[Requirements for the Induction of Broadly Neutralizing Antibodies against HIV-1 by Vaccination].

A study of the induction of broadly neutralizing antibodies (bNAbs) in HIV-infected patients and vaccinated subjects revealed the main criteria for the formation of bNAbs (the duration of exposure to a viral antigen, the effect of the diversity of HIV variants, and the removal of barriers associated with the Env-dependent defense mechanisms of HIV-1). Modified trimers of the HIV-1 envelope protein (Env) exposed on virus-like particles (VLP) have unique properties: they (i) modulate the exposure of binding sites (bs) of the CD4 receptor and co-receptor; (ii) create steric restrictions for contact with bNAbs; (iii) increase the Env presentation density, thus enhancing the immune response; (iv) form stable trimers that do not induce off-target immune responses; and (v) allow additional modifications to their structure and construction of a platform with immunostimulating molecules. Immunization using a heterologous subtype-cross prime-boost regime with modified trimeric Env is capable of inducing somatic hypermutation levels necessary for the formation of bNAbs.

Enhanced expression of trim14 gene suppressed Sindbis virus reproduction and modulated the transcription of a large number of genes of innate immunity.

In the present research, we have studied an influence of enhanced expression TRIM14 on alphavirus Sindbis (SINV, Togaviridae family) infection. In the HEK293 cells transfected with human trim14 gene (HEK-trim14), SINV yield after infection was decreased 1000-10,000 times (3-4 lg of TCD50/ml) at 24 h p.i. and considerably less (1-2 lg of TCD50/ml) at 48 h p.i. Analysis of the expression of 43 genes directly or indirectly involved in innate immune machine in HEK-trim14 non-infected cells comparing with the control (non-transfected) HEK293 cells revealed that stable trim14 transfection in HEK293 cells caused increased transcription of 18 genes (ifna, il6 (ifnß2), isg15, raf-1, NF-kB (nf-kb1, rela, nf-kb2, relb), grb2, grb3-3, traf3ip2, junB, c-myb, pu.1, akt1, tyk2, erk2, mek2) and lowered transcription of 3 genes (ifnγ, gata1, il-17a). The similar patterns of genes expression observe in SINV-infected non-transfected HEK293 cells. However, SINV infection of HEK-trim14 cells caused inhibition of the most interferon cascade genes as well as subunits of transcription factor NF-κB. Thus, stable enhanced expression of trim14 gene in cells activates the transcription of many immunity genes and suppresses the SINV reproduction, but SINV infection of HEK-trim14 cells promotes inhibition of some genes involved in innate immune system.

Contamination of cell cultures with bovine viral diarrhea virus (BVDV).

The incidence of contamination of cell strains used in biological and virological studies and of fetal calf sera (FCS) manufactured by Russian and foreign companies used for cell culturing with noncytocidal bovine viral diarrhea virus (BVDV; Pestivirus, Flaviviridae) was analyzed. The virus was detected by reverse transcription PCR and indirect immunofluorescence with monoclonal antibodies to BVDV virion envelope glycoprotein in 25% of 117 cell strains and 45% of 35 tested FCS lots. The virus multiplied and persisted in a wide spectrum of human cell strains and in monkey, swine, sheep, rabbit, dog, cat, and other animal cells. The levels of BVDV genome RNA in contaminated cell cultures reached 10(2)-10(3) g-eq/cell and in serum samples 10(3)-10(7) g-eq/ml. These facts necessitate testing of cells and FCS for BVDV reproduced in cells without signs of infection detectable by light microscopy. The molecular mechanisms of long-term virus persistence in cells without manifestation of cell destruction are unknown.

[CMV-induced cell death and fas gene expression in resting and proliferating human fibroblasts].

Cytomegalovirus (CMV) infection development and mRNA fas transcription levels (CD95) in resting (GO) and proliferating (S-phase) human lung embryo fibroblasts (HLEF-110044 line) were studied. In GO cells accumulation of infectious CMV was high and cell death was very quick, and fas gene expression was inhibited in early period of infection. In cells infected during S-phase CMV synthesis was lower and total cell death was detected only after 5 days; fas gene activity remained on high levels and increased during 6-48 hours. Death of CMV-infected fibroblasts occurred through apoptosis with cytopathic effect and detachment of cells in early stage, but without changes of cell membrane permeability and internucleosome fragmentation of DNA during later stages. In another HLEF-977 line CMV-induced apoptosis correlated with increased levels of fas gene transcription in resting cells. Positive association of activation Fas-receptor pathway and cell proliferation as well as different effect of CMV on activity of fas gene in 2 HLEF lines are discussed.

[Regulation of human fibrolastic 2',5'-oligoadenylate synthetase gene activity in cytomegalovirus infected fibroblast cells].

Great differences were found in the level of 2',5'-oligoadenylate synthetase 40-46 kDa (OAS1) mRNA in relation to the proliferative state of human diploid fibroblasts at the moment of cytomegalovirus (CMV) (the strain AD169) infection. In the phase of synthesis of cellular cycle DNA (S), CMV induced OAS1 mRNA transcription by 10-100 times stronger than then in phase Go infection. The level of viral induction OAS1 mRNA peaked by hour 12 postinfection. The high gene activity correlated with suppressed DNA synthesis, a slowing-down mitotic cycle, markedly inhibited CMV replication, and delayed cell death. When the cells were infected in phase Go, the stimulation of OAS1 gene activity was less and it was attended by intensive viral replication and rapid cell death. There was a direct relationship between the resistance of cells and the constitutive level of OAS1 gene expression: in the low CMV-sensitive cells, the activity of OAS1 gene was more than 10 times greater than that in the highly sensitive cells. The inhibitors of the enzymatic OAS activity induced by IFN and dsRNA were found in the cytoplasm of the CMV-infected cells.

[Individual changes of gene expression in the interferon system in human blood cells due to amixin and cycloferon].

The action of amixin and cycloferon on the expression of genes in the systems of interferon (IF) and cell apoptosis (CA) was studied by semi-quantitative RT-PCR in human blood microsamples before and after the administration of the drugs. Individual changes were determined in the transcription activity of genes of IF (alpha, beta, gamma), enzymes 2',5' oligoadenylatesynthetase (OAS), RNSase L, dsRNA-dependent proteinkinase (dsPK) and of CA effectors (FasAg, bcl-2, gamma-actin) registered dynamically in 24 h and 48 h. The activity parameters of IF genes were compared with the results of biological titration of IF activity in blood samples in vivo and in vitro. A pronounced ability of cycloferon to stimulate selectively the activity of genes of human IF, type I (beta IF--by 100 times and alpha IF--by 10 times), without affecting essentially the activity of other genes in blood cells, was detected. Amixin was found to inhibit the titration of genes with high activity levels. (alpha-, beta-IF, RNAases L, bcl-2 and gamma-actin). The antiviral and IF-induced properties of the drug are explained to a great extent by the apoptotic effect (activation of genes Fas, gamma-IF, OAS and affected transcription of gene bcl-2). A positive correlation was observed between the processes of activation of IF-genes transcription and the production of the total circulating IF. Antagonistic relations between type I and II IFs in human blood cells were shown.

[Gene expression of the interferon and cell-immunity systems in human blood samples].

The interferon (IF) and cell-apoptosis (CA) systems are interrelated and regulate the protective reactions in body by means of a complex of biologically active proteins. The transcription levels of mRNA were determined by the RT-PCR semi-quantitative method in order to compare the constitutive expression levels of IF (alpha, beta, gamma) genes, IF-dependent enzymes of 2'5'-oligoadenylatesynthetyase (OAS), RNAase L, dsRNA-protein kinase (dsPK) and CA effectors (Fas-Ag, bcl-2 and gamma-actin) in human blood microsamples. cDNA dilutions, different numbers of amplification cycles (PCR with specific pairs of primers) as well as PCR-products' dilutions for dot-hybridization with specific probes were made use of to detect and evaluate levels of 9 mRNAs. The constitutive levels of gene expression of the IF and CA systems were found to differ essentially from others (1000-fold). The studied mRNA types were shared between 5 groups according to their transcription levels: very high--alpha-IF, high--RNAase L, medium--gamma-actin and bcl-2, low--beta-IF and very low (detectable after induction only)--gamma-IF, OAS, dsPK and Fas-Ag. The used detection method has a sufficiently high sensitivity and can be recommended for studies of IF inductors with unknown action mechanisms.

[Comparative analysis of expression of genes for 2',5'-oligoadenylate synthetase and mRNA for dsRNA-protein kinase in human fibroblasts infected with the Sindbis alphavirus and induced by alpha-interferon]. / Sravnitel'nyi analiz ékspressii genov fermentov 2',5'-oligoadenilatsintetazy i dsRNK-proteinkinazy v fibroblastakh cheloveka, infitsirovannykh al'fa-virusom Sindbis i indutsirovannykh alpha-interferonom.

Expression of 2,5-OAS and ds-protein kinase (ds-PK) in human fibroblast culture following treatment with alpha-IF in a dose of 1000 U or challenge with Karelian fever virus (KFV) (5 PFU/cell, 4 h.p.i.) was compared. A highly sensitive and rapid method, coupled RT-PCR, was used to detect mRNA transcription levels. Alpha-IF had an expressed stimulating effect on 2,5-OAS mRNA level, whereas the virus markedly inhibited the synthesis of ds-PK mRNA.

[Recombination of alpha-viruses during mixed infection in cultured cells. Formation of hybrid forms of RNA of Venezuelan equine encephalomyelitis virus and Karelian fever virus in the region of genes for nucleocapsid and envelope proteins]. / Rekombinatsiia al'favirusov pri smeshannoi infektsii v kul'turakh kletok. Obrazovanie gibridnykh form RNK virusov venesuél'skogo éntsefalomielita loshadei i karel'skoi likhoradki v oblasti genov belkov nukleokapsida i obolochki.

A recombination of the genomes of VEE and KF viruses, two alphaviruses of different serotypes, takes place in mixed infection of chick embryo fibroblasts and Vero cells with vaccine strain VEE-230 and KF-9298 strain. This recombination resulted in the production of recombinant virions with altered antigenic properties. Polymerase chain reaction (PCR) rapidly and effectively detected the hybrid sites of the genome in recombinant viruses. Short nucleotide sequences (approximately 20 n. p.) of a known primary structure in the RNA genomes, specifically reacting with the complementary PCR primers, were chosen as genetic markers. These data are the first experimental validation of the probable recombinant formation of alphaviruses in nature. They are in good agreement with findings of the recombinant structure of Western equine encephalomyelitis virus genome.

[Comparative analysis of primary structure of nucleocapsid protein from Western equine encephalomyelitis virus and other alphaviruses]. / Sravnitel'nyi analiz pervichnoi struktury belka nukleokapsida virusa zapadnogo éntsefalomielita loshadei n drugikh al'favirusov.

Part of WEE virus (strain 16310-5614) genome coding for the nucleocapsid (C) protein was cloned and sequenced in two independent clones. The C gene of WEE virus is composed of 77 nucleotides, both for the BFS and 16310 strains, and is 48, 24, 33, 15, 3, and 3 nucleotides shorter than that of Venezuelan equine encephalomyelitis (VEE), Semliki Forest, Ross River, Sindbis, O'Nyong-Nyong, and Eastern equine encephalomyelitis (EEE) viruses, respectively. It contains 16 nucleotide changes in comparison with the BFS-1703 strain, four of which are significant: Ser57(BFS)-->Ala(16310), Gly63-->Cys, Lys74-->Glu, Gly97-->Trp. Amino acid composition, charges, hydropathic profiles, and location of potential functional sites in C proteins of the heretofore studied alphaviruses have been compared. High positively charged N-domain of the nucleocapsid is the most variable in all alphaviruses and is characterized by an irregular secondary structure due to high Pro content (25.5%). Positively charged Lys (10.8% of total) and Arg (6.9% of total) are presented 18 and 11 times, respectively, in the N-domain of WEE virus (16310) protein, and clusters thereof possibly form the initial sites for interaction with RNA. Only Sindbis virus (HRSP and Ock) nucleocapsids do not contain Cys, while others do contain several residues. This part of C protein includes overlapping nuclear transport signals predicted for several cellular proteins and repeated 4, 7, and 2 times in WEE, EEE, and Sindbis viruses, respectively. There is a highly conservative region (96-113 as residues) in the C protein structure of all studied alphaviruses, which potentially binds to a large ribosomal subunit as it was shown for Sindbis virus by Wengler et al. (1992), and a consensus motif K/R95-P-X-K/R-X-R-M could be a main part of the nucleoprotein ribosome binding site. The W186HHGAVQ (WEE virus) is absolutely conservative for all alphaviruses and with the invariant Asn222 could have a common function, including C protein lateral interaction (Choi et al., 1991). The origination of WEE virus C protein from EEE virus is confirmed by very high (92.7%) similarity of this protein's C domain in the WEE/EEE pair and low (64.8%) in the WEE/Sindbis pair. Determination of C gene and protein type in the Sindbis/WEE virus serocomplex might be useful in the differential identification of this virus group.