Inside and outside of virus-like particles HBc and HBc/4M2e: A comprehensive study of the structure.

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.

Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins.

The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of HA2 (76-130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2e tandem copies (Flg-4M2e-HA2) provides partial α-helix formation nontypical of native M2e on the virion surface. The C-terminal position of the M2e tandem copies (Flg-HA2-4M2e) largely retained its native M2e conformation. These conformational differences in the structure of the two chimeric proteins were shown to affect their immunogenic properties. Different antibody levels induced by the chimeric proteins were detected. The protein Flg-HA2-4M2e was more immunogenic as compared to Flg-4M2e-HA2, with the former offering full protection to mice against a lethal challenge. We obtained evidence suggesting that the order of linkage of target antigens in a fusion protein may influence the 3D conformation of the chimeric construct, which leads to changes in immunogenicity and protective potency.

CROSS-PROTECTIVE PROPERTIES OF AN INFLUENZA VACCINE BASED ON HBC4M2E RECOMBINANT PROTEIN.

One of the main problems in the area of influenza prophylaxis and pandemic prevention is the development of cross-reactive vaccines, i.e. vaccines directed against all subtypes of human influenza viruses. Such vaccines are being developed in many countries for more than 10 years. A number of vaccines are presently undergoing clinical trials. We created Uniflu candidate vaccine based on recombinant HBc4M2e protein consisting of 4 tandem-connected copies of the highly conserved ectodomain of M2 protein of the influenza A virus. These 4 copies were genetically fused to the carrier protein, namely hepatitis B core antigen. Commercially available Derinat was used as adjuvant in the candidate vaccine. Preclinical studies on laboratory animals (mice, ferrets) demonstrated that immunization with Uniflu leads to significantly higher level of specific immunoglobulins in the blood and bronchoalveolar lavages. Moreover, it produces immunoglobulins belonging to subtype IgG2a that is the most important mediator of antibody-dependent cytotoxicity. The vaccine under review stimulates the proliferation of T-lymphocytes, as well as the formation of CD4+ and CD8+ T-cells synthesizing ɣ-IFN. When infected with the lethal doses (5 LD50) of influenza A viruses of the subtypes H1N1, H2N2, H3N2, and H1N1pdm09, immunized animals typically developed mild form of illness. This kept them alive in 90-100% of cases, which demonstrated almost complete protection from death. Replication of the virus in the lungs of immunized mice was reduced by 1.8-4.8 log10. High immunogenicity of the vaccine, and reduced clinical symptoms following experimental infection, were demonstrated in ferrets as well. The developed recombinant vaccine Uniflu has high specific activity and cross-protection. Uniflu can be proposed as pre-pandemic vaccine, provided that it passes clinical trials.

NEUROPHYSIOLOGICAL STATE OF HUMANS IN LONG-DURATION ISOLATION.

During the 520-d imitation of isolation and confinementin a long-duration exploration mission, the neuroufunctional state of 6 crewmembers was studied by synchronous registration of cerebral electrical (EEG) and energy (permanent potential level (PPL)) activities of the brain. Two baseline investigations predicted high adaptability of all crew members and low probability of depletion of the adaptation reserve in the experiment. During isolation, dynamics of neurofunctional correlates of the cerebral activity was generally proportionate to the factors the crewmembers were affected by. Events epochal to the crew gave rise to physiological reactions both of the metabolic and electrical activity of the brain. On week 54, crew physiological adaptation to the experimental environment was manifested by decreases in PPL without interhemispheric asymmetry and absolute a-power. All through the experiment none of the crew members showed signs of a distinct stress-reaction (a-activity asymmetry with a significant PPL decrease) supporting our prediction of low probability of adaptation reserve depletion in participants in the 520-day isolation and confinement study.

[Immunogenicity of recombinant proteins including ectodomain of M2 influenza virus A].

Two recombinant proteins with three copies of the ectodomain of the conserved influenza protein M2 (M2e) of influenza viruses were developed: A (H1N1)pdm09, A/Kurgan/05/05 (H5N1), and M2e consensus sequence of the human influenza A virus (H1N1, H2N2, H3N2) based on flagellin and core antigen of hepatitis B (HBc). The first recombinant protein comprised flagellin fused to three tandem copies of M2e, the second preparation was based on non-covalent interaction between M2e peptides and HBc. The immunogenicity of two preparations was comparatively tested. A covalent linkage of flagellin with M2e significant increased the immunogenicity of the target antigen compared with non-covalent interaction M2e and HBc. Flagellin as a protein carrier of M2e induced mainly IgG1 subclass, whereas HBc stimulated more balanced Th1/Th2 response. Our study showed a decrease in the viral titers in lung tissues of immunized mice after lethal challenge of A/PR/8/34 (H1N1). The study revealed a possibility to obtain a vaccine preparation with equal immunogenicity both against human influenza viruses and highly pathogenic avian influenza viruses.

[Microcirculation stream modeling: hydromechanic and acoustic models].

The objective was to attempt mathematical modeling of ultrasonic scanning tissue section in order to discern signals from erythrocytes and leucocytes that is displayed as Doppler images. Hydromechanic and acoustic microcirculation models have been constructed for a 20 MHz ultrasonic sensor. Results of the modeling showed that ultrasonic blood cells differentiation will require complex analysis of amplitude and frequency parameters of echoed signal.

[Myxosporeans (Cnidospora: Myxosporea) of Antarctic fishes]. / Miksosporidii (Cnidospora: Myxosporea) antarkticheskikh ryb.

Data on 13 species of fish myxosporeans from the Antarctic part of the Atlantic Ocean is present. Among them, 8 new species are described: Zschokkella australis sp. n., Davisia nototheniae sp. n., Ceratomyxa orthospora sp. n., C. ellipsoidea sp. n., C. antarctica sp. n., Leptotheca ampla sp. n., Pseudalataspora squamifrons sp. n., P. meridionalis sp. n.). Four species were found for the first time in the region and four species were found in new hosts.

[Davisia amoena sp. n. (Myxosporidia, Sinuolineidae), a parasite of plaice in the northwestern Atlantic]. / Davisia amoena sp. n. (Myxosporidia, Sinuolineidae)--parazit kambalovykh ryb severo-zapadnoi Atlantiki.

Davisia amoena sp. n. has been recorded from Hippoglossoides platessoides from northwestern Atlantic (in 36% of fishes). It differs from closely related D. longibrachia in the structure of vegetative forms, a smaller size of polar capsules and longer (1.5 times) lateral appendages. D. newfoundlandia Yoshino a. Noble, 1973 firstly recorded from Coryphaenoides holotrachys in the region of Falkland Islands (in 4.5% of fishes) is redescribed.

[2 new species of myxosporidians of the genus Myxoproteus from fish of the Celtic sea]. / Dva novykh vida miksosporidii roda Myxoproteus ot ryb Kel'tskogo moria.

Two new species of Myxosporidia of the genus Myxoproteus from the Celtic Sea fishes are described. M. scoleciformis was found in the biliary bladder of the John dory, Zeus faber (on 1 from 4 fishes), M. formosus was found in the urinary bladder of the whiting Merlangius merlangus (on 2 from 9 fishes).

[New species of trematodes from fishes of the Falkland-Patagonian region (South-Western Atlantic)]. / Novye vidy trematod ryb Folklendsko-Patagonskogo raiona (Iugo-Zapadnaia Atlantika).

Four new species of trematodes are described from fishes of the Falkland--Patagonian region as follows: Infundibulostomum patagonicum from Notothenia ramzay; Stenacron mancopsetti from Mancopsetta maculata; Neolepidapedon argentinensis from Salilota australis; Hirudinelloides elongatus from Thyrsites atun. For the latter species a new genus, Hirudinelloides, has been erected. Representatives of the genera Infundibulostomum, Stenacron and Neolepidapedon are first reported from this region.