Combined virus-like particle-based polyepitope DNA/protein HIV-1 vaccine design, immunogenicity and toxicity studies.

We have previously described designing of polyepitope immunogens TBI and TCI, to stimulate the humoral and cellular immune responses to HIV-1. Here, immunogens TBI and TCI were used to create new vaccine construct named CombiHIVvac (Combined HIV-1 vaccine). CombiHIVvac is a virus-like particles (VLP) containing the DNA vaccine pcDNA-TCI as a core encapsulated within a spermidine-polyglucin-TBI conjugate. The immunogenic and toxic properties of the candidate vaccine CombiHIVvac have been studied. CombiHIVvac induces a strong humoral and CTL responses in mice; the antibodies are highly specific and are able to neutralize HIV-1 in vitro. Preclinical study demonstrated that CombiHIVvac does not cause long-term changes in physiological, biochemical and morphological parameters in immunized animals and thus can be recommended for clinical trials.

The complete genomic sequence of strain ROS/HUVLV-100, a representative Russian Crimean Congo hemorrhagic fever virus strain.

The complete genomic sequence (minus primer-generated ends) of the laboratory-adapted Crimean Congo hemorrhagic fever virus (CCHFV) strain ROS/HUVLV-100, isolated in 2003 from the blood of a deceased female from the Rostov region of southern European Russia, was determined by direct sequencing of overlapping reverse transcription/polymerase chain reaction amplified products. The size of the ROS/HUVLV-100 genome is 19.2 kilobases--individual genome segments are similar in size and sequence features to previously reported "Europe-1" group CCHFV strains. The low-passage ROS/HUVLV-100 strain is the first Russian Crimean Congo hemorrhagic fever virus isolate for which complete sequence information is available, and this work reports the first complete genomic CCHFV sequence determined from a single viral RNA preparation in the same laboratory.

A variable region in the Crimean-Congo hemorrhagic fever virus L segment distinguishes between strains isolated from different geographic regions.

Alignment of Crimean-Congo hemorrhagic fever virus (CCHFV) L genome segment full-length sequences reveals an overall high level of conservation among strains, with greater than 90% of translated amino acid residues strictly conserved. However, a region of marked variability identified previously, corresponding to L polyprotein amino acid positions 760-810, shares only 40% overall identity between strains. The variable regions sequences of 16 laboratory-adapted CCHFV strains were determined, including 11 strains from European Russia, one strain from Bulgaria, and four strains from the Central Asian countries of Tajikistan, Turkmenistan, and Uzbekistan. Phylogenetic analysis demonstrates this L segment variable region sequence divides CCHFV strains into similar geographically-defined groupings observed for S segment-derived trees, but with higher bootstrap support and a much smaller character set required for analysis.

Designing and engineering of DNA-vaccine construction encoding multiple CTL-epitopes of major HIV-1 antigens.

A synthetic T cell immunogen (TCI) has been designed as a candidate DNA-based vaccine against Human immunodeficiency virus (HIV)-1 using cytotoxic T lymphocytes (CD8(+) CTL) and T-helper lymphocytes (CD4(+) Th) epitopes retrieved from the Los Alamos HIV Molecular Immunology Database. The protein 392 amino acids in length contains about eighty CTL-epitopes, many of which are overlapping and are totally restricted by ten different HLA class I molecules. To be able to detect CTL responses induced by a DNA vaccine in experimental animals, additional epitopes, restricted by mouse and Macaque rhesus major histocompatibility complex (MHC) class I molecules, were included in the target immunogen. The gene encoding the TCI protein was assembled, cloned into vector plasmids and expressed in a prokaryotic and a eukaryotic system. The presence of HIV-1 protein fragments in the immunogen structure was ascertained by ELISA and immunoblotting using panels of HIV-1-positive sera and monoclonal antibodies to p24. It has been demonstrated that DNA vaccine can induce both specific T cell responses (CTL and blast transformation) and specific antibodies in mice immunized with pcDNA-TCI.

Comparative analysis using a mouse model of the immunogenicity of artificial VLP and attenuated Salmonella strain carrying a DNA-vaccine encoding HIV-1 polyepitope CTL-immunogen.

Two systems have been examined for delivery of DNA-vaccine encoding a HIV-1 polyepitope CTL-immunogen (TCI). One is intended for i.m. injection and is in the form of an artificial virus like particle containing eukaryotic expression plasmid pcDNA-TCI encapsulated within a spermidine-polyglucin conjugate. The other is intended for mucosal immunization and is based on attenuated Salmonella typhimurium strain 7207, which can deliver pcDNA-TCI directly into professional antigen-presenting cells (APC). After immunization, the artificial VLP and recombinant Salmonella induced an enhanced HIV specific serum antibody, proliferative and CTL responses compared to those induced by naked pcDNA-TCI. The most significant responses were produced when pcDNA-TCI was delivered by Salmonella.

Genetic characterization of the M RNA segment of Crimean-Congo hemorrhagic fever virus strains isolated in Russia and Tajikistan.

The data on the structure of the M genome segment of CCHF virus strains from Russia and Central Asia (Tajikistan) are presented. Data obtained have been compared with other available published sequences of the middle segment of strains from China, Nigeria, and Pakistan. It has been found that all the known strains can be divided into four genetic groups, based on the nucleotide sequence of the M genome segment and an amino acid sequence of the glycoprotein precursor it encodes, whereas VLG/TI29414 and STV/HU29223 strains from Russia form a separate group. The CCHF virus strain from Tajikistan, TADJ/HU8966, was genetically related to strains 7803 and 75024 from China, and together with these and the Nigerian IbAr 10200 strain, it forms another group.