[Hepatitis C Virus Nonstructural Protein 3 Increases Secretion of Interleukin-lbeta in HEK293T Cells with a Reconstructed NLRP3 Inflammasome].

The pathology of diseases arising from infections by viruses of Flaviviridae is largely determined by the development of systemic inflammation. The cytokines interleukin-1beta and interleukin-18 play a key role in triggering inflammation. Their secretion from cells, in its turn, is induced upon activation of inflammasomes. Activation of NLRP3 (NLR pyrin domain-containing family 3) inflammasomes was detected in cells infected with Flaviviridae. Some nonstructural proteins of these viruses have been shown to be able to activate or to inhibit the NLRP3 inflammasome, in particular, through interaction with its components. In this study, a functional NLRP3 inflammasome was reconstructed in human HEK293T cells and the effect of some nonstructural proteins of individual Flaviviridae viruses on it was studied. This model did not reveal any impact of nonstructural NS1 proteins of the West Nile virus, NS3 of hepatitis C virus, or NS5 of tick-borne encephalitis virus on the inflammasome components content. At the same time, in the presence of the NS1 of the West Nile virus and NS5 of the tick-borne encephalitis virus, the level of secretion of interleukin-1beta did not change, whereas in the presence of the NS3 protein of the hepatitis C virus, it increased by 1.5 times. Thus, NS3 can be considered as one of the factors of NLRP3 inflammasome activation and inflammatory pathogenesis in chronic hepatitis C virus infection.

[A DNA Construct That Encodes the Rabies Virus Consensus Glycoprotein with a Proteasome Degradation Signal Induces Antibody Production with IgG2A Subtype Predominance].

The possibility of enhancing the immunogenicity of the rabies virus glycoprotein antigen encoded by a DNA vaccine has been investigated. Ubiquitin-like protein FAT10 has been attached to the N-terminus of the glycoprotein to target it to the proteasome and stimulate its presentation by MHC class I. Two forms of the protein, chimeric and original, have been detected in cells transfected with the DNA construct encoding the chimeric protein. The presence of the glycoprotein on the cell surface has been detected by immunostaining of transfected cells. The production of IgG and IgG2a antibodies has been more efficiently induced in mice immunized with the plasmid that encodes the chimeric protein than in those immunized with the plas-mid that encodes unmodified glycoprotein. Moreover, the level of IgG2a antibodies exceeded the level of IgG1 antibodies, which indicates a preferential increase in the Th1 component of the immune response. The proposed DNA construct that encodes a modified glycoprotein with a proteasome degradation signal maybe a promising DNA vaccine immunogen for post-exposure prophylaxis of rabies.

Codon optimization and improved delivery/immunization regimen enhance the immune response against wild-type and drug-resistant HIV-1 reverse transcriptase, preserving its Th2-polarity.

DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid ("surrogate challenge"). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199-220 and aa 528-543. Drug-resistance mutations disrupted the epitope at aa 205-220, while the CTL epitope at aa 202-210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.

[Rabies Virus Glycoprotein with a Consensus Amino Acid Sequence and a Lysosome Targeting Signal Causes Effective Production of Antibodies in DNA-Immunized Mice].

Safe and effective anti-rabies vaccines are intensely sought worldwide. DNA vaccines have already shown their efficacy and safety and have occupied a special place in the field. Two prototype anti-rabies DNA vaccines were compared for the potential to induce virus-specific antibody production. One vector contained a codon-optimized gene with a territory-adapted consensus sequence of the rabies virus glycoprotein. The other one expressed the same glycoprotein in fusion with a c-CD63 lysosome targeting motif at the C terminus. ELISA of serum samples from immunized mice showed that the c-CD63 variant induced more efficient antibody production and shifted the IgG2a/IgG1 ratio towards the Th2-type immune response. The results gave grounds to believe that the approach successfully applied to the rabies glycoprotein may help to develop new-generation anti-rabies vaccines.

Luciferase Expression Allows Bioluminescence Imaging But Imposes Limitations on the Orthotopic Mouse (4T1) Model of Breast Cancer.

Implantation of reporter-labeled tumor cells in an immunocompetent host involves a risk of their immune elimination. We have studied this effect in a mouse model of breast cancer after the orthotopic implantation of mammary gland adenocarcinoma 4T1 cells genetically labelled with luciferase (Luc). Mice were implanted with 4T1 cells and two derivative Luc-expressing clones 4T1luc2 and 4T1luc2D6 exhibiting equal in vitro growth rates. In vivo, the daughter 4T1luc2 clone exhibited nearly the same, and 4T1luc2D6, a lower growth rate than the parental cells. The metastatic potential of 4T1 variants was assessed by magnetic resonance, bioluminescent imaging, micro-computed tomography, and densitometry which detected 100-µm metastases in multiple organs and bones at the early stage of their development. After 3-4 weeks, 4T1 generated 11.4 ± 2.1, 4T1luc2D6, 4.5 ± 0.6; and 4T1luc2, <1 metastases per mouse, locations restricted to lungs and regional lymph nodes. Mice bearing Luc-expressing tumors developed IFN-γ response to the dominant CTL epitope of Luc. Induced by intradermal DNA-immunization, such response protected mice from the establishment of 4T1luc2-tumors. Our data show that natural or induced cellular response against the reporter restricts growth and metastatic activity of the reporter-labelled tumor cells. Such cells represent a powerful instrument for improving immunization technique for cancer vaccine applications.

[C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein].

The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.

[Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence].

An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen.

[Rabies vaccines: Current status and prospects for development].

Rabies is an infectious disease among humans and animals that remains incurable, despite its longstanding research history. The only way to prevent the disease is prompt treatment, including vaccination as an obligatory component and administration of antirabies immunoglobulin as a supplement. Since the first antirabies vaccination performed in the 19th century, a large number of different rabies vaccines have been developed. Progress in molecular biology and biotechnology enabled the development of effective and safe technologies of vaccine production. Currently, new-generation vaccines are being developed based on recombinant rabies virus strains or on the production of an individual recombinant rabies antigen-glycoprotein (G protein), either as a component of nonpathogenic viruses, or in plants, or in the form of DNA vaccines. In this review, the main modern trends in the development of rabies vaccines have been discussed.

Fusion to the Lysosome Targeting Signal of the Invariant Chain Alters the Processing and Enhances the Immunogenicity of HIV-1 Reverse Transcriptase.

Intracellular processing of the antigen encoded by a DNA vaccine is one of the key steps in generating an immune response. Immunization with DNA constructs targeted to the endosomal-lysosomal compartments and to the MHC class II pathway can elicit a strong immune response. Herein, the weakly immunogenic reverse transcriptase of HIV-1 was fused to the minimal lysosomal targeting motif of the human MHC class II invariant chain. The motif fused to the N-terminus shifted the enzyme intracellular localization and accelerated its degradation. Degradation of the chimeric protein occurred predominantly in the lysosomal compartment. BALB/c mice immunized with the plasmid encoding the chimeric protein demonstrated an enhanced immune response, in the form of an increased antigen-specific production of Th1 cytokines, INF-γ and IL-2, by mouse splenocytes. Moreover, the majority of the splenocytes secreted both cytokines; i.e., were polyfunctional. These findings suggest that retargeting of the antigen to the lysosomes enhances the immune response to DNA vaccine candidates with low intrinsic immunogenicity.