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.

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.

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.

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.

The role of the 5'-cap structure in viral ribonucleoproteins assembly from potato virus X coat protein and RNAs.

The potato virus X (PVX) virion can be reconstituted in vitro from the virus coat protein (CP) and RNA; heterologous RNAs may be used as well. In our recent study, structure and properties of cognate and heterologous viral ribonucleoproteins (vRNPs) were demonstrated to be similar to those of native virions. The assembly was found to be initiated at the 5' terminus of an RNA and was not dependent on RNA sequence. The aim of the present study was to search for a signal or an essential structural element that directs packaging of viral genetic material into vRNPs. vRNPs were formed by incubation of the PVX CP with heterologous capped RNAs, their functional fragments lacking the cap structure, as well as the capped and uncapped transcripts corresponding to the 5'-terminal region of the genomic PVX RNA. Experimental data show that the presence of the cap structure at the 5' end of a nucleic acid is an important condition for vRNP assembly from RNA and CP. Presumably, the 5'-cap affects conformational state of the RNA region responsible for the efficient interaction with CP and creates conformational encapsidation signal for vRNP assembly.

Characterization of Alternanthera mosaic virus and its Coat Protein.

A new isolate of Alternantheramosaic virus (AltMV-MU) was purified from Portulaca grandiflora plants. It has been shown that the AltMV-MU coat protein (CP) can be efficiently reassembled in vitro under different conditions into helical RNA-free virus-like particles (VLPs) antigenically related to native virus. The AltMV-MU and VLPs were examined by atomic force and transmission electron microscopies. The encapsidated AltMV-MU RNA is nontranslatable in vitro. However, it can be translationally activated by CP phosphorylation or by binding to the TGB1protein from the virus-coded movement triple gene block.

Linear remodeling of helical virus by movement protein binding.

Previously we have shown that encapsidated potato virus X (PVX) RNA was nontranslatable in vitro, but could be converted into a translatable form by binding of the PVX-coded movement protein (termed TGBp1) to one end of a polar helical PVX virion. We reported that binding of TGBp1 to coat protein (CP) subunits located at one extremity of the helical particles induced a linear destabilization of the CP helix, which was transmitted along the whole particle. Two model structures were used: (i) native PVX and (ii) artificial polar helical PVX-like particles lacking intact RNA (PVX(RNA-DEG)). Binding of TGBp1 to the end of either of these particles led to their destabilization, but no disassembly of the CP helix occurred. Influence of additional factors was required to trigger rapid disassembly of TGBp1-PVX and TGBp1-PVX(RNA-DEG) complexes. Thus: (i) no disassembly was observed unless TGBp1-PVX complex was translated. A novel phenomenon of TGBp1-dependent, ribosome-triggered disassembly of PVX was described: initiation of translation and few translocation steps were needed to trigger rapid (and presumably cooperative) disassembly of TGBp1-PVX into protein subunits and RNA. Importantly, the whole of the RNA molecule (including its 3'-terminal region) was released. The TGBp1-induced linear destabilization of CP helix was reversible, suggesting that PVX in TGBp1-PVX complex was metastable; (ii) entire disassembly of the TGBp1-PVX(RNA-DEG) complex (but not of the TGBp1-free PVX(RNA-DEG) particles) into 2.8S subunits was triggered under influence of a centrifugal field. To our knowledge, transmission of the linear destabilization along the polar helical protein array induced by a foreign protein binding to the end of the helix represents a novel phenomenon. It is tempting to suggest that binding of TGBp1 to the end of the PVX CP helix induced conformational changes in terminal CP subunits that can be linearly transferred along the whole helical particle, i.e. that intersubunit conformational changes may be transferred along the CP helix.

AFM study of potato virus X disassembly induced by movement protein.

Recently we have reported that a selective binding of potato virus X (PVX)-coded movement protein (termed TGBp1 MP) to one end of a polar coat protein (CP) helix converted viral RNA into a translatable form and induced a linear destabilization of the whole helical particle. Here, the native PVX virions, RNase-treated (PVX(RNA-DEG)) helical particles lacking intact RNA and their complexes with TGBp1 (TGBp1-PVX and TGBp1-PVX(RNA-DEG)), were examined by atomic force microscopy (AFM). When complexes of the TGBp1 MP with PVX were examined by means of AFM in liquid, no structural reorganization of PVX particles was observed. By contrast, the products of TGBp1-dependent PVX degradation termed "beads-on-string" were formed under conditions of AFM in air. The AFM images of PVX(RNA-DEG) were indistinguishable from images of native PVX particles; however, the TGBp1-dependent disassembly of the CP-helix was triggered when the TGBp1-PVX(RNA-DEG) complexes were examined by AFM, regardless of the conditions used (in air or in liquid). Our data supported the idea that binding of TGBp1 to one end of the PVX CP-helix induced linear destabilization of the whole helical particle, which may lead to its disassembly under conditions of AFM.