery efficiency and good safety profile due to their unique structural and physicochemical properties, which provides many possibilities for their clinical applications for nucleic acid delivery in the future. This review focused on the characteristics of nucleic acid drugs and their delivery barriers, and discussed the approved nucleic acid drugs to illustrate the key aspects of the success of their delivery carrier system. In addition, problems to be solved in the field were highlighted.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.

Proteins get $210 million gift

The institute intends to provide scientists with synthetic antibodies and other protein tools to help illuminate fundamental biological processes and therapeutic leads. BIOMEDICINE ;Immunologist Timothy Springer, a founder of vaccinemaker Moderna, this week announced he will give $210 million to a nonprofit research center he created to develop the use of proteins for medical research. [Extracted from the article] Copyright of Science is the property of American Association for the Advancement of Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Subacute thyroiditis and thyrotoxicosis after SARS-CoV-2 vaccine: report of 2 cases / Tiroiditis subaguda y tirotoxicosis posterior a vacuna contra SARS-CoV-2: reporte de 2 casos

La enfermedad por coronavirus SARS-CoV-2 que surgió en el año 2019 (COVID-19), ha obligado al rápido desarrollo de vacunas para prevenir su propagación e intentar controlar la pandemia. Dentro de las vacunas desarrolladas, las primeras en ser aprobadas con una tecnología nueva en el campo de la vacunación, fueron las vacunas basadas en ARNm (ácido ribonucleico mensajero), que lograron tasas de efectividad cercanas al 95 % para la prevención de la enfermedad COVID-19 grave. Los eventos adversos comunes son reacciones locales leves, pero ha habido varios informes de pacientes que desarrollaron tiroiditis subaguda y disfunción tiroidea después de recibir la vacuna contra SARS-CoV-2. Este artículo presenta dos casos de tiroiditis subaguda poco después de recibir la vacuna contra COVID-19

The SARS-CoV-2 coronavirus disease which emerged in 2019 (COVID-19), has forced the rapid development of vaccines to prevent the spread of infection and attempt to control the pandemic. Among the vaccines developed, one of the first to be approved with a new technology in the field of vaccination, was the mRNA (messenger ribonucleic acid) vaccine, with rates of effectiveness close to 95% for the prevention of severe COVID-19 disease. Common adverse events are mild local reactions, but there have been some reports of patients developing sub-acute thyroiditis and thyroid dysfunction after receiving the SARS-CoV-2 vaccine. This article presents two case reports of subacute thyroiditis shortly after receiving the COVID-19 vaccine

Strategic construction of mRNA vaccine derived from conserved and experimentally validated epitopes of avian influenza type A virus: a reverse vaccinology approach.

Purpose: The development of vaccines that confer protection against multiple avian influenza A (AIA) virus strains is necessary to prevent the emergence of highly infectious strains that may result in more severe outbreaks. Thus, this study applied reverse vaccinology approach in strategically constructing messenger RNA (mRNA) vaccine construct against avian influenza A (mVAIA) to induce cross-protection while targeting diverse AIA virulence factors. Materials and Methods: Immunoinformatics tools and databases were utilized to identify conserved experimentally validated AIA epitopes. CD8+ epitopes were docked with dominant chicken major histocompatibility complexes (MHCs) to evaluate complex formation. Conserved epitopes were adjoined in the optimized mVAIA sequence for efficient expression in Gallus gallus. Signal sequence for targeted secretory expression was included. Physicochemical properties, antigenicity, toxicity, and potential cross-reactivity were assessed. The tertiary structure of its protein sequence was modeled and validated in silico to investigate the accessibility of adjoined B-cell epitope. Potential immune responses were also simulated in C-ImmSim. Results: Eighteen experimentally validated epitopes were found conserved (Shannon index <2.0) in the study. These include one B-cell (SLLTEVETPIRNEWGCR) and 17 CD8+ epitopes, adjoined in a single mRNA construct. The CD8+ epitopes docked favorably with MHC peptide-binding groove, which were further supported by the acceptable ΔGbind (-28.45 to -40.59 kJ/mol) and Kd (<1.00) values. The incorporated Sec/SPI (secretory/signal peptidase I) cleavage site was also recognized with a high probability (0.964814). Adjoined B-cell epitope was found within the disordered and accessible regions of the vaccine. Immune simulation results projected cytokine production, lymphocyte activation, and memory cell generation after the 1st dose of mVAIA. Conclusion: Results suggest that mVAIA possesses stability, safety, and immunogenicity. In vitro and in vivo confirmation in subsequent studies are anticipated.

Coronavirus murine hepatitis virus strain 3 induces intestinal injury via caspase 3 dependent apoptosis

Background: Diarrhea was typical symptoms of the coronavirus disease 2019 (COVID-19). However, the underlying mechanism had not been fully understood. Aim(s): The study aimed to explore the mechanism of intestinal injury during COVID-19 in a coronavirus murine hepatitis virus strain 3 (MHV-3) induced acute mouse model. Method(s): MHV-3 induced acute infection Balb/cJ mice model was established. Intestine samples were collected at indicated time points as 0 h, 24 h, 48 h and 60 h post infection. The mRNA and protein expression of IL1b, TNFalpha, IL6, caspase 3 and cleaved caspase 3 were examined by real-time quantitative PCR (qPCR) and western blot respectively. The intestine injury and apoptosis were measured by HE staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Moreover, Z-DEVD-FMK (caspase 3 inhibitor) pre-treated MHV-3 infection mice model were established, in which the apoptosis of intestine was evaluated as well. Meanwhile, the murine intestinal cell MODE-K was infected by MHV-3 in vitro for evaluation of virus induced apoptosis. Result(s): Post MHV-3 infection, the histopathology of intestine tissue showed extraordinary injury with time dependence, as well as high level of TUNEL positivity. The mRNA levels of inflammatory cytokine IL1b, TNFalpha and IL6 were significantly increased. The protein expressions of caspase 3 and cleaved caspase 3 in the intestine was found significantly elevated from 24 to 48 h post MHV-3 infection. Z-DEVD-FMK pretreatment inhibited caspase 3 and cleaved caspase 3 expression and decreased TUNEL positivity. Meanwhile, alleviated gut injury and inhibited TNFalpha expression were observed. In vitro treated by MHV-3, intestinal cell line MODE-K showed nine-fold increase of apoptosis by comparison with saline treated ones. The expressions of apoptosis crucial protein caspase3 and cleaved caspase3 significantly elevated, as well as TNFalpha. Conclusion(s): Coronavirus murine hepatitis virus strain 3 induces intestinal injury via caspase 3 dependent apoptosis, which might shed light on the treatment of intestinal complications in COVID-19.

Third Booster Dose Vaccine against Sars-Cov-2 in Primary Sjogren's Syndrome: Longitudinal Cohort from the Safer Study

Objectives: Few studies evaluate the immunogenicity and safety of different COVID-19 vaccine platforms in patients with primary Sjogren's Syndrome (pSS). The present study aims to assess the immunogenicity through anti-spike IgG antibodies after the COVID-19 vaccine dose in heterologous groups compared to homologous regimen in patients with pSS. Method(s): These data are from the SAFER study: 'Safety and efficacy of the COVID-19 vaccine in rheumatic disease', a real-life phase IV multicenter longitudinal study, evaluating patients since before the first dose. Pregnant women, those with a history of serious adverse events prior to any vaccine, and those with other causes of immunosuppression were excluded. Patients with pSS > 18 years, classified according to ACR/EULAR 2016 classification criteria were included. Antibodies against the Receptor Binding Domain - RBD portion of the Spike protein of SARS-CoV-2 (IgG-S) were measured by chemiluminescence (Architect SARS-CoV-2 Quanti II, Abbott), before the first dose and 28 days after the 2nd and 3rd dose. Seropositivity was defined as IgG-Spike titers >=7.1 BAU/mL. Patients received adenoviral vector (ChAdOx1, Astrazeneca), mRNA (Pfizer) or inactivated SARS-COV-2 (Coronavac). Non-parametric methods were used. The alpha level of significance was set at 5%. Result(s): 56 participants received 3 doses, 46 +/- 11 years old, disease duration 7.62 years, 92.9% female, 41.1% White and 55.4% Mixed. The homologous third-booster dose group (n = 15, all ChAdOx1) and heterologous group (n = 41) were homogeneous for age, sex, ethnicity, comorbidities, medication and baseline IgG-S median [IQR] titers. After primary vaccination (2 doses) IgG-S median and titers [IQR] were similar in homologous and heterologous groups (373.03 [179.58, 843.92] vs. 473.36 [119.05, 1059.60], p = 0.705). Third-booster dose induced higher IgG-S median [IQR] titers compared to only 2 doses (1229.54 [333.55, 4365.47] vs 464.95 [140.42, 1015.25], p alpha 0.001). Heterologous 3rd-booster induced higher IgG-S median [IQR] titers than homologous scheme with ChAdOx1 (1779.52 [335.83, 4523.89] vs 730.76 [303.37, 1858.98], p = 0.150), Fig 1 and 2, although not statistically significant. Conclusion(s): Third booster dose induced higher humoral immune response compared to two doses whichmay improve protection against COVID-19 in patients with pSS. Although not statistically significant, the response to the heterologous scheme tended to be better than the response to the homologous booster vaccination, which heterologous booster scheme tended to respond better than homologous booster vaccination, which is relevant in this immunosuppressed population. Increasing the sample size will help clarify this issue. .

IMMUNE RESPONSES TO MRNA AND INACTIVATED COVID-19 VACCINES AMONG HEALTHCARE WORKERS IN HONG KONG

COVID-19 vaccines against the earlier strains of SARS-CoV-2 are now available. However, breakthrough infections can still occur due to waning antibodies and immune escape by new variants. We assessed humoral immune responses to the mRNA (BNT162b2) and inactivated (CoronaVac) vaccines in our healthcare worker cohort (HCW). We recruited HCWs from public and private healthcare institutions across Hong Kong and collected blood samples at enrolment and every 6 months from June 2020 to June 2022. A subset of volunteers provided blood samples between 10 – 42 days after each dose of vaccine. Immune responses to vaccination were measured as SARS-CoV-2 binding antibodies detected by an enzyme-linked immunosorbent assay (ELISA) and SARS-CoV-2 neutralising antibodies by surrogate virus neutralization test (sVNT) and plaque reduction neutralization test (PRNT). Among the 1,736 HCWs enrolled in our cohort, 252 HCWs provided pre- and post-vaccination blood samples after each dose of either vaccine. Two doses of BNT162b2 generated levels of neutralizing antibodies (sVNT inhibition = 96.8%, range = 42.8%, 98.2%) comparable to those generated by natural infections in the first wave (sVNT inhibition = 84.0%, range = 32.9%, 93.8%). Similar levels were achieved with three doses of CoronaVac (sVNT inhibition = 95.3%, range = 64.7%, 98.3%) and heterologous vaccination with two doses of CoronaVac followed by a booster dose of BNT162b2 vaccine (sVNT inhibition = 97.0%, range = 85.8%, 97.7%). These antibody levels waned faster after second doses and slower after third doses for both vaccines. The BNT162b2 vaccine and CoronaVac vaccines can generate robust antibody responses comparable to natural infections. Three doses of the CoronaVac vaccine, or a heterologous boost with the BNT162b2 vaccine following two doses of the CoronaVac vaccine are required to achieve similar levels of neutralising antibodies in vaccinees who received two doses of the BNT162b2 vaccine. [ FROM AUTHOR] Copyright of International Journal of Infectious Diseases is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Allergic reactions of COVID-19 vaccine based on mRNA-LNP and its pharmacokinetics in vivo

Vaccination has been proved to be the most effective strategy to prevent the Corona Virus Disease 2019 (COVID-19). The mRNA vaccine based on nano drug delivery system (NDDS) - lipid nanoparticles (LNP) has been widely used because of its high effectiveness and safety. Although there have been reports of severe allergic reactions caused by mRNA-LNP vaccines, the mechanism and components of anaphylaxis have not been completely clarified yet. This review focuses on two mRNA-LNP vaccines, BNT162b2 and mRNA-1273. After summarizing the structural characteristics, potential allergens, possible allergic reaction mechanism, and pharmacokinetics of mRNA and LNP in vivo, this article then reviews the evaluation methods for patients with allergic history, as well as the regulations of different countries and regions on people who should not be vaccinated, in order to promote more safe injection of vaccines. LNP has become a recognized highly customizable nucleic acid delivery vector, which not only shows its value in mRNA vaccines, but also has great potential in treating rare diseases, cancers and other broad fields in the future. At the moment when mRNA-LNP vaccines open a new era of nano medicine, it is expected to provide some inspiration for safety research in the process of research, development and evaluation of more nano delivery drugs, and promote more nano drugs successfully to market.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.