Non-industrial production of therapeutic lentiviral vectors: How to provide vectors to academic CAR-T.

Bringing effective cancer therapy in the form of chimeric antigen receptor technology to untapped markets faces numerous challenges, including a global shortage of therapeutic lentiviral or retroviral vectors on which all current clinical therapies using genetically modified T cells are based. Production of these lentiviral vectors in academic settings in principle opens the way to local production of therapeutic cells, which is the only economically viable approach to make this therapy available to patients in developing countries. The conditions for obtaining and concentrating lentiviral vectors have been optimized and described. The calcium phosphate precipitation method was found to be suitable for transfecting high cell-density cultures, a prerequisite for high titers. We describe protocols for gradually increasing production from 6-well plates to P100 plates, T-175 flasks, and 5-layer stacks while maintaining high titers, >108 transducing units. Concentration experiments using ultracentrifugation revealed the advantage of lower centrifugation speeds compared to competing protocols. The resulting batches of lentiviral vectors had a titer of 1010 infectious particles and were used to transduce primary human T lymphocytes generating chimeric antigen receptor T cells, the quality of which was checked and found potential applicability for treatment.

Tilorone and Cridanimod Protect Mice and Show Antiviral Activity in Rats despite Absence of the Interferon-Inducing Effect in Rats.

The synthetic compounds, Tilorone and Cridanimod, have the antiviral activity which initially had been ascribed to the capacity to induce interferon. Both drugs induce interferon in mice but not in humans. This study investigates whether these compounds have the antiviral activity in mice and rats since rats more closely resemble the human response. Viral-infection models were created in CD-1 mice and Wistar rats. Three strains of Venezuelan equine encephalitis virus were tested for the performance in these models. One virus strain is the molecularly cloned attenuated vaccine. The second strain has major virulence determinants converted to the wild-type state which are present in virulent strains. The third virus has wild-type virulence determinants, and in addition, is engineered to express green fluorescent protein. Experimentally infected animals received Tilorone or Cridanimod, and their treatment was equivalent to the pharmacopoeia-recomended human treatment regimen. Tilorone and Cridanimod show the antiviral activity in mice and rats and protect the mice from death. In rats, both drugs diminish the viremia. These drugs do not induce interferon-alpha or interferon-beta in rats. The presented observations allow postulating the existence of an interferon-independent and species-independent mechanism of action.

Highly pathogenic avian influenza virus of the A/H5N8 subtype, clade 2.3.4.4b, caused outbreaks in Kazakhstan in 2020.

Background: Large poultry die-offs happened in Kazakhstan during autumn of 2020. The birds' disease appeared to be avian influenza. Northern Kazakhstan was hit first and then the disease propagated across the country affecting eleven provinces. This study reports the results of full-genome sequencing of viruses collected during the outbreaks and investigation of their relationship to avian influenza virus isolates in the contemporary circulation in Eurasia. Methods: Samples were collected from diseased birds during the 2020 outbreaks in Kazakhstan. Initial virus detection and subtyping was done using RT-PCR. Ten samples collected during expeditions to Northern and Southern Kazakhstan were used for full-genome sequencing of avian influenza viruses. Phylogenetic analysis was used to compare viruses from Kazakhstan to viral isolates from other world regions. Results: Phylogenetic trees for hemagglutinin and neuraminidase show that viruses from Kazakhstan belong to the A/H5N8 subtype and to the hemagglutinin H5 clade 2.3.4.4b. Deduced hemagglutinin amino acid sequences in all Kazakhstan's viruses in this study contain the polybasic cleavage site (KRRKR-G) indicative of the highly pathogenic phenotype. Building phylogenetic trees with the Bayesian phylogenetics results in higher statistical support for clusters than using distance methods. The Kazakhstan's viruses cluster with isolates from Southern Russia, the Russian Caucasus, the Ural region, and southwestern Siberia. Other closely related prototypes are from Eastern Europe. The Central Asia Migratory Flyway passes over Kazakhstan and birds have intermediate stops in Northern Kazakhstan. It is postulated that the A/H5N8 subtype was introduced with migrating birds. Conclusion: The findings confirm the introduction of the highly pathogenic avian influenza viruses of the A/Goose/Guangdong/96 (Gs/GD) H5 lineage in Kazakhstan. This virus poses a tangible threat to public health. Considering the results of this study, it looks justifiable to undertake measures in preparation, such as install sentinel surveillance for human cases of avian influenza in the largest pulmonary units, develop a human A/H5N8 vaccine and human diagnostics capable of HPAI discrimination.

Fluorescent tagging the NS1 protein in yellow fever virus: Replication-capable viruses which produce the secretory GFP-NS1 fusion protein.

Yellow fever virus, the prototype in the genus Flavivirus, was used to develop viruses in which the nonstructural protein NS1 is genetically fused to GFP in the context of viruses capable of autonomous replication. The GFP-tagging of NS1 at the amino-terminus appeared possible despite the presence of a small and functionally important domain at the NS1's amino-terminus which can be distorted by such fusing. GFP-tagged NS1 viruses were rescued from DNA-launched molecular clones. The initially produced GFP-tagged NS1 virus was capable of only poor replication. Sequential passages of the virus in cell cultures resulted in the appearance of mutations in GFP, NS4A, NS4B and NS5. The mutations which change amino acid sequences of GFP, NS4A and NS5 have the adaptive effect on the replication of GFP-tagged NS1 viruses. The pattern of GFP-fluorescence indicates that the GFP-NS1 fusion protein is produced into the endoplasmic reticulum. The intracellular GFP-NS1 fusion protein colocalizes with dsRNA. The discovered forms of extracellular GFP-NS1 possibly include tetramers and hexamers.