Engineering an Escherichia coli based in vivo mRNA manufacturing platform.

Synthetic mRNA is currently produced in standardized in vitro transcription systems. However, this one-size-fits-all approach has associated drawbacks in supply chain shortages, high reagent costs, complex product-related impurity profiles, and limited design options for molecule-specific optimization of product yield and quality. Herein, we describe for the first time development of an in vivo mRNA manufacturing platform, utilizing an Escherichia coli cell chassis. Coordinated mRNA, DNA, cell and media engineering, primarily focussed on disrupting interactions between synthetic mRNA molecules and host cell RNA degradation machinery, increased product yields >40-fold compared to standard "unengineered" E. coli expression systems. Mechanistic dissection of cell factory performance showed that product mRNA accumulation levels approached theoretical limits, accounting for ~30% of intracellular total RNA mass, and that this was achieved via host-cell's reallocating biosynthetic capacity away from endogenous RNA and cell biomass generation activities. We demonstrate that varying sized functional mRNA molecules can be produced in this system and subsequently purified. Accordingly, this study introduces a new mRNA production technology, expanding the solution space available for mRNA manufacturing.

Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 µM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach.

Lipid nanoparticles (LNPs) are the leading technology for RNA delivery, given the success of the Pfizer/BioNTech and Moderna COVID-19 mRNA (mRNA) vaccines, and small interfering RNA (siRNA) therapies (patisiran). However, optimization of LNP process parameters and compositions for larger RNA payloads such as self-amplifying RNA (saRNA), which can have complex secondary structures, have not been carried out. Furthermore, the interactions between process parameters, critical quality attributes (CQAs), and function, such as protein expression and cellular activation, are not well understood. Here, we used two iterations of design of experiments (DoE) (definitive screening design and Box-Behnken design) to optimize saRNA formulations using the leading, FDA-approved ionizable lipids (MC3, ALC-0315, and SM-102). We observed that PEG is required to preserve the CQAs and that saRNA is more challenging to encapsulate and preserve than mRNA. We identified three formulations to minimize cellular activation, maximize cellular activation, or meet a CQA profile while maximizing protein expression. The significant parameters and design of the response surface modeling and multiple response optimization may be useful for designing formulations for a range of applications, such as vaccines or protein replacement therapies, for larger RNA cargoes.

Unique human immune signature of Ebola virus disease in Guinea.

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4(+) and CD8(+) T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

SARS-CoV-2 variant detection from wastewater: rapid spread of B.1.1.7 lineage in Hungary.

Wastewater-based epidemiology (WBE) is a recognised tool for tracking community transmission of COVID-19. From the second half of 2020, the emergence of new, highly infective, more pathogenic or vaccine-escape SARS-CoV-2 variants is the major public health concern. Variant analysis in sewage might assist the early detection of new mutations. Weekly raw sewage samples from 22 wastewater treatment plants (WWTPs) in Hungary (representing 40% of the population) were analysed between December 2020 and March 2021 for signature mutations N501Y and del H69/V70 of B.1.1.7 lineage by melting point genotyping and RT-digital droplet PCR (RT-ddPCR). The latter method proved to be more efficient in parallel detection of different variants and also provides quantitative information. Wastewater surveillance indicated that the B.1.1.7 variant first emerged in Budapest in early January 2021 and rapidly became dominant in the entire country. Results are in close agreement with the available clinical data (Pearson's correlation coefficient, R = 0.9153). RT-ddPCR was confirmed to be a reliable tool for tracking emerging variant ratios in wastewaters. It is a rapid and cost-effective method compared to whole-genome sequencing, but only applicable for the detection of known mutations. Efficient variant surveillance might require the combination of multiple methods.

Topography, Spike Dynamics, and Nanomechanics of Individual Native SARS-CoV-2 Virions.

SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, displays a corona-shaped layer of spikes which play a fundamental role in the infection process. Recent structural data suggest that the spikes possess orientational freedom and the ribonucleoproteins segregate into basketlike structures. How these structural features regulate the dynamic and mechanical behavior of the native virion are yet unknown. By imaging and mechanically manipulating individual, native SARS-CoV-2 virions with atomic force microscopy, here, we show that their surface displays a dynamic brush owing to the flexibility and rapid motion of the spikes. The virions are highly compliant and able to recover from drastic mechanical perturbations. Their global structure is remarkably temperature resistant, but the virion surface becomes progressively denuded of spikes upon thermal exposure. The dynamics and the mechanics of SARS-CoV-2 are likely to affect its stability and interactions.

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

Tissue-Specific Accumulation and Isomerization of Valuable Phenylethanoid Glycosides from Plantago and Forsythia Plants.

A comparative phytochemical study on the phenylethanoid glycoside (PhEG) composition of the underground organs of three Plantago species (P. lanceolata, P. major, and P. media) and that of the fruit wall and seed parts of Forsythia suspensa and F. europaea fruits was performed. The leaves of these Forsythia species and six cultivars of the hybrid F. × intermedia were also analyzed, demonstrating the tissue-specific accumulation and decomposition of PhEGs. Our analyses confirmed the significance of selected tissues as new and abundant sources of these valuable natural compounds. The optimized heat treatment of tissues containing high amounts of the PhEG plantamajoside (PM) or forsythoside A (FA), which was performed in distilled water, resulted in their characteristic isomerizations. In addition to PM and FA, high amounts of the isomerization products could also be isolated after heat treatment. The isomerization mechanisms were elucidated by molecular modeling, and the structures of PhEGs were identified by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HR-MS) techniques, also confirming the possibility of discriminating regioisomeric PhEGs by tandem MS. The PhEGs showed no cytostatic activity in non-human primate Vero E6 cells, supporting their safe use as natural medicines and allowing their antiviral potency to be tested.

Protection by humoral elements independent of virus neutralization activity against an influenza virus challenge.

To investigate the protective efficacy of a seasonal trivalent inactivated whole virion influenza vaccine (TIV) adjuvanted with aluminum phosphate (Fluval AB, referred to here as TIV+Al), we immunized mice with the TIV+Al, and as controls, with TIV, TIV+Al combined with Freund adjuvant (TIV+Al+F), inactivated A/PR/8/34(H1N1) (PR8) strain or PBS, and challenged them with a lethal dose of a mouse-adapted PR8 virus. Serum pools from immunized mice were passively transferred to recipient mice that were then challenged similarly. All actively immunized mice survived the challenge. Of recipient mice receiving serum from mice actively immunized with TIV, TIV+Al or TIV+Al+F, 20%, 80%, and 100% survived, respectively. Rates of mortality and morbidity of recipient mice were inversely proportional to the hemagglutination inhibition (HI) antibody level to the vaccine virus in the absence of detectable PR8-specific HI, neuraminidase inhibition (NI) and virus neutralization (VN) antibodies. No cross-reactivity was observed between vaccine and PR8 strains in in vitro HI, NI or VN assays. In splenocytes from TIV+Al-immunized mice production of IFN-γ or granzyme-B protein and mRNA expression increased (p<0.05). Thus, antibodies play a major role in the protection against a mismatched challenge infection independent of HI, NI and VN activity, but cellular immune responses may contribute to full protection in actively immunized mice.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

The redesigned neutron imaging facility, NORMA at BNC, Budapest.

The Budapest Neutron Center operates the cold neutron beam imaging station, Neutron Optics and Radiography for Material Analysis (NORMA), for non-destructive testing. For the NORMA station, there have been increasing requests to reach higher spatial resolution and the ability to follow time-dependent processes. Therefore, the system used successfully so far was completely redesigned and installed for a variety of tasks. The new system is based on the principle of three independent modules, allowing for highly configurable settings. It is to find the right balance between the necessary spatial resolution, a sufficiently shorter or longer temporal resolution, and a large enough field of view. The systematic study of the setups clearly shows the parameters' effects, helping to make the right choice for the measurement tasks. Among the rarely investigated parameters, we studied both the effect of the pixel binning and the change in the lens f-stop value on the spatial resolution. The newly improved NORMA facility allows the acquisition of high-quality neutron images for planned observations, e.g., local water kinetics in fuel cells.

Quality by Digital Design for Developing Platform RNA Vaccine and Therapeutic Manufacturing Processes.

Quality by digital design (QbDD) utilizes data-driven, mechanistic, or hybrid models to define and optimize a manufacturing design space. It improves upon the QbD approach used extensively in the pharmaceutical industry. The computational models developed in this approach identify and quantify the relationship between the product's critical quality attributes (CQAs) and the critical process parameters (CPPs) of unit operations within the manufacturing process. This chapter discusses the QbDD approach in developing and optimizing unit operations such as in vitro transcription, tangential flow filtration, affinity chromatography, and lipid nanoparticle (LNP) formulation in mRNA vaccine manufacturing. QbDD can be an efficient framework for developing a production process for a disease-agnostic product that requires extensive experimental and model-based process-product interaction characterization during the early process development phase.

Strategic Planning of a Joint SARS-CoV-2 and Influenza Vaccination Campaign in the UK.

The simultaneous administration of SARS-CoV-2 and influenza vaccines is being carried out for the first time in the UK and around the globe in order to mitigate the health, economic, and societal impacts of these respiratory tract diseases. However, a systematic approach for planning the vaccine distribution and administration aspects of the vaccination campaigns would be beneficial. This work develops a novel multi-product mixed-integer linear programming (MILP) vaccine supply chain model that can be used to plan and optimise the simultaneous distribution and administration of SARS-CoV-2 and influenza vaccines. The outcomes from this study reveal that the total budget required to successfully accomplish the SARS-CoV-2 and influenza vaccination campaigns is equivalent to USD 7.29 billion, of which the procurement costs of SARS-CoV-2 and influenza vaccines correspond to USD 2.1 billion and USD 0.83 billion, respectively. The logistics cost is equivalent to USD 3.45 billion, and the costs of vaccinating individuals, quality control checks, and vaccine shipper and dry ice correspond to USD 1.66, 0.066, and 0.014, respectively. The analysis of the results shows that the choice of rolling out the SARS-CoV-2 vaccine during the vaccination campaign can have a significant impact not only on the total vaccination cost but also on vaccine wastage rate.

First Broad-Range Serological Survey of Crimean-Congo Hemorrhagic Fever among Hungarian Livestock.

(1) Background: Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne disease endemic in Africa, Asia, the Middle East, and the Balkan and Mediterranean regions of Europe. Although no human CCHF cases have been reported, based on vector presence, serological evidence among small vertebrates, and the general human population, Hungary lies within high evidence consensus for potential CCHF introduction and future human infection. Thus, the aim of our pilot serosurvey was to assess CCHF seropositivity among cattle and sheep as indicator animals for virus circulation in the country. (2) Methods: In total, 1905 serum samples taken from free-range cattle and sheep in 2017 were tested for the presence of anti-CCHF virus IgG antibodies using commercial ELISA and commercial and in-house immunofluorescent assays. (3) Results: We found a total of eleven reactive samples (0.58%) from five administrative districts of Hungary comprising 8 cattle and 3 sheep. The most affected regions were the south-central and northwestern parts of the country. (4) Conclusions: Based on these results, more extended surveillance is advised, especially in the affected areas, and there should be greater awareness among clinicians and other high-risk populations of the emerging threat of CCHF in Hungary and Central Europe.

Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development.

mRNA technology has recently demonstrated the ability to significantly change the timeline for developing and delivering a new vaccine from years to months. The potential of mRNA technology for rapid vaccine development has recently been highlighted by the successful development and approval of two mRNA vaccines for COVID-19. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, e.g., treatments for cancer, metabolic disorders, cardiovascular conditions, and autoimmune diseases. There is currently significant demand for the development of improved manufacturing processes for the production of mRNA therapeutics in an effort to increase their yield and quality. The development of suitable analytical methods for the analysis of mRNA therapeutics is critical to underpin manufacturing development and the characterisation of the drug product and drug substance. In this study we have developed a high-throughput, high-performance liquid chromatography (HPLC) workflow for the rapid analysis of mRNA generated using in vitro transcription (IVT). We have optimised anion exchange (AEX) HPLC for the analysis of mRNA directly from IVT. Chromatography was performed in under 6 min enabling separation of all of the key components in the IVT, including nucleoside triphosphates (NTPs), Cap analogue, plasmid DNA and mRNA product. Moreover, baseline separation of the NTPs was achieved, which facilitates accurate quantification of each NTP such that their consumption may be determined during IVT reactions. Furthermore, the HPLC method was used to rapidly assess the purification of the mRNA product, including removal of NTPs/Cap analogue and other contaminants after downstream purification, including solid phase extraction (SPE), oligo deoxythymidine (oligo-dT) affinity chromatography and tangential flow filtration (TFF). Using the developed method excellent precision was obtained with calibration curves for an external mRNA standard and NTPs giving correlation coefficients of 0.98 and 1.0 respectively. Intra- and inter-day studies on retention time stability of NTPs, showed a relative standard deviation ≤ 0.3% and ≤1.5% respectively. The mRNA retention time variability was ≤0.13%. This method was then utilised to monitor the progress of an IVT reaction for the production of Covid spike protein (C-Spike) mRNA to measure the increasing yield of mRNA alongside the consumption of NTPs during the reaction.

Quality by Design Framework Applied to GMMA Purification.

In recent years, Generalized Modules for Membrane Antigens (GMMA) have received increased attention as an innovative vaccine platform against bacterial pathogens, particularly attractive for low- and middle-income countries because of manufacturing simplicity. The assessment of critical quality attributes (CQAs), product-process interactions, identification of appropriate in process analytical methods, and process modeling is part of a robust quality by design (QbD) framework to support further development and control of manufacturing processes. QbD implementation in the context of the GMMA platform will ensure robust manufacturing of batches with desired characteristics, facilitating technical transfer to local manufacturers, regulatory approval, and commercialization of vaccines based on this technology. Here, we summarize the methodology suggested, applied to a first step of GMMA manufacturing process.

Protection of Chinese painted quails (Coturnix chinensis) against a highly pathogenic H5N1 avian influenza virus strain after vaccination.

Chinese painted quails immunized with a single dose (6 µg HA) of inactivated H5N1 (clade 1) influenza vaccine NIBRG-14 and challenged with 100 LD50 of the heterologous A/Swan/Nagybaracska/01/06(H5N1) (clade 2.2) strain were protected, whereas unvaccinated quails died after challenge. No viral antigens or RNA were detected in cloacal swabs from immunized animals. Sera obtained post-immunization gave low titres in serological assays against the vaccine and the challenge viruses. Our results demonstrate the protective efficacy of the NIBRG-14 strain against the challenge virus and the usefulness of these small birds in protection studies of influenza vaccines.

Vaccines on demand, part II: future reality.

INTRODUCTION: Prior to the emergence of SARS-CoV-2, the potential use of mRNA vaccines for a rapid pandemic response had been well described in the scientific literature, however during the SARS-CoV-2 outbreak we witnessed the large-scale deployment of the platform in a real pandemic setting. Of the three RNA platforms evaluated in clinical trials, including 1) conventional, non-amplifying mRNA (mRNA), 2) base-modified, non-amplifying mRNA (bmRNA), which incorporate chemically modified nucleotides, and 3) self-amplifying RNA (saRNA), the bmRNA technology emerged with superior clinical efficacy. AREAS COVERED: This review describes the current state of these mRNA vaccine technologies, evaluates their strengths and limitations, and argues that saRNA may have significant advantages if the limitations of stability and complexities of manufacturing can be overcome. EXPERT OPINION: The success of the SARS-CoV-2 mRNA vaccines has been remarkable. However, several challenges remain to be addressed before this technology can successfully be applied broadly to other disease targets. Innovation in the areas of mRNA engineering, novel delivery systems, antigen design, and high-quality manufacturing will be required to achieve the full potential of this disruptive technology.

A novel ACE2 decoy for both neutralization of SARS-CoV-2 variants and killing of infected cells.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2-infected animal models but also for (ii) clearance of virus-infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule, which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor-modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Stability Modelling of mRNA Vaccine Quality Based on Temperature Monitoring throughout the Distribution Chain.

The vaccine distribution chains in several low- and middle-income countries are not adequate to facilitate the rapid delivery of high volumes of thermosensitive COVID-19 mRNA vaccines at the required low and ultra-low temperatures. COVID-19 mRNA vaccines are currently distributed along with temperature monitoring devices to track and identify deviations from predefined conditions throughout the distribution chain. These temperature readings can feed into computational models to quantify mRNA vaccine critical quality attributes (CQAs) and the remaining vaccine shelf life more accurately. Here, a kinetic modelling approach is proposed to quantify the stability-related CQAs and the remaining shelf life of mRNA vaccines. The CQA and shelf-life values can be computed based on the conditions under which the vaccines have been distributed from the manufacturing facilities via the distribution network to the vaccination centres. This approach helps to quantify the degree to which temperature excursions impact vaccine quality and can also reduce vaccine wastage. In addition, vaccine stock management can be improved due to the information obtained on the remaining shelf life of mRNA vaccines. This model-based quantification of mRNA vaccine quality and remaining shelf life can improve the deployment of COVID-19 mRNA vaccines to low- and middle-income countries.