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.

Multimodal OCT Control for Early Histological Signs of Vulvar Lichen Sclerosus Recurrence after Systemic PDT: Pilot Study.

Photodynamic therapy (PDT) is a modern treatment for severe or treatment-resistant vulvar lichen sclerosus (VLS). The chronic and recurrent nature of VLS requires control of recurrences at an early stage. In this paper, a non-invasive multimodal optical coherence tomography (OCT) method was used to control for early histological signs of VLS recurrence after systemic PDT using Photodithazine®. To interpret the OCT data, a histological examination was performed before PDT and 3 months after PDT. Two groups of patients were identified: with early histological signs of VLS recurrence (Group I, n = 5) and without histological signs of VLS recurrence (Group II, n = 6). We use structural OCT, OCT angiography, and OCT lymphangiography throughout 6 months after PDT to visually assess the skin components and to quantitatively assess the dermis by calculating the depth-resolved attenuation coefficient and the density of blood and lymphatic vessels. The OCT data assessment showed a statistically significant difference between the patient groups 3 months after PDT. In Group II, all the studied OCT parameters reached maximum values by the 3rd month after PDT, which indicated recovery of the skin structure. At the same time, in Group I, the values of OCT parameters did not approach the values those in Group II even after 6 months. The obtained results of multimodal OCT can be used for non-invasive control of early histological recurrence of VLS after systemic PDT and for adjusting treatment tactics in advance, without waiting for new clinical manifestations of the disease.

Vaccination potential of B and T epitope-enriched NP and M2 against Influenza A viruses from different clades and hosts.

To avoid outbreaks of influenza virus epidemics and pandemics among human populations, modern medicine requires the development of new universal vaccines that are able to provide protection from a wide range of influenza A virus strains. In the course of development of a universal vaccine, it is necessary to consider that immunity must be generated even against viruses from different hosts because new human epidemic virus strains have their origins in viruses of birds and other animals. We have enriched conserved viral proteins-nucleoprotein (NP) and matrix protein 2 (M2)-by B and T-cell epitopes not only human origin but also swine and avian origin. For this purpose, we analyzed M2 and NP sequences with respect to changes in the sequences of known T and B-cell epitopes and chose conserved and evolutionarily significant epitopes. Eventually, we found consensus sequences of M2 and NP that have the maximum quantity of epitopes that are 100% coincident with them. Consensus epitope-enriched amino acid sequences of M2 and NP proteins were included in a recombinant adenoviral vector. Immunization with Ad5-tet-M2NP induced strong CD8 and CD4 T cells responses, specific to each of the encoded antigens, i.e. M2 and NP. Eight months after immunization with Ad5-tet-M2NP, high numbers of M2- and NP-responding "effector memory" CD44posCD62neg T cells were found in the mouse spleens, which revealed a long-term T cell immune memory conferred by the immunization. In all, the challenge experiments showed an extraordinarily wide-ranging efficacy of protection by the Ad5-tet-M2NP vaccine, covering 5 different heterosubtypes of influenza A virus (2 human, 2 avian and 1 swine).

Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2).

This work continues a series of recently published studies that employ recombinant single-domain antibody (sdAb, or nanobody®) generation technologies to battle viruses by a passive immunization approach. As a proof of principle, we describe a modified technique to efficiently generate protective molecules against a particular strain of influenza virus within a reasonably short period of time. This approach starts with the immunization of a camel (Camelus bactrianus) with the specified antigen-enriched material presented in as natural a form as possible. An avian influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2) adapted for mice was used as a model source of antigens for both the immunization and phage display-based selection procedures. To significantly increase activities of initially selected monovalent single-domain antibodies, we propose a new type of sdAb formatting that involves the addition of a special type of coiled-coil sequence, the isoleucine zipper domain (ILZ). Presumably, the ILZ-containing peptides adopt trimeric parallel conformations. After the formatting, the biological activities (virus neutralization) of the initially selected anti-influenza virus (H5N2) sdAbs were significantly increased. Intraperitoneal or intranasal administration of the formatted sdAb at 2h before or 24h after viral challenge specifically protects mice from lethal infection with influenza virus. We hope that the described approach combined with the selection focused on particular conservative epitopes will lead to the generation of sdAb-based molecules protective against a broad spectrum of influenza virus subtypes.

Passive immunization with a recombinant adenovirus expressing an HA (H5)-specific single-domain antibody protects mice from lethal influenza infection.

One effective method for the prevention and treatment of influenza infection is passive immunization. In our study, we examined the feasibility of creating an antibody-based preparation with a prolonged protective effect against influenza virus. Single-domain antibodies (sdAbs) specific for influenza virus hemagglutinin were generated. Experiments in mouse models showed 100% survivability for both intranasal sdAbs administration 24h prior to influenza challenge and 24h after infection. sdAb-gene delivery by an adenoviral vector led to gene expression for up to 14days. Protection by a recombinant adenovirus containing the sdAb gene was observed in cases of administration prior to influenza infection (14d-24h). We also demonstrated that the single administration of a combined preparation containing sdAb DNA and protein expanded the protection time window from 14d prior to 48h after influenza infection. This approach and the application of a broad-spectrum sdAbs will allow the development of efficient drugs for the prevention and treatment of viral infections produced by pandemic virus variants and other infections.