Protective Activity of Inactivated Rabies Vaccine Using Flagellin-Based Adjuvant.

Rabies is a zoonotic disease with high lethality. Most human deaths are associated with the bites received from dogs and cats. Vaccination is the most effective method of preventing rabies disease in both animals and humans. In this study, the ability of an adjuvant based on recombinant Salmonella typhimurium flagellin to increase protective activity of the inactivated rabies vaccine in mice was evaluated. A series of inactivated dry culture vaccine for dogs and cats "Rabikan" (strain Shchelkovo-51) with addition of an adjuvant at various dilutions were used. The control preparation was a similar series of inactivated dry culture vaccine without an adjuvant. Protective activity of the vaccine preparations was evaluated by the NIH potency test, which is the most widely used and internationally recommended method for testing effectiveness of the inactivated rabies vaccines. The value of specific activity of the tested rabies vaccine when co-administered with the adjuvant was significantly higher (48.69 IU/ml) than that of the vaccine without the adjuvant (3.75 IU/ml). Thus, recombinant flagellin could be considered as an effective adjuvant in the composition of future vaccine preparations against rabies virus.

Novel Universal Recombinant Rotavirus A Vaccine Candidate: Evaluation of Immunological Properties.

Rotavirus infection is a leading cause of severe dehydrating gastroenteritis in children under 5 years of age. Although rotavirus-associated mortality has decreased considerably because of the introduction of the worldwide rotavirus vaccination, the global burden of rotavirus-associated gastroenteritis remains high. Current vaccines have a number of disadvantages; therefore, there is a need for innovative approaches in rotavirus vaccine development. In the current study, a universal recombinant rotavirus antigen (URRA) for a novel recombinant vaccine candidate against rotavirus A was obtained and characterised. This antigen included sequences of the VP8* subunit of rotavirus spike protein VP4. For the URRA, for the first time, two approaches were implemented simultaneously-the application of a highly conserved neutralising epitope and the use of the consensus of the extended protein's fragment. The recognition of URRA by antisera to patient-derived field rotavirus isolates was proven. Plant virus-based spherical particles (SPs), a novel, effective and safe adjuvant, considerably enhanced the immunogenicity of the URRA in a mouse model. Given these facts, a URRA + SPs vaccine candidate is regarded as a prospective basis for a universal vaccine against rotavirus.

Effect of the Coat Protein N-Terminal Domain Structure on the Structure and Physicochemical Properties of Virions of Potato Virus X and Alternanthera Mosaic Virus.

The amino acid sequences of the coat proteins (CPs) of the potexviruses potato virus X (PVX) and alternanthera mosaic virus (AltMV) share ~40% identity. The N-terminal domains of these proteins differ in the amino acid sequence and the presence of the N-terminal fragment of 28 residues (ΔN peptide) in the PVX CP. Here, we determined the effect of the N-terminal domain on the structure and physicochemical properties of PVX and AltMV virions. The circular dichroism spectra of these viruses differed significantly, and the melting point of PVX virions was 10-12°C higher than that of AltMV virions. Alignment of the existing high-resolution 3D structures of the potexviral CPs showed that the RMSD value between the Cα-atoms was the largest for the N-terminal domains of the two compared models. Based on the computer modeling, the ΔN peptide of the PVX CP is fully disordered. According to the synchrotron small-angle X-ray scattering (SAXS) data, the structure of CPs from the PVX and AltMV virions differ; in particular, the PVX CP has a larger portion of crystalline regions and, therefore, is more ordered. Based on the SAXS data, the diameters of the PVX and AltMV virions and helix parameters in solution were calculated. The influence of the conformation of the PVX CP N-terminal domain and its position relative to the virion surface on the virion structure was investigated. Presumably, an increased thermal stability of PVX virions vs. AltMV is provided by the extended N-terminal domain (ΔN peptide, 28 amino acid residues), which forms additional contacts between the adjacent CP subunits in the PVX virion.

Identification of the Channelrhodopsin Genes in the Green and Cryptophytic Algae from the White and Black Seas.

Due to the unique capability of modulating cell membrane potential upon photoactivation, channelrhodopsins of green (Chlorophyta) and cryptophytic (Cryptophyta) algae are widely employed in optogenetics, a modern method of light-dependent regulation of biological processes. To enable the search for new genes perspective for optogenetics, we have developed the PCR tests for the presence of genes of the cation and anion channelrhodopsins. Six isolates of green algae Haematococcus and Bracteacoccus from the White Sea region and 2 specimens of Rhodomonas sp. (Cryptophyta) from the regions of White and Black Seas were analyzed. Using our PCR test we have demonstrated the known Haematococcus rhodopsin genes and have discovered novel rhodopsin genes in the genus of Bracteacoccus. Two distantly homologous genes of anion channelrhodopsins were also identified in the cryptophytic Rhodomonas sp. from the White and Black Seas. These results indicate that the developed PCR tests might be useful tool for a broad-range screening of the Chlorophyta and Cryptophyta algae to identify unique channelrhodopsin genes.

New formulation of a recombinant anthrax vaccine stabilised with structurally modified plant viruses.

Anthrax is a disease caused by Bacillus anthracis. The most promising approach to the development of anthrax vaccine is use of the anthrax protective antigen (PA). At the same time, recombinant PA is a very unstable protein. Previously, the authors have designed a stable modified recombinant anthrax protective antigen with inactivated proteolytic sites and substituted deamidation sites (rPA83m). As a second approach to recombinant PA stabilisation, plant virus spherical particles (SPs) were used as a stabiliser. The combination of these two approaches was shown to be the most effective. Here, the authors report the results of a detailed study of the stability, immunogenicity and protectiveness of rPA83m + SPs compositions. These compositions were shown to be stable, provided high anti-rPA83m antibody titres in guinea pigs and were able to protect them from a fully virulent 81/1 Bacillus anthracis strain. Given these facts, the formulation of rPA83m + SPs compositions is considered to be a prospective anthrax vaccine candidate.

Structurally Modified Plant Viruses and Bacteriophages with Helical Structure. Properties and Applications.

Structurally modified virus particles can be obtained from the rod-shaped or filamentous virions of plant viruses and bacteriophages by thermal or chemical treatment. They have recently attracted attention of the researchers as promising biogenic platforms for the development of new biotechnologies. This review presents data on preparation, structure, and properties of the structurally modified virus particles. In addition, their biosafety for animals is considered, as well as the areas of application of such particles in biomedicine. A separate section is devoted to one of the most relevant and promising areas for the use of structurally modified plant viruses - design of vaccine candidates based on them.

Designing Stable Bacillus anthracis Antigens with a View to Recombinant Anthrax Vaccine Development.

Anthrax is a disease caused by Bacillus anthracis that affects mammals, including humans. Recombinant B. anthracis protective antigen (rPA) is the most common basis for modern anthrax vaccine candidates. However, this protein is characterised by low stability due to proteolysis and deamidation. Here, for the first time, two modification variants leading to full-size rPA stabilisation have been implemented simultaneously, through deamidation-prone asparagine residues substitution and by inactivation of proteolysis sites. Obtained modified rPA (rPA83m) has been demonstrated to be stable in various temperature conditions. Additionally, rPA1+2 containing PA domains I and II and rPA3+4 containing domains III and IV, including the same modifications, have been shown to be stable as well. These antigens can serve as the basis for a vaccine, since the protective properties of PA can be attributed to individual PA domains. The stability of each of three modified anthrax antigens has been considerably improved in compositions with tobacco mosaic virus-based spherical particles (SPs). rPA1+2/rPA3+4/rPA83m in compositions with SPs have maintained their antigenic specificity even after 40 days of incubation at +37 °C. Considering previously proven adjuvant properties and safety of SPs, their compositions with rPA83m/rPA1+2/rPA3+4 in any combinations might be suitable as a basis for new-generation anthrax vaccines.

Vaccine Candidate Against COVID-19 Based on Structurally Modified Plant Virus as an Adjuvant.

A recombinant vaccine candidate has been developed based on the major coronaviruses' antigen (S protein) fragments and a novel adjuvant-spherical particles (SPs) formed during tobacco mosaic virus thermal remodeling. The receptor-binding domain and the highly conserved antigenic fragments of the S2 protein subunit were chosen for the design of recombinant coronavirus antigens. The set of three antigens (Co1, CoF, and PE) was developed and used to create a vaccine candidate composed of antigens and SPs (SPs + 3AG). Recognition of SPs + 3AG compositions by commercially available antibodies against spike proteins of SARS-CoV and SARS-CoV-2 was confirmed. The immunogenicity testing of these compositions in a mouse model showed that SPs improved immune response to the CoF and PE antigens. Total IgG titers against both proteins were 9-16 times higher than those to SPs. Neutralizing activity against SARS-CoV-2 in serum samples collected from hamsters immunized with the SPs + 3AG was demonstrated.

Chilling Upregulates Expression of the PsbS and LhcSR Genes in the Chloroplasts of the Green Microalga Lobosphaera incisa IPPAS C-2047.

Non-photochemical quenching (NPQ) of excited chlorophyll states is essential for protecting the photosynthetic apparatus (PSA) from the excessive light-induced damage in all groups of oxygenic photosynthetic organisms. The key component of the NPQ mechanism in green algae and some other groups of algae and mosses is the LhcSR protein of the light harvesting complex (LHC) protein superfamily. In vascular plants, LhcSR is replaced by PsbS, another member of the LHC superfamily and a subunit of photosystem II (PSII). PsbS also performs the photoprotective function in mosses. For a long time, PsbS had been believed to be nonfunctional in green algae, although the corresponding gene was discovered in the genome of these organisms. The first evidence of the PsbS accumulation in the model green alga Chlamydomonas reinhardtii in response to the increase in irradiance was obtained only six years ago. However, the observed increase in the PsbS content was short-termed (on an hour-timescale). Here, we report a significant (more than three orders of magnitude) and prolonged (four days) upregulation of PsbS expression in response to the chilling-induced high-light stress followed by a less significant (~ tenfold) increase in the PsbS expression for nine days. This is the first evidence for the long-term upregulation of the PsbS expression in green alga (Chlorophyta) in response to stress. Our data indicate that the role of PsbS in the PSA of Chlorophyta is not limited to the first-line defense against stress, as it was previously assumed, but includes full-scale participation in the photoprotection of PSA from the environmental stress factors.

The Effect of Chilling on the Photosynthetic Apparatus of Microalga Lobosphaera incisa IPPAS C-2047.

Photosynthetic organisms have developed a set of mechanisms aimed at preventing photo-oxidative reactions in the photosynthetic apparatus (PSA) initiated by excessively absorbed light energy. Along with high irradiance, other stressors, e.g., chilling temperatures, can lead to the absorption of the excess of light energy and hence to photo-oxidative stress. Here, we studied induction of photoprotective mechanisms in response to chilling (0°C) at a low irradiance (50 µmol PAR photons m-2·s-1) in the cells of microalga Lobosphaera incisa IPPAS C-2047. After 4 days of incubation at a low temperature, L. incisa IPPAS C-2047 cells showed a notable decrease in the photochemical activity of photosystem II (PSII) and in the efficiency of photosynthetic electron transport, as well as a significant increase in the thermal dissipation of the absorbed light energy in the light-harvesting antenna. In contrast, most conventional markers of PSA acclimation to excess light energy [total chlorophyll and carotenoid content; violaxanthin cycle pigment content and de-epoxidation state; photosynthetic antenna, PSII, and photosystem I (PSI) ratio] remained virtually unchanged. The content of major unsaturated fatty acids also remained almost unaffected, except for arachidonic acid (increased by 40%) recently assumed to activate violaxanthin de-epoxidase by adjusting its lipid microenvironment. Significant changes (4-7-fold increase) were observed in the expression of the gene encoding protective protein LhcSR. Pre-conditioning at 5°C prior to the acclimation to 0°C augmented the PSA photochemical activity. Our data show that the mid-term (4-d) acclimation of L. incisa IPPAS C-2047 to a chilling temperature at a low irradiance triggers the PSA response resembling, in part, the response to high light but relying mostly on the LhcSR protein-dependent quenching of excitation in the photosynthetic antenna.

Novel antigen panel for modern broad-spectrum recombinant rotavirus A vaccine.

PURPOSE: Recombinant rotavirus A vaccines are being developed as an alternative to existing live oral attenuated vaccines. One of the main problems in the production of such vaccines is the genetic diversity of the strains that are in circulation. The goal of this study was to create an antigen panel for modern broad-spectrum recombinant rotavirus A vaccine. MATERIALS AND METHODS: The antigens of rotavirus were cloned and expressed in Escherichia coli. Antigenic specificity was investigated by Western blot analysis, which was performed using commercial polyclonal antisera to several RVA strains. Phylogenetic analysis was based on the amino acid sequences of the VP8* protein fragment of human RVA isolates representing genotypes P[4], P[6], and P[8]. RESULTS: A universal panel of antigens was established, including consensus and conserved sequences of structural proteins VP8*, VP5*, and VP7, which are the main targets of neutralizing antibodies. For the first time, a consensus approach was used in the design of extended antigens based on VP8* (genotypes P[4], P[6], and P[8]) and VP5* (genotype P[8]) proteins' fragments. In addition, a gene coding the protein (ep-875) containing several copies of conserved short neutralizing epitopes of VP8*, VP7, and VP5* was created. Western blot analysis demonstrated that three synthetic VP8*-based antigens were not recognized by commercial antiserum against rotavirus strains isolated more than 35 years ago, but the specific activity of the VP5* and ep-875 antigens was confirmed. The problems of serological mismatch of vaccine strains and antigens with currently circulating strains are discussed. CONCLUSION: Five antigens representing sequences of structural proteins belonging to different genotypes can be used in various combinations (from mono- to pentavalent mixtures) for the development of an effective broad-spectrum rotavirus vaccine.

Thermal remodelling of Alternanthera mosaic virus virions and virus-like particles into protein spherical particles.

The present work addresses the thermal remodelling of flexible plant viruses with a helical structure and virus-like particles (VLPs). Here, for the first time, the possibility of filamentous Alternanthera mosaic virus (AltMV) virions' thermal transition into structurally modified spherical particles (SP) has been demonstrated. The work has established differences in formation conditions of SP from virions (SPV) and VLPs (SPVLP) that are in accordance with structural data (on AltMV virions and VLPs). SP originate from AltMV virions through an intermediate stage. However, the same intermediate stage was not detected during AltMV VLPs' structural remodelling. According to the biochemical analysis, AltMV SPV consist of protein and do not include RNA. The structural characterisation of AltMV SPV/VLP by circular dichroism, intrinsic fluorescence spectroscopy and thioflavin T fluorescence assay has been performed. AltMV SPV/VLP adsorption properties and the availability of chemically reactive surface amino acids have been analysed. The revealed characteristics of AltMV SPV/VLP indicate that they could be applied as protein platforms for target molecules presentation and for the design of functionally active complexes.

Two approaches for the stabilization of Bacillus anthracis recombinant protective antigen.

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacteria Bacillus anthracis. There is a need for safe, highly effective, long-term storage vaccine formulations for mass vaccination. However, the development of new subunit vaccines based on recombinant protective antigen (rPA) faces the problem of vaccine antigen instability. Here, the potential of simultaneous application of two different approaches to stabilize rPA was demonstrated. Firstly, we employed spherical particles (SPs) obtained from the tobacco mosaic virus (TMV). Previously, we had reported that SPs can serve as an adjuvant and platform for antigen presentation. In the current work, SPs were shown to increase the stability of the full-size rPA without loss of its antigenic properties. The second direction was site-specific mutagenesis of asparagine residues to avoid deamidation that causes partial protein degradation. The modified recombinant protein comprising the PA immunogenic domains 3 and 4 (rPA3 + 4) was stable during storage at 4 and 25°C. rPA3 + 4 interacts with antibodies to rPA83 both individually and as a part of a complex with SPs. The results obtained can underpin the development of a recombinant vaccine with a full-size modified rPA (with similar amino acid substitutions that stabilize the protein) and SPs.

Various Adjuvants Effect on Immunogenicity of Puumala Virus Vaccine.

Various adjuvant effects on the immunogenicity of the candidate inactivated Puumala virus vaccine were detected in BALB/c mice. Adjuvants under study were: aluminum hydroxide, spherical particles of Tobacco mosaic virus coat protein, B subunit of heat-labile enterotoxin of Escherichia coli, and low endotoxic lipopolysaccharide of Shigella sonnei. Aluminum hydroxide (1 mg/ml) did not affect neutralizing antibodies' induction and vaccine stability during storage compared to immunization with the vaccine without adjuvant. B subunit of heat-labile enterotoxin (0.2 µg/ml), low endotoxic lipopolysaccharide (50 µg/ml), and plant virus-based spherical particles (300 µg/ml) significantly enhance the humoral immune response of vaccine (p < 0.0001). Pronounced stimulation of IL-12 and IFN-É£ was observed when mice were immunized with vaccines both with adjuvants (except of aluminum hydroxide) and without adjuvants. It has been shown that low endotoxic lipopolysaccharide contributes not only to enhance the immune response but also to stabilize vaccine immunogenicity during at least 1 year storage.

Plant virus particles with various shapes as potential adjuvants.

Plant viruses are biologically safe for mammals and can be successfully used as a carrier/platform to present foreign epitopes in the course of creating novel putative vaccines. However, there is mounting evidence that plant viruses, their virus-like and structurally modified particles may also have an immunopotentiating effect on antigens not bound with their surface covalently. Here, we present data on the adjuvant properties of plant viruses with various shapes (Tobacco mosaic virus, TMV; Potato virus X, PVX; Cauliflower mosaic virus, CaMV; Bean mild mosaic virus, BMMV) and structurally modified TMV spherical particles (SPs). We have analysed the effectiveness of immune response to individual model antigens (ovalbumin, OVA/hen egg lysozyme, HEL) and to OVA/HEL in compositions with plant viruses/SPs, and have shown that CaMV, TMV and SPs can effectively induce total IgG titers to model antigen. Some intriguing data were obtained when analysing the immune response to the plant viruses/SPs themselves. Strong immunity was induced to CaMV, BMMV and PVX, whereas TMV and SPs stimulated considerably lower self-IgG titers. Our results provide new insights into the immunopotentiating properties of plant viruses and can be useful in devising adjuvants based on plant viruses.

Vaccines against anthrax based on recombinant protective antigen: problems and solutions.

Introduction: Anthrax is a dangerous bio-terror agent because Bacillus anthracis spores are highly resilient and can be easily aerosolized and disseminated. There is a threat of deliberate use of anthrax spores aerosol that could lead to serious fatal diseases outbreaks. Existing control measures against inhalation form of the disease are limited. All of this has provided an impetus to the development of new generation vaccines. Areas сovered: This review is devoted to challenges and achievements in the design of vaccines based on the anthrax recombinant protective antigen (rPA). Scientific databases have been searched, focusing on causes of PA instability and solutions to this problem, including new approaches of rPA expression, novel rPA-based vaccines formulations as well as the simultaneous usage of PA with other anthrax antigens. Expert opinion: PA is a central anthrax toxin component, playing a key role in the defense against encapsulated and unencapsulated strains. Subunit rPA-based vaccines have a good safety and protective profile. However, there are problems of PA instability that are greatly enhanced when using aluminum adjuvants. New adjuvant compositions, dry formulations and resistant to proteolysis and deamidation mutant PA forms can help to handle this issue. Devising a modern anthrax vaccine requires huge efforts.

Surface characterization of the thermal remodeling helical plant virus.

Previously, we have reported that spherical particles (SPs) are formed by the thermal remodeling of rigid helical virions of native tobacco mosaic virus (TMV) at 94°C. SPs have remarkable features: stability, unique adsorption properties and immunostimulation potential. Here we performed a comparative study of the amino acid composition of the SPs and virions surface to characterize their properties and take an important step to understanding the structure of SPs. The results of tritium planigraphy showed that thermal transformation of TMV leads to a significant increase in tritium label incorporation into the following sites of SPs protein: 41-71 а.a. and 93-122 a.a. At the same time, there was a decrease in tritium label incorporation into the N- and C- terminal region (1-15 a.a., 142-158 a.a). The use of complementary physico-chemical methods allowed us to carry out a detailed structural analysis of the surface and to determine the most likely surface areas of SPs. The obtained data make it possible to consider viral protein thermal rearrangements, and to open new opportunities for biologically active complex design using information about SPs surface amino acid composition and methods of non-specific adsorption and bioconjugation.

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen.

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

Structure and properties of virions and virus-like particles derived from the coat protein of Alternanthera mosaic virus.

Plant viruses and their virus-like particles (VLPs) have a lot of advantages for biotechnological applications including complete safety for humans. Alternanthera mosaic virus (AltMV) is a potentially promising object for design of novel materials. The 3D structures of AltMV virions and its VLPs were obtained by single particle EM at ~13Å resolution. The comparison of the reconstructions and a trypsin treatment revealed that AltMV CPs possesses a different fold in the presence (virions) and absence of viral RNA (VLPs). For the first time, the structure of morphologically similar virions and virus-like particles based on the coat protein of a helical filamentous plant virus is shown to be different. Despite this, both AltMV virions and VLPs are stable in a wide range of conditions. To provide a large amount of AltMV for biotechnology usage the isolation procedure was modified.

Study of rubella candidate vaccine based on a structurally modified plant virus.

A novel rubella candidate vaccine based on a structurally modified plant virus - spherical particles (SPs) - was developed. SPs generated by the thermal remodelling of the tobacco mosaic virus are promising platforms for the development of vaccines. SPs combine unique properties: biosafety, stability, high immunogenicity and the effective adsorption of antigens. We assembled in vitro and characterised complexes (candidate vaccine) based on SPs and the rubella virus recombinant antigen. The candidate vaccine induced a strong humoral immune response against rubella. The IgG isotypes ratio indicated the predominance of IgG1 which plays a key role in immunity to natural rubella infection. The immune response was generally directed against the rubella antigen within the complexes. We suggest that SPs can act as a platform (depot) for the rubella antigen, enhancing specific immune response. Our results demonstrate that SPs-antigen complexes can be an effective and safe candidate vaccine against rubella.