Evaluation of the Interactions between Mumps Virus and Guinea Pig.

Mumps is a highly contagious viral disease that can be prevented by vaccination. In the last decade, we have encountered repeated outbreaks of mumps in highly vaccinated populations, which call into question the effectiveness of available vaccines. Animal models are crucial for understanding virus-host interactions, and viruses such as mumps virus (MuV), whose only natural host is the human, pose a particular challenge. In our study, we examined the interaction between MuV and the guinea pig. Our results present the first evidence that guinea pigs of the Hartley strain can be infected in vivo after intranasal and intratesticular inoculation. We observed a significant viral replication in infected tissues up to 5 days following infection and induction of cellular and humoral immune responses as well as histopathological changes in infected lungs and testicles, without clinical signs of disease. Transmission of the infection through direct contact between animals was not possible. Our results demonstrate that guinea pigs and guinea pig primary cell cultures represent a promising model for immunological and pathogenetic studies of the complex MuV infection. IMPORTANCE Understanding of mumps virus (MuV) pathogenesis and the immune responses against MuV infection is limited. One of the reasons is the lack of relevant animal models. This study explores the interaction between MuV and the guinea pig. We demonstrated that all tested guinea pig tissue homogenates and primary cell cultures are highly susceptible to MuV infection and that α2,3-sialylated glycans (MuV cellular receptors) are being abundantly expressed at their surface. The virus remains in the guinea pig lungs and trachea for up to 4 days following intranasal infection. Although asymptomatic, MuV infection strongly activates both humoral and cellular immune response in infected animals and provides protection against virus challenge. Infection of the lungs and testicles after intranasal and intratesticular inoculation, respectively, is also supported by histopathological changes in these organs. Our findings give perspective for application of guinea pigs in research on MuV pathogenesis, antiviral response, and vaccine development and testing.

ChAdOx1-S adenoviral vector vaccine applied intranasally elicits superior mucosal immunity compared to the intramuscular route of vaccination.

COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete protection against breakthrough infections. This could be due to suboptimal mucosal immunity at the site of virus entry, given that all currently approved vaccines are administered via the intramuscular route. In this study, we assessed humoral and cellular immune responses in BALB/c mice after intranasal and intramuscular immunization with adenoviral vector ChAdOx1-S expressing full-length Spike protein of SARS-CoV-2. We showed that both routes of vaccination induced a potent IgG antibody response, as well as robust neutralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in the sera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunized mice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularly immunized group. Moreover, substantially higher percentages of epitope-specific CD8 T cells exhibiting a tissue resident phenotype were found in the lungs of intranasally immunized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1-S efficiently protected the mice after the challenge with recombinant herpesvirus expressing the Spike protein. Our results demonstrate that intranasal application of adenoviral vector ChAdOx1-S induces superior mucosal immunity and therefore could be a promising strategy for putting the COVID-19 pandemic under control.

Multi-Detection Size Exclusion Chromatography as an Advanced Tool for Monitoring Enzyme-Antibody Conjugation Reaction and Quality Control of a Final Product.

The multi-detection size exclusion chromatography (SEC) has been recognized as an advanced analytical technique for the characterization of macromolecules and process control, as well as the manufacturing and formulation of biotechnology products. It reveals reproducible molecular characterization data, such as molecular weight and its distribution, and the size, shape, and composition of the sample peaks. The aim of this work was to investigate the potential and suitability of the multi-detection SEC as a tool for surveillance over the molecular processes during the conjugation reaction between the antibody (IgG) and horseradish peroxidase (HRP), and demonstrate the plausibility of its application in the quality control of the final product, the IgG-HRP conjugate. Guinea pig anti-Vero IgG-HRP conjugate was prepared using a modified periodate oxidation method, based on periodate oxidation of the carbohydrate side chains of HRP, followed by the formation of Schiff bases between the activated HRP and amino groups of the IgG. The quantitative molecular characterization data of the starting samples, intermediates, and final product were obtained by multi-detection SEC. Titration of the prepared conjugate was performed by the ELISA and its optimal working dilution was determined. This methodology proved to be a promising and powerful technology for the IgG-HRP conjugate process control and development, as well as for the quality control of the final product, as verified by the analysis of several commercially available reagents.

Development of Improved High-Performance Liquid Chromatography Method for the Determination of Residual Caprylic Acid in Formulations of Human Immunoglobulins.

Quality control of human immunoglobulin formulations produced by caprylic acid precipitation necessitates a simple, rapid, and accurate method for determination of residual caprylic acid. A high-performance liquid chromatography method for that purpose was developed and validated. The method involves depletion of immunoglobulins, the major interfering components that produce high background noise, by precipitation with acetonitrile (1:1, v/v). Chromatographic analysis of caprylic acid, preserved in supernatant with no loss, was performed using a reverse-phase C18 column (2.1 × 150 mm, 3 µm) as a stationary phase and water with 0.05% TFA-acetonitrile (50:50, v/v) as a mobile phase at a flow rate of 0.2 mL/min and run time of 10 min. The developed method was successfully validated according to the ICH guidelines. The validation parameters confirmed that method was linear, accurate, precise, specific, and able to provide excellent separation of peaks corresponding to caprylic acid and the fraction of remaining immunoglobulins. Furthermore, a 24-1 fractional factorial design was applied in order to test the robustness of developed method. As such, the method is highly suitable for the quantification of residual caprylic acid in formulations of human immunoglobulins for therapeutic use, as demonstrated on samples produced by fractionation of convalescent anti-SARS-CoV-2 human plasma at a laboratory scale. The obtained results confirmed that the method is convenient for routine quality control.

Compassionate mesenchymal stem cell treatment in a severe COVID-19 patient: a case report.

COVID-19 presentations range from cold-like symptoms to severe symptoms with the development of acute respiratory distress syndrome (ARDS). We report on a severe COVID-19 patient who was mechanically ventilated and who developed ARDS and bacterial infection. Because of rapid clinical deterioration and the exhaustion of other treatment options, the family and attending physicians requested a compassionate use of adult allogeneic bone marrow-derived mesenchymal stem cells (MSC) in addition to commonly used immunosuppressive, antiviral, and supportive therapy. The clinical course is discussed thoroughly, with a special emphasis on the safety and effect of MSC therapy. Compassionate MSC treatment, given in three rounds, affected ARDS regression. The patient was discharged from the intensive care unit after 31 days and from hospital after 49 days in a good general condition. MSC treatment was not associated with any side effects and was well tolerated in a three-week period; therefore, it should be studied in larger trials and considered for compassionate use.

Mass spectrometry-based investigation of measles and mumps virus proteome.

BACKGROUND: Measles (MEV) and mumps virus (MUV) are enveloped, non-segmented, negative single stranded RNA viruses of the family Paramyxoviridae, and are the cause of measles and mumps, respectively, both preventable by vaccination. Aside from proteins coded by the viral genome, viruses are considered to contain host cell proteins (HCPs). The presence of extracellular vesicles (ECVs), which are often co-purified with viruses due to their similarity in size, density and composition, also contributes to HCPs detected in virus preparations, and this has often been neglected. The aim was to identify which virus-coded proteins are present in MEV and MUV virions, and to try to detect which HCPs, if any, are incorporated inside the virions or adsorbed on their outer surface, and which are more likely to be a contamination from co-purified ECVs. METHODS: MUV, MEV and ECVs were purified by ultracentrifugation, hydrophobic interaction chromatography and immunoaffinity chromatography, proteins in the samples were resolved by SDS-PAGE and subjected to identification by MALDI-TOF/TOF-MS. A comparative analysis of HCPs present in all samples was carried out. RESULTS: By proteomics approach, it was verified that almost all virus-coded proteins are present in MEV and MUV particles. Protein C in MEV which was until now considered to be non-structural viral protein, was found to be present inside the MeV virions. Results on the presence of HCPs in differently purified virus preparations imply that actin, annexins, cyclophilin A, moesin and integrin ß1 are part of the virions. CONCLUSIONS: All HCPs detected in the viruses are present in ECVs as well, indicating their possible function in vesicle formation, or that most of them are only present in ECVs. Only five HCPs were constantly present in purified virus preparations, regardless of the purification method used, implying they are likely the integral part of the virions. The approach described here is helpful for further investigation of HCPs in other virus preparations.

Identification of mumps virus protein and lipid composition by mass spectrometry.

BACKGROUND: Mumps virus is a negative-sense, single stranded RNA virus consisting of a ribonucleocapsid core enveloped by a lipid membrane derived from host cell, which causes mumps disease preventable by vaccination. Since virus lipid envelope and glycosylation pattern are not encoded by the virus but dependent on the host cell at least to some extent, the aim of this work was to analyse L-Zagreb (L-Zg) mumps virus lipids and proteins derived from two cell types; Vero and chicken embryo fibroblasts (CEF). Jeryl Lynn 5 (JL5) mumps strain lipids were also analysed. METHODS: Virus lipids were isolated by organic phase extraction and subjected to 2D-high performance thin layer chromatography followed by lipid extraction and identification by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Virus samples were also subjected to gel electrophoresis under denaturating conditions and protein bands were excised, in-gel trypsinized and identified by MS as well as tandem MS. RESULTS: Results showed that lipids of both mumps virus strains derived from Vero cells contained complex glycolipids with up to five monosaccharide units whereas the lipid pattern of mumps virus derived from CEF was less complex. Mumps virus was found to contain expected structural proteins with exception of fusion (F) protein which was not detected but on the other hand, V protein was detected. Most interesting finding related to the mumps proteins is the detection of several forms of nucleoprotein (NP), some of which appear to be C-terminally truncated. CONCLUSIONS: Differences found in lipid and protein content of mumps virus demonstrated the importance of detailed biochemical characterization of mumps virus and the methodology described here could provide a means for a more comprehensive quality control in vaccine production.

Stability, biophysical properties and effect of ultracentrifugation and diafiltration on measles virus and mumps virus.

Measles virus and mumps virus (MeV and MuV) are enveloped RNA viruses used for production of live attenuated vaccines for prophylaxis of measles and mumps disease, respectively. For biotechnological production of and basic research on these viruses, the preparation of highly purified and infectious viruses is a prerequisite, and to meet that aim, knowledge of their stability and biophysical properties is crucial. Our goal was to carry out a detailed investigation of the stability of MeV and MuV under various pH, temperature, shear stress, filtration and storage conditions, as well as to evaluate two commonly used purification techniques, ultracentrifugation and diafiltration, with regard to their efficiency and effect on virus properties. Virus titers were estimated by CCID50 assay, particle size and concentration were measured by Nanoparticle tracking analysis (NTA) measurements, and the host cell protein content was determined by ELISA. The results demonstrated the stability of MuV and MeV at pH <9 and above pH 4 and 5, respectively, and aggregation was observed at pH >9. Storage without stabilizer did not result in structural changes, but the reduction in infectivity after 24 hours was significant at +37 °C. Vortexing of the viruses resulted in significant particle degradation, leading to lower virus titers, whereas pipetting had much less impact on virus viability. Diafiltration resulted in higher recovery of both total and infectious virus particles than ultracentrifugation. These results provide important data for research on all upstream and downstream processes on these two viruses regarding biotechnological production and basic research.

Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology.

Tetanus toxoid (TTd) is a highly immunogenic, detoxified form of tetanus toxin, a causative agent of tetanus disease, produced by Clostridium tetani. Since tetanus disease cannot be eradicated but is easily prevented by vaccination, the need for the tetanus vaccine is permanent. The aim of this work was to investigate the possibility of optimizing TTd purification, i.e., ammonium sulfate precipitation process. The influence of the percentage of ammonium sulfate, starting amount of TTd, buffer type, pH, temperature, and starting purity of TTd on the purification process were investigated using optimal design for response surface models. Responses measured for evaluation of the ammonium sulfate precipitation process were TTd amount (Lf/mL) and total protein content. These two parameters were used to calculate purity (Lf/mgPN) and the yield of the process. Results indicate that citrate buffer, lower temperature, and lower starting amount of TTd result in higher purities of precipitates. Gel electrophoresis combined with matrix-assisted laser desorption ionization-mass spectrometric analysis of precipitates revealed that there are no inter-protein cross-links and that all contaminating proteins have pIs similar to TTd, so this is most probably the reason for the limited success of purification by precipitation.

Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

An Unconventional Case Study of Neoadjuvant Oncolytic Virotherapy for Recurrent Breast Cancer.

Intratumoural oncolytic virotherapy may have promise as a means to debulk and downstage inoperable tumours in preparation for successful surgery. Here, we describe the unique case of a 50-year-old self-experimenting female virologist with locally recurrent muscle-invasive breast cancer who was able to proceed to simple, non-invasive tumour resection after receiving multiple intratumoural injections of research-grade virus preparations, which first included an Edmonston-Zagreb measles vaccine strain (MeV) and then a vesicular stomatitis virus Indiana strain (VSV), both prepared in her own laboratory. The intratumoural virus therapy was well tolerated. Frequent imaging studies and regular clinical observations documenting size, consistency and mobility of the injected tumour demonstrate that both the MeV- and VSV-containing parts of the protocol contributed to the overall favourable response. Two months after the start of the virus injections, the shrunken tumour was no longer invading the skin or underlying muscle and was surgically excised. The excised tumour showed strong lymphocytic infiltration, with an increase in CD20-positive B cells, CD8-positive T cells and macrophages. PD-L1 expression was detected in contrast to the baseline PD-L1-negative phenotype. The patient completed one-year trastuzumab adjuvant therapy and remains well and recurrence-free 45 months post-surgery. Although an isolated case, it encourages consideration of oncolytic virotherapy as a neoadjuvant treatment modality.

Snake Antivenoms-Toward Better Understanding of the Administration Route.

Envenomations induced by animal bites and stings constitute a significant public health burden. Even though a standardized protocol does not exist, parenterally administered polyclonal antivenoms remain the mainstay in snakebite therapy. There is a prevailing opinion that their application by the i.m. route has poor efficacy and that i.v. administration should preferentially be chosen in order to achieve better accomplishment of the antivenom therapeutic activity. Recently, it has been demonstrated that neutralization not only in the systemic circulation but also in the lymphatic system might be of great importance for the clinical outcome since it represents another relevant body compartment through which the absorption of the venom components occurs. In this review, the present-day and summarized knowledge of the laboratory and clinical findings on the i.v. and i.m. routes of antivenom administration is provided, with a special emphasis on the contribution of the lymphatic system to the process of venom elimination. Until now, antivenom-mediated neutralization has not yet been discussed in the context of the synergistic action of both blood and lymph. A current viewpoint might help to improve the comprehension of the venom/antivenom pharmacokinetics and the optimal approach for drug application. There is a great need for additional dependable, practical, well-designed studies, as well as more practice-related experience reports. As a result, opportunities for resolving long-standing disputes over choosing one therapeutic principle over another might be created, improving the safety and effectiveness of snakebite management.

What can neutralizing antibodies tell us about the quality of immunity in COVID-19 convalescents and vaccinees?

During the SARS-CoV-2 pandemic, the lack of standardized measurements of the immune response after vaccination or recovery from COVID-19 resulted in incomparable results and hindered correlation establishment. Prioritizing reliable and standardized methods to monitor pathogen-specific immunity is crucial, not only during the COVID-19 pandemic but also for future outbreaks. During our study of the humoral immune response, we used a SARS-CoV-2 wild-type neutralization assay, ensuring the measurement of the immune response directed to all SARS-CoV-2 antigens in their proper conformation. A head-to-head comparison of the neutralizing antibody (NAb) responses elicited by four vaccines used in Europe during 2021 (BNT162b2, mRNA-1273, ChAdOx nCoV-19, and Ad26.COV2.S) and their comparison to NAb responses in convalescents showed that while the amount was comparable, NAbs induced by natural infection were of higher quality. Namely, NAbs produced by disease were better activators of the complement system than NAbs induced by vaccination. Furthermore, the contribution of spike protein-specific IgGs to the SARS-CoV-2 neutralization was lower in convalescents compared to vaccinees, indicating that those who recovered from COVID-19 were armed with antibodies of additional specificities and/or classes that contributed to virus neutralization. These findings suggest that a higher stringency of public policy measures targeting individuals who have recovered from COVID-19, in comparison to those who have been vaccinated, may not have been fully justified.

Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins.

The ammodytoxins (Atxs) are neurotoxic phospholipases which occur in Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms, A, B, and C, which differ in only five amino acid positions at the C-terminus but differ substantially in their toxicity. The objective of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method to assess a procedure for purification of the most toxic phospholipase, AtxA, from the venom. Isolation procedure for AtxA consisted of isolation of Atx-cross-reactive material (proteins recognized by anti-Atx antibodies), by use of an affinity column, then cation exchange on CIM (Convective Interaction Media) disks. The purification procedure was monitored by means of reversed-phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of the pure isoforms enabled separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate the Atx isoforms, whereas an MS based approach proved to be more powerful. Peptides resulting from tryptic digestion of Atxs which enable differentiation between the three isoforms were successfully detected and their sequences were confirmed by post-source decay (PSD) fragmentation. Separation of Atx isoforms by ion-exchange chromatography is most presumably prevented by Atxs heterodimer formation. The tendency of Atxs to form homodimers and heterodimers of similar stability was confirmed by molecular modeling.

Roughness of Production Conditions: Does It Really Affect Stability of IgG-Based Antivenoms?

Antivenoms contain either pure animal IgGs or their fragments as an active substance, and are the only specific therapeutics against envenomation arising from snakebites. Although they are highly needed, the low sustainability of such preparations' manufacture causes constant global shortages. One reason for this is the stability of the product, which contributes not only to the manufacture sustainability, but the product safety as well. It has been hypothesized that the roughness of conditions to which IgGs are exposed during downstream purification disturbs their conformation, making them prone to aggregation, particularly after exposure to secondary stress. The aim of this research was to investigate how the roughness of the downstream purification conditions influences the stability properties of purified IgGs. For this purpose, equine IgGs were extracted from unique hyperimmune plasma by two mild condition-based operational procedures (anion-exchange chromatography and caprylic acid precipitation) and three rougher ones (ammonium sulphate precipitation, cation-exchange chromatography and protein A affinity chromatography). The stability of the refined preparations was studied under non-optimal storage conditions (37 °C, 42 °C, and a transiently lower pH) by monitoring changes in the aggregate content and thermal stability of the pure IgGs. Mild purification protocols generated IgG samples with a lower aggregate share in comparison to the rougher ones. Their tendency for further aggregation was significantly associated with the initial aggregate share. The thermal stability of IgG molecules and the aggregate content in refined samples were inversely correlated. Since the initial proportion of aggregates in the samples was influenced by the operating conditions, we have shown a strong indication that each of them also indirectly affected the stability of the final preparations. This suggests that mild condition-based refinement protocols indeed generate more stable IgGs.

Efficient and Sustainable Platform for Preparation of a High-Quality Immunoglobulin G as an Urgent Treatment Option During Emerging Virus Outbreaks.

During the pre-vaccine era of the COVID-19 pandemic convalescent plasma has once again emerged as a major potential therapeutic form of passive immunization that in specific cases still represents irreplaceable treatment option. There is a growing concern that variable concentration of neutralizing antibodies, present in convalescent plasma which originates from different donors, apparently affects its effectiveness. The drawback can be overcome through the downstream process of immunoglobulin fraction purification into a standardized product of improved safety and efficacy. All modern procedures are quite lengthy processes. They are also based on fractionation of large plasma quantities whose collection is not attainable during an epidemic. When outbreaks of infectious diseases are occurring more frequently, there is a great need for a more sustainable production approach that would be goal-oriented towards assuring easily and readily available immunoglobulin of therapeutic relevance. We propose a refinement strategy for the IgG preparation achieved through simplification and reduction of the processing steps. It was designed as a small but scalable process to offer an immediately available treatment option that would simultaneously be harmonized with an increased availability of convalescent plasma over the viral outbreak time-course. Concerning the ongoing pandemic status of the COVID-19, the proof of concept was demonstrated on anti-SARS-CoV-2 convalescent plasma but is likely applicable to any other type depending on the current needs. It was guided by the idea of persistent keeping of IgG molecules in the solution, so that protection of their native structure could be assured. Our manufacturing procedure provided a high-quality IgG product of above the average recovery whose composition profile was analyzed by mass spectrometry as quality control check. It was proved free from IgA and IgM as mediators of adverse transfusion reactions, as well as of any other residual impurities, since only IgG fragments were identified. The proportion of S protein-specific IgGs remained unchanged relative to the convalescent plasma. Undisturbed IgG subclass composition was accomplished as well. However, the fractionation principle affected the final product's capacity to neutralize wild-type SARS-CoV-2 infectivity, reducing it by half. Decrease in neutralization potency significantly correlated with the amount of IgM in the starting material.

Is Better Standardization of Therapeutic Antibody Quality in Emerging Diseases Epidemics Possible?

During the ongoing COVID-19 epidemic many efforts have gone into the investigation of the SARS-CoV-2-specific antibodies as possible therapeutics. Currently, conclusions cannot be drawn due to the lack of standardization in antibody assessments. Here we describe an approach of establishing antibody characterisation in emergent times which would, if followed, enable comparison of results from different studies. The key component is a reliable and reproducible assay of wild-type SARS-CoV-2 neutralisation based on a banking system of its biological components - a challenge virus, cells and an anti-SARS-CoV-2 antibody in-house standard, calibrated to the First WHO International Standard immediately upon its availability. Consequently, all collected serological data were retrospectively expressed in an internationally comparable way. The neutralising antibodies (NAbs) among convalescents ranged from 4 to 2869 IU mL-1 in a significant positive correlation to the disease severity. Their decline in convalescents was on average 1.4-fold in a one-month period. Heat-inactivation resulted in 2.3-fold decrease of NAb titres in comparison to the native sera, implying significant complement activating properties of SARS-CoV-2 specific antibodies. The monitoring of NAb titres in the sera of immunocompromised COVID-19 patients that lacked their own antibodies evidenced the successful transfusion of antibodies by the COVID-19 convalescent plasma units with NAb titres of 35 IU mL-1 or higher.

Modern venomics-Current insights, novel methods, and future perspectives in biological and applied animal venom research.

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.

Production- and Purification-Relevant Properties of Human and Murine Cytomegalovirus.

There is a large unmet need for a prophylactic vaccine against human cytomegalovirus (HCMV) to combat the ubiquitous infection that is ongoing with this pathogen. A vaccination against HCMV could protect immunocompromised patients and prevent birth defects caused by congenital HCMV infections. Moreover, cytomegalovirus (CMV) has a number of features that make it a very interesting vector platform for gene therapy. In both cases, preparation of a highly purified virus is a prerequisite for safe and effective application. Murine CMV (MCMV) is by far the most studied model for HCMV infections with regard to the principles that govern the immune surveillance of CMVs. Knowledge transfer from MCMV and mice to HCMV and humans could be facilitated by better understanding and characterization of the biological and biophysical properties of both viruses. We carried out a detailed investigation of HCMV and MCMV growth kinetics as well as stability under the influence of clarification and different storage conditions. Further, we investigated the possibilities to concentrate and purify both viruses by ultracentrifugation and ion-exchange chromatography. Defective enveloped particles were not separately analyzed; however, the behavior of exosomes was examined during all experiments. The effectiveness of procedures was monitored using CCID50 assay, Nanoparticle tracking analysis, ELISA for host cell proteins, and quantitative PCR for host cell DNA. MCMV generally proved to be more robust in handling. Despite its greater sensitivity, HCMV was efficiently (100% recovery) purified and concentrated by anion-exchange chromatography using QA monolithic support. The majority of the host genomic DNA as well as most of the host cell proteins were removed by this procedure.

Impact of complement and difference of cell-based assay and ELISA in determination of neutralization capacity against mumps and measles virus.

Neutralizing antibodies against mumps and measles virus are considered a correlate of protection against these diseases. Measurement of neutralizing antibodies is mostly performed using plaque reduction neutralization assay or 50% cell culture infective dose (CCID50) neutralization assay, but there are attempts for measuring neutralizing antibodies using enzyme-linked immunosorbent assay (ELISA) which is simpler, but the literature data regarding its convenience are diverse. The role of complement and antibodies in neutralizing capacity of sera is not completely defined. Here, CCID50 neutralization assay and ELISA were used to determine the neutralization capacity against mumps and measles virus in human sera and therapeutic immunoglobulins (IVIGs). Results showed no correlation of neutralization titers obtained by CCID50 neutralization assay and IgG content obtained by ELISA for mumps or measles in human sera. Data showed some neutralization activity against measles virus and quite high against mumps virus of naïve guinea pig serum and that its addition increases neutralization capacity of IVIG and human sera against mumps and measles viruses. Heat inactivation of human sera reduced neutralization capacity against measles to small extent, and substantially against mumps virus. There is a significant impact of complement in measurement of neutralization capacity against mumps virus.