Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcγRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcγRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcγRI-expressing cells was confirmed by flow cytometry. The fusion protein induced a strong signal transduction on human monocytes via FcγRI. The expression vector pTriEx-3 Neo containing the described construct was used as a naked DNA vaccine and introduced directly to experimental humanized NOD SCID gamma mice with or without boosting with the expressed fusion protein. Immunization with the generated DNA chimeric molecules and prime-boost with the expressed recombinant proteins induced significant serum levels of anti-influenza immunoglobulin G antibodies and strong cytotoxic T lymphocyte activity against influenza virus-infected cells in humanized animals.

Highly purified, subunit, or synthetic viral antigens are known to be weakly immunogenic and potentate only the antibody, rather than cell-mediated immune responses. An alternative approach for inducing protective immunity with small viral peptides would be the direct targeting of viral epitopes to the immunocompetent cells by DNA vaccines encoding antibody fragments specific to activating cell surface co-receptor molecules. Here, we are exploring as a new genetic vaccine, a DNA chimeric molecule encoding a T and B cell epitope-containing influenza A virus hemagglutinin peptide joined to sequences encoding a single-chain variable fragment antibody fragment specific for the costimulatory B cell complement receptors 1 and 2. This recombinant DNA molecule was inserted into eukaryotic expression vector and used as a naked DNA vaccine in WT and CR1/2 KO mice. The intramuscular administration of the DNA construct resulted in the in vivo expression of an immunogenic chimeric protein, which cross-links cell surface receptors on influenza-specific B cells. The DNA vaccination was followed by prime-boosting with the protein-engineered replica of the DNA construct, thus delivering an activation intracellular signal. Immunization with an expression vector containing the described construct and boosting with the protein chimera induced a strong anti-influenza cytotoxic response, modulation of cytokine profile, and a weak antibody response in Balb/c mice. The same immunization scheme did not result in generation of influenza-specific response in mice lacking the target receptor, underlining the molecular adjuvant effect of receptor targeting.

The phylogeny of the latest recognized domain, Archaea, is still complicated and it is largely based on environmental sequences. A culture independent molecular phylogenetic analysis revealed high Archaea diversity in a terrestrial hot spring, village Varvara, Bulgaria. A total of 35 archaeal operational taxonomic units (OTUs) belonging to three of the classified five Archaea phyla were identified. Most of the sequences were affiliated with the phylum Crenarchaeota (23), grouped in four branches. The rest of the sequences showed highest similarity to the unidentified archaeal clones (9), Euryarchaeota (2), and "Korarchaeota " (1). Eight (23%) of the sequenced 16S rDNAs didn't have known close relatives and represented new and diverse OTUs, four of them forming a new archaeal subgroup without close described sequences or culturable relatives. A sequence affiliated with "Korarchaeota " showed low similarity (90%) to the closest neighbor and both sequences formed unique branch in this phylum. Consequently, the constructed archaeal libraries are characterized by (1) high proportion of OTUs representing uncultivated archaeal phylogroups, (2) the abundance of novel phylotype sequences, (3) the presence of high proportions of Crenarchaeota phylotypes unrelated to cultivated organisms and (4) the presence of a sequence only distantly related to "Korarchaeota " phylum.