Recombinant Particles Research Articles

Self-Replicating RNA Viruses for Vaccine Development against Infectious Diseases and Cancer.

Alphaviruses, flaviviruses, measles viruses and rhabdoviruses are enveloped single-stranded RNA viruses, which have been engineered for recombinant protein expression and vaccine development. Due to the presence of RNA-dependent RNA polymerase activity, subgenomic RNA can replicate close to 106 copies per cell for translation in the cytoplasm providing extreme transgene expression levels, which is why they are named self-replicating RNA viruses. Expression of surface proteins of pathogens causing infectious disease and tumor antigens provide the basis for vaccine development against infectious diseases and cancer. Self-replicating RNA viral vectors can be administered as replicon RNA at significantly lower doses than conventional mRNA, recombinant particles, or DNA plasmids. Self-replicating RNA viral vectors have been applied for vaccine development against influenza virus, HIV, hepatitis B virus, human papilloma virus, Ebola virus, etc., showing robust immune response and protection in animal models. Recently, paramyxovirus and rhabdovirus vector-based SARS-CoV-2 vaccines as well as RNA vaccines based on self-amplifying alphaviruses have been evaluated in clinical settings. Vaccines against various cancers such as brain, breast, lung, ovarian, prostate cancer and melanoma have also been developed. Clinical trials have shown good safety and target-specific immune responses. Ervebo, the VSV-based vaccine against Ebola virus disease has been approved for human use.

Phage display as a tool for identifying HIV-1 broadly neutralizing antibodies.

Combinatorial biology methods offer a good solution for targeting interactions of specif ic molecules by a high-throughput screening and are widely used for drug development, diagnostics, identif ication of novel monoclonal antibodies, search for linear peptide mimetics of discontinuous epitopes for the development of immunogens or vaccine components. Among all currently available techniques, phage display remains one of the most popular approaches. Despite being a fairly old method, phage display is still widely used for studying protein-protein, peptide-protein and DNA-protein interactions due to its relative simplicity and versatility. Phage display allows highly representative libraries of peptides, proteins or their fragments to be created. Each phage particle in a library displays peptides or proteins fused to its coat protein and simultaneously carries the DNA sequence encoding the displayed peptide/protein in its genome. The biopanning procedure allows isolation of specif ic clones for almost any target, and due to the physical link between the genotype and the phenotype of recombinant phage particles it is possible to determine the structure of selected molecules. Phage display technology continues to play an important role in HIV research. A major obstacle to the development of an effective HIV vaccine is an extensive genetic and antigenic variability of the virus. According to recent data, in order to provide protection against HIV infection, the so-called broadly neutralizing antibodies that are cross-reactive against multiple viral strains of HIV must be induced, which makes the identif ication of such antibodies a key area of HIV vaccinology. In this review, we discuss the use of phage display as a tool for identif ication of HIV-specif ic antibodies with broad neutralizing activity. We provide an outline of phage display technology, brief ly describe the design of antibody phage libraries and the affinity selection procedure, and discuss the biology of HIV-1-specif ic broadly neutralizing antibodies. Finally, we summarize the studies aimed at identif ication of broadly neutralizing antibodies using various types of phage libraries.

Lentiviral transduction of neonatal rat ventricular myocytes preserves ultrastructural features of genetically modified cells

Preserving morphological features that are important for cell function and structure is a critical parameter for in vitro experiments with rat cardiomyocytes. Lentiviral vectors are commonly used as gene transfer tool because of its high flexibility, efficiency to deliver expression cassettes and versatility of transducing quiescent cells. The tropism of the recombinant viral particle can be determined depending on the virus envelope, which shows a specific binding to cell surface receptors on the target cell. The combination of promoter arrangement and viral envelope must be optimized to achieve a greater transduction efficiency and a higher transgene expression. In this study we explored the optimization of promoters and heterologous envelopes to transduce primary culture of neonatal rat ventricular myocytes. Our results suggest a robust expression driven by the cytomegalovirus promoter, and high efficiency transduction mediated by VSV-G envelope with no apparent compromising ultrastructural features of genetically modified cells.

A β-Hairpin Motif in the Envelope Protein E2 Mediates Receptor Binding of Bovine Viral Diarrhea Virus.

Pestivirus envelope protein E2 is crucial to virus infection and accomplishes virus-receptor interaction during entry. However, mapping of E2 residues mediating these interactions has remained unexplored. In this study, to investigate the structure-function relationship for a β-hairpin motif exposed to the solvent in the crystal structure of bovine viral diarrhea virus (BVDV) E2, we designed two amino acidic substitutions that result in a change of electrostatic potential. First, using wild type and mutant E2 expressed as soluble recombinant proteins, we found that the mutant protein had reduced binding to susceptible cells compared to wild type and diminished ability to inhibit BVDV infection, suggesting a lower affinity for BVDV receptors. We then analyzed the effect of β-hairpin mutations in the context of recombinant viral particles. Mutant viruses recovered from cell culture supernatant after transfection of recombinant RNA had almost completely inhibited ability to re-infect susceptible cells, indicating an impact of mutations on BVDV infectivity. Finally, sequential passaging of the mutant virus resulted in the selection of a viral population in which β-hairpin mutations reverted to the wild type sequence to restore infectivity. Taken together, our results show that this conserved region of the E2 protein is critical for the interaction with host cell receptors.

Call for More Effective Regulation of Clinical Trials with Advanced Therapy Medicinal Products Consisting of or Containing Genetically Modified Organisms in the European Union.

Advanced therapy medicinal products (ATMPs), such as gene therapies that consist of or contain genetically modified organisms (GMOs) need to comply with the European Union (EU) GMO legislation, as implemented in each EU Member State, before a clinical trial can commence. Complying with GMO requirements is complex, varies significantly across EU Member States and is leading to delays to clinical trials with ATMPs. Such delays and varying implementation of the GMO legislation makes the EU less attractive as a region to conduct clinical trials with investigational gene therapies. This is detrimental to EU patients, since their timely access to these transformative potentially curative medicines is delayed. Despite recent initiatives coordinated by the European Commission (EC) to facilitate and reduce discrepancies across the EU regarding the application of the GMO requirements, it remains particularly difficult to conduct multicenter clinical trials with ATMPs containing or consisting of GMOs involving several EU Member States. The recent decision for the EC to temporarily derogate potential coronavirus disease 2019 treatments and vaccines from some provisions of the GMO requirements was made on the basis of a clear recognition of such complexities and resulting delays to clinical development. The Alliance for Regenerative Medicine, the European Federation of Pharmaceutical Industries and Associations, and the European Association for Bioindustries call upon the EC, together with national competent authorities, to exempt ATMPs containing or consisting of GMOs from the GMO legislation. Such a simplification will eliminate the delays currently reported to occur when submitting environmental risk assessments and GMO applications to the national competent authorities. An exemption from GMO requirements will make the EU a more attractive region for clinical development of gene therapies and could accelerate European patients' access to these potentially life-saving medicines. Maintaining a system for GMO assessment that is different across countries may also prevent ATMPs from realizing the full benefits of a harmonized clinical trial approval process under the Clinical Trials Regulation. The undersigned organizations to this publication urge the EC to use its right of initiative to put forward a legislative proposal to exempt ATMPs in clinical development from the EU GMO legislation, within the timeframe proposed in the 2020 EU Pharmaceutical Strategy (by 2022). Implementation of a GMO exemption scheme before the end of the transition period for the Clinical Trial Regulation (the end of 2023) is important to avoid new Clinical Trial Application submissions for ATMPs under the Clinical Trial Regulation having to conduct the whole GMO assessment process in parallel. It is considered that ATMPs pose negligible risk to the environment. Such ATMPs include the following: human somatic cells modified ex vivo; recombinant virus-based vectors, including those containing genome editing nucleic acid sequences (which may also be delivered nonvirally); and bacterial vectors. Outside of controlled storage conditions, gene therapies cannot survive for any appreciable length of time. Upon clinical administration, any recombinant gene therapy viral vector particles that do not enter host cells are diluted within the body and if excreted are in such low multiplicity to no longer be viable or considered infectious to persons, animals, or living organisms within the environment. Any nucleic acids released into the environment are rapidly degraded.

Monoclonal antibody against VP0 recognizes a broad range of human parechoviruses

Parechoviruses (PeVs) are common viruses that cause mild gastrointestinal or respiratory symptoms to severe central nervous system infections. In infants, parechovirus infection is one of the leading causes of life-threatening viral disease. High-quality antibodies with broad binding specificities are essential to improve accurate parechovirus diagnosis in diagnostic laboratories. Such antibodies have potential in the development of rapid antigen detection assay against PeVs. In the present study, VP4 and VP2 genes from human parechovirus A1 (PeV-A1) were cloned and VP0 fusion protein produced to develop monoclonal antibodies against PeVs. Two pan-parechovirus antibodies, one IgG and one IgM isotype, were isolated. The properties of IgG1/κ monoclonal (designated as Mab-PAR-1) was studied further. Mab-PAR-1 was shown to be functional in western blot against denatured recombinant protein and viral particles. In immunofluorescence assay, the antibody tested positive for nineteen PeV-A1 isolates while showing no cross-reactivity to fourteen entero- and rhinovirus types. In addition, Mab-PAR-1 showed positive reactivity against five other cultivable parechovirus types 2−6. A unique Mab-PAR-1 epitope located in the junction of the three capsid proteins VP0, VP1, and VP3 was identified using a peptide library screen. This study demonstrates that PeV-A1-VP0 protein is functional antigen for developing monoclonal antibody for diagnosis of broad range of parechovirus infections.

Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors.

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03323-6.

RNA Interference Screening Reveals Requirement for Platelet-Derived Growth Factor Receptor Beta in Japanese Encephalitis Virus Infection

Mosquito-borne Japanese encephalitis virus (JEV) causes serious illness worldwide and is associated with high morbidity and mortality. To identify potential host therapeutic targets, a high-throughput receptor tyrosine kinase small interfering RNA library screening was performed with recombinant JEV particles. Platelet-derived growth factor receptor beta (PDGFRβ) was identified as a hit after two rounds of screening. Knockdown of PDGFRβ blocked JEV infection and transcomplementation of PDGFRβ could partly restore its infectivity. The PDGFRβ inhibitor imatinib, which has been approved for the treatment of malignant metastatic cancer, protected mice against JEV-induced lethality by decreasing the viral load in the brain while abrogating the histopathological changes associated with JEV infection. These findings demonstrated that PDGFRβ is important in viral infection and provided evidence for the potential to develop imatinib as a therapeutic intervention against JEV infection.

The expression in plants of an engineered VP2 protein of Infectious Bursal Disease Virus induces formation of structurally heterogeneous particles that protect from a very virulent viral strain.

Infectious Bursal Disease Virus (IBDV), the etiological agent of Gumboro disease, causes mortality and immunosuppression in chickens and major losses to poultry industry worldwide. The IBDV major capsid protein VP2 is considered the best candidate for the production of novel subunit vaccines. This structural protein contains the major conformational epitopes responsible for the induction of IBDV neutralizing antibodies in chickens and has been demonstrated able to form supramolecular structures in yeast and insect cells. The aim of this study was to express an engineered version of the VP2 protein (His-pVP2) to verify its ability to self-assemble into virus-like particles in plants. The recombinant VP2 was transiently expressed by agroinfiltration in Nicotiana benthamiana and transmission electron microscopy of sucrose density gradient fractions revealed the presence of a mixed population of differently shaped particles ranging from spherical capsids, with a diameter between ~25 and ~70 nm, to tubular structures, with variable length (from 100 to 400 nm). The recombinant VP2-based particles when used for the intramuscular immunization of specific-pathogen-free chicks resulted able to induce the production of anti-IBDV specific antibodies at titers comparable to those induced by a commercial vaccine. Moreover, all the immunized birds survived to the challenge with a Moroccan very virulent IBDV strain with no major histomorphological alterations of the Bursa of Fabricius, similarly to what obtained with the commercial inactivated vaccine.

The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients

In order to control the COVID-19 pandemic caused by SARS-CoV-2 infection, serious progress has been made to identify infected patients and to detect patients with a positive immune response against the virus. Currently, attempts to generate a vaccine against the coronavirus are ongoing. To understand SARS-CoV-2 immunoreactivity, we compared the IgG antibody response against SARS-CoV-2 in infected versus control patients by dot blot using recombinant viral particle proteins: N (Nucleocapsid), M (Membrane) and S (Spike). In addition, we used different protein fragments of the N and S protein to map immune epitopes. Most of the COVID-19 patients presented a specific immune response against the full length and fragments of the N protein and, to lesser extent, against a fragment containing amino acids 300–685 of the S protein. In contrast, immunoreactivity against other S protein fragments or the M protein was low. This response is specific for COVID-19 patients as very few of the control patients displayed immunoreactivity, likely reflecting an immune response against other coronaviruses. Altogether, our results may help develop method(s) for measuring COVID-19 antibody response, selectivity of methods detecting such SARS-CoV-2 antibodies and vaccine development.

Bone formation potential of collagen type I-based recombinant peptide particles in rat calvaria defects.

IntroductionThis study aimed to examine the bone-forming ability of medium-cross-linked recombinant collagen peptide (mRCP) particles developedbased on human collagen type I, contains an arginyl-glycyl-aspartic acid-rich motif, fabricated as bone filling material, compared to that of the autologous bone graft.MethodsCalvarial bone defects were created in immunodeficient rats though a surgical procedure. The rats were divided into 2 groups: mRCP graft and tibia bone graft (bone graft). The bone formation potential of mRCP was evaluated by micro-computed tomography and hematoxylin-eosin staining at 1, 2, 3, and 4 weeks after surgery, and the data were analyzed and compared to those of the bone graft.ResultsThe axial volume-rendered images demonstrated considerable bony bridging with the mRCP graft, but there was no significant difference in the bone volume and bone mineral density between the mRCP graft and bone graft at 4 weeks. The peripheral new bone density was significantly higher than the central new bone density and the bottom side score was significantly higher than the top side score at early stage in the regenerated bone within the bone defects.ConclusionThese results indicate that mRCP has a high potential of recruiting osteogenic cells, comparable to that of autologous bone chips.

Fabrication of α-S/CdS Composites for Photocatalytic Hydrogen Production Under Visible Light

Energy issue is the one of the environmental problems. In particular, dependence on fossil fuels such as oil, coal, and natural gas should be resolved due to depletion and greenhouse gas emission such as CO2 after energy conversion. Hydrogen is attracting attention as one of the energies as a substitute for fossil fuels because of not emission CO2, abundant resources, and high energy efficiency. However, in the steam reforming, the current main method of hydrogen generation, fossil fuels are used as the raw material, so CO2 is emitted. Therefore, establishment of environmentally friendly generation method is desired.Photocatalysts are substances that absorb light energy and proceed various chemical reactions to proceed. One of the functions of photocatalyst is the generation of hydrogen from water. If the light energy can be supplemented by sunlight, it will be possible to generate hydrogen from natural energy, which is a very attractive hydrogen generation method. Therefore, ideal photocatalysts should at least have the characteristics of absorbing visible light, which accounts for the majority of solar energy, suitable band edges for targeted reactions, good stability in water, and low cost.α-sulfur (α-S) crystals of cyclo-octasulfur (S8) are a visible-light-responsive elemental photocatalyst. Although, ionic sulfur is widely used in sulfide photocatalysts or as a dopant in oxide photocatalysts, the possibility of using elemental sulfur as a photocatalyst has been rarely reported. The α-S crystals were found to have the ability not only to generate・OH radicals to degradation of organic dye but also to generate hydrogen by split water in a photoelectrochemical process under both UV−vis and visible-light irradiation. Although the hydrogen production efficiency was low because of fast charge recombination, poor hydrophilicity and large particle size of the α-S crystals studied, there is great potential for increasing the activity with the assistance of known strategies such as surface modification, nanoscaling, doping, and coupling with other photocatalysts.In this study, we focused on coupling with cadmium sulfide (CdS) to improve the hydrogen production efficiency because CdS is one of the popular visible-light-responsive photocatalyst, hydrophilicity, and has the band structure suitable for charge separation to α-S.α-S is synthesized by the decomposition of sodium thiosulfate in acetic acid solution. CdS is synthesized by the simple method of adding sodium sulfide (Na2S) to a cadmium nitrate solution and drying overnight. In addition, α-S/CdS composite is synthesized by replacing Na2S used in CdS synthesis process with potassium sulfide (K2S). The sample of α-S8/CdS composite were named X:Y. (X and Y are molar ratio of Na2S and K2S )The identification and structure of the prepared sample were analyzed by XRD and IR. Absorbed light wavelength was measured by UV-vis DRS. Surface condition was observed by SEM and TEM. The hydrogen generation experiment was conducted in a closed circulation system using the reaction solution with sulfide sacrificial reagent, and 300 W Xe lamp was used with a cut filter to irradiate visible light. (λ≧420nm)From the hydrogen generation experiment, 30:70 sample showed about 1300 and 2.6 times higher hydrogen generation efficiency than those of pure α-S and CdS. Based on the measured band gaps and the flat band potentials of the pure materials, a mechanism for electron and hole separation in α-S/CdS is proposed as shown in below figure. The valence band level of CdS is higher than the corresponding bands in α-S, so oxidation reaction of sulfur compounds is carried out at valence band of CdS. In addition, the level of the conduction band of α-S is lower than that of CdS, so hydrogen production reaction is carried out at conduction band of α-S.Through this study, we succeeded in preparing a new composite photocatalyst of α-S/CdS by a simple method and confirming improve of hydrogen production efficiency of α-S by coupling with other photocatalysts. CdS exhibits decomposition sulfur compounds such as hydrogen sulfide, so we aim for hydrogen sulfide decomposition in tandem with hydrogen generation in this composite photocatalysts. Furthermore, using this model, we expect that even more composite photocatalysts with even higher efficiencies will be discovered. Figure 1

The race for a coronavirus vaccine

The international race to find a preventive vaccine and effective treatments against COVID 19 has been influenced by two fundamental factors. Firstly, by the molecular characterization of the causative virus and the pathology it produces, and secondly, by access to this information, mostly free of charge by the international scientific community causing a synergy to obtain results in such a short time. Several vaccines preparations against Covid19 have entered Phase 3 clinical trials. Although it is uncertain the degree of protection that they will achieve, preliminary data from Phase 1 and 2 trials and studies in animals indicate that they trigger an antiviral immune response without serious side effects. The current formulations include viral vectors, RNA vaccines, inactivated viruses, and recombinant proteins particles. They all have advantages and disadvantages, but only the results of Phase 3 clinical trials will ultimately decide the best candidates for vaccination campaigns. The tremendous impact of the SARS-CoV-2 in our society has triggered an unprecedented effort to find a vaccine to control the pandemic. Billions of dollars have already been invested in multiple vaccination schemes. According to the WHO, more than 170 vaccines were in different phases of development in August 2020. Here is a summary of the advantages and disadvantages of the front runner strategies categorized according to their delivery method.

Alphavirus-Based Antigen Preparation.

Alphavirus-based vectors present an efficient approach for antigen preparation applied for vaccine development. Semliki Forest virus, Sindbis virus, and Venezuelan equine encephalitis virus have been engineered for high-level expression of antigens targeting infectious diseases and tumors. Alphaviruses possess a large application range as vectors can be delivered as naked RNA replicons, recombinant viral particles, and layered DNA plasmids. Immunization studies in animal models have provided protection against challenges with lethal doses of pathogenic infectious agents and tumor cells. So far, a limited number of clinical trials have been conducted for alphavirus vectors in humans.

A Scalable Topical Vectored Vaccine Candidate against SARS-CoV-2.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) caused an ongoing unprecedented global public health crises of coronavirus disease in 2019 (CoVID-19). The precipitously increased death rates, its impact on livelihood and trembling economies warrant the urgent development of a SARS-CoV-2 vaccine which would be safe, efficacious and scalable. Owing to unavailability of the vaccine, we propose a de novo synthesized avian orthoavulavirus 1 (AOaV-1)-based topical respiratory vaccine candidate against CoVID-19. Avirulent strain of AOaV-1 was engineered to express full length spike (S) glycoprotein which is highly neutralizing and a major protective antigen of the SARS-CoV-2. Broad-scale in vitro characterization of a recombinant vaccine candidate demonstrated efficient co-expression of the hemagglutinin-neuraminidase (HN) of AOaV-1 and S protein of SARS-CoV-2, and comparable replication kinetics were observed in a cell culture model. The recombinant vaccine candidate virus actively replicated and spread within cells independently of exogenous trypsin. Interestingly, incorporation of S protein of SARS-CoV-2 into the recombinant AOaV-1 particles attributed the sensitivity to anti-SARS-CoV-2 antiserum and more prominently to anti-AOaV-1 antiserum. Finally, our results demonstrated that the recombinant vaccine vector stably expressed S protein after multiple propagations in chicken embryonated eggs, and this expression did not significantly impact the in vitro growth characteristics of the recombinant. Taken together, the presented respiratory vaccine candidate is highly attenuated in primates per se, safe and lacking pre-existing immunity in human, and carries the potential for accelerated vaccine development against CoVID-19 for clinical studies.

TWIST1 upregulates matrix metalloproteinase (MMP) genes family in esophageal squamous carcinoma cells

Twist-related protein 1 (TWIST1), a highly conserved basic helix-loop-helix transcription factor, stimulates epithelial-mesenchymal transition (EMT) and plays a crucial role in the regulation of the extracellular matrix (ECM) and cell-cell adhesion. Our aim in this study was to evaluate the functional correlation between TWIST1 and MMP genes in human ESCC cell lines, KYSE-30 and YM-1. To generate recombinant retroviral particles, the Pruf-IRES-GFP-hTWIST1 was co-transfected into HEK293T along with pGP and pMD2. G as well as Pruf-IRES-GFP control plasmid. Stably transduced high-expressing GFP-hTWIST1 and GFP-control KYSE-30 cells were generated. The produced retroviral particles were transduced into the KYSE-30 and YM-1 ESCC cells. Ectopic expression of TWIST1 mRNA and expression of the MMP genes (MMP-2, MMP-3, MMP-7, MMP-9, and MMP-10) were examined by relative comparative real-time PCR. In silico analysis of the MMP markers and their promoter elements was explored. Moreover, the scratch wound assay was used to evaluate the migration of TWIST1-induced cells. TWIST1 level was up-regulated by nearly 5-fold and 7.4-fold in GFP-hTWIST1 KYSE-30 and YM-1 cells compared to GFP control cells, respectively. Interestingly, this enforced expression of TWIST1 subsequently caused significant overexpression of transcripts for selected MMP genes in GFP-hTWIST1 in comparison with GFP control cells in both ESCC cell lines. Also, the scratch assay indicated that TWIST1 expression effectively increased the migration of GFP-TWIST1 KYSE-30 cells against GFP KYSE-30 control cells in vitro. The present findings illuminate that TWIST1 may contribute broadly to ESCC development in concert with up-regulation of MMPs expression and further suggest the potential advantage of exerting TWIST1/MMPs signaling axis as a framework from which to expand our understanding about the mechanisms of ESCC tumorigenesis.

Self-Amplifying RNA Viruses as RNA Vaccines

Single-stranded RNA viruses such as alphaviruses, flaviviruses, measles viruses and rhabdoviruses are characterized by their capacity of highly efficient self-amplification of RNA in host cells, which make them attractive vehicles for vaccine development. Particularly, alphaviruses and flaviviruses can be administered as recombinant particles, layered DNA/RNA plasmid vectors carrying the RNA replicon and even RNA replicon molecules. Self-amplifying RNA viral vectors have been used for high level expression of viral and tumor antigens, which in immunization studies have elicited strong cellular and humoral immune responses in animal models. Vaccination has provided protection against challenges with lethal doses of viral pathogens and tumor cells. Moreover, clinical trials have demonstrated safe application of RNA viral vectors and even promising results in rhabdovirus-based phase III trials on an Ebola virus vaccine. Preclinical and clinical applications of self-amplifying RNA viral vectors have proven efficient for vaccine development and due to the presence of RNA replicons, amplification of RNA in host cells will generate superior immune responses with significantly reduced amounts of RNA delivered. The need for novel and efficient vaccines has become even more evident due to the global COVID-19 pandemic, which has further highlighted the urgency in challenging emerging diseases.

Identification of SARS-CoV-2 in a Proficiency Testing Program.

ObjectivesAt the onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in the United States, testing was limited to the Centers for Disease Control and Prevention–developed reverse transcription polymerase chain reaction assay. The urgent and massive demand for testing prompted swift development of assays to detect SARS-CoV-2. The objective of this study was to assess the accuracy of these newly developed tests.MethodsThe American Proficiency Institute sent 2 test samples to 346 clinical laboratories in order to assess the accuracy of SARS-CoV-2 assays. The positive sample, containing 5,175 viral copies/mL, was fully extractable with SARS-CoV-2 viral capsid protein and RNA. The negative sample, with 3,951 viral copies/mL, contained recombinant virus particles with sequences for targeting human RNAase P gene sequences.ResultsOf the laboratories submitting results, 97.4% (302/310) correctly detected the virus when present and 98.3% (296/301) correctly indicated when the virus was not present. Among incorrect results reported in this proficiency challenge, 76.9% (10/13) were likely related to clerical error. This accounts for 1.6% (10/611) of all reported results.ConclusionsOverall performance in this SARS-CoV-2 RNA detection challenge was excellent, providing confidence in the results of these new molecular tests and assurance for the clinical and public health decisions based on these test results.

Recombinant Retroviral Particles: Technology of Poduction and Application as Positive Controls for PCR Diagnostics of Dangerous Viral Infections

Objective. Construction of positive control samples based on recombinant retroviral particles and their application in RT-PCR diagnostic assays for RNA detection of agents of dangerous and particularly dangerous viral infections.Materials and methods. Molecular biological, genetic engineering, and immunological methods were used: polymerase chain reaction, restriction, ligation, cloning, transformation, transfection, flow cytometry.Results and discussion. Technology of positive control samples producing based on recombinant virions has been developed and tested. It includes construction of retroviral vector with cloned diagnostic sequence of the viral genome; obtaining a packaging cell line producing chimeric retroviral particles; determination of recombinant virions titer by flow cytometry and polymerase chain reaction; application of the obtained preparation as a control sample for PCR diagnostics of infectious agents. Positive controls based on retroviral vectors as carriers of genomic RNA fragments of pathogenic viruses were used in the development of PCR diagnostic kits for dangerous and particularly dangerous viral infections. Their application increased the kits quality and made it possible to exclude the work with concentrated hazardous infectious agents (Lassa virus, tick-borne encephalitis virus, lymphocytic choriomeningitis virus, Puumala virus).

Human proteins incorporated into tick-borne encephalitis virus revealed by in situ proximity ligation

Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs. The technique allows highly selective and sensitive antibody-based detection of viral and host proteins on the surface of individual virions. We detected recombinant noninfectious sub-viral particles (SVPs) of tick-borne encephalitis virus (TBEV) immobilized in microtiter wells as fluorescent particles detected by regular fluorescence microscopy. Counting the particles in the images enabled us to estimate individual TBEV-SVP counts in different samples. Using isPLA we detected individual calnexin-, CD9-, CD81-, CD29- and CD59-positive SVPs among the viral particles. Our data suggests that a diversity of host proteins may be incorporated into TEBV, illustrating that isPLA with digital counting enables single-virus analysis of host protein incorporation.