SARS-CoV-2 RBD Conjugated to Polyglucin, Spermidine, and dsRNA Elicits a Strong Immune Response in Mice.

Despite the rapid development and approval of several COVID vaccines based on the full-length spike protein, there is a need for safe, potent, and high-volume vaccines. Considering the predominance of the production of neutralizing antibodies targeting the receptor-binding domain (RBD) of S-protein after natural infection or vaccination, it makes sense to choose RBD as a vaccine immunogen. However, due to its small size, RBD exhibits relatively poor immunogenicity. Searching for novel adjuvants for RBD-based vaccine formulations is considered a good strategy for enhancing its immunogenicity. Herein, we assess the immunogenicity of severe acute respiratory syndrome coronavirus 2 RBD conjugated to a polyglucin:spermidine complex (PGS) and dsRNA (RBD-PGS + dsRNA) in a mouse model. BALB/c mice were immunized intramuscularly twice, with a 2-week interval, with 50 µg of RBD, RBD with Al(OH)3, or conjugated RBD. A comparative analysis of serum RBD-specific IgG and neutralizing antibody titers showed that PGS, PGS + dsRNA, and Al(OH)3 enhanced the specific humoral response in animals. There was no significant difference between the groups immunized with RBD-PGS + dsRNA and RBD with Al(OH)3. Additionally, the study of the T-cell response in animals showed that, unlike adjuvants, the RBD-PGS + dsRNA conjugate stimulates the production of specific CD4+ and CD8+ T cells in animals.

Self-Assembled Particles Combining SARS-CoV-2 RBD Protein and RBD DNA Vaccine Induce Synergistic Enhancement of the Humoral Response in Mice.

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.

Delivery of mRNA Vaccine against SARS-CoV-2 Using a Polyglucin:Spermidine Conjugate.

One of the key stages in the development of mRNA vaccines is their delivery. Along with liposome, other materials are being developed for mRNA delivery that can ensure both the safety and effectiveness of the vaccine, and also facilitate its storage and transportation. In this study, we investigated the polyglucin:spermidine conjugate as a carrier of an mRNA-RBD vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The conditions for the self-assembling of mRNA-PGS complexes were optimized, including the selection of the mRNA:PGS charge ratios. Using dynamic and electrophoretic light scattering it was shown that the most monodisperse suspension of nanoparticles was formed at the mRNA:PGS charge ratio equal to 1:5. The average hydrodynamic particles diameter was determined, and it was confirmed by electron microscopy. The evaluation of the zeta potential of the investigated complexes showed that the particles surface charge was close to the zero point. This may indicate that the positively charged PGS conjugate has completely packed the negatively charged mRNA molecules. It has been shown that the packaging of mRNA-RBD into the PGS envelope leads to increased production of specific antibodies with virus-neutralizing activity in immunized BALB/c mice. Our results showed that the proposed polycationic polyglucin:spermidine conjugate can be considered a promising and safe means to the delivery of mRNA vaccines, in particular mRNA vaccines against SARS-CoV-2.

Cationic Polymers for the Delivery of the Ebola DNA Vaccine Encoding Artificial T-Cell Immunogen.

BACKGROUND: According to current data, an effective Ebola virus vaccine should induce both humoral and T-cell immunity. In this work, we focused our efforts on methods for delivering artificial T-cell immunogen in the form of a DNA vaccine, using generation 4 polyamidoamine dendrimers (PAMAM G4) and a polyglucin:spermidine conjugate (PG). METHODS: Optimal conditions were selected for obtaining complexes of previously developed DNA vaccines with cationic polymers. The sizes, mobility and surface charge of the complexes with PG and PAMAM 4G have been determined. The immunogenicity of the obtained vaccine constructs was investigated in BALB/c mice. RESULTS: It was shown that packaging of DNA vaccine constructs both in the PG envelope and the PAMAM 4G envelope results in an increase in their immunogenicity as compared with the group of mice immunized with the of vector plasmid pcDNA3.1 (a negative control). The highest T-cell responses were shown in mice immunized with complexes of DNA vaccines with PG and these responses significantly exceeded those in the groups of animals immunized with both the combination of naked DNAs and the combination DNAs coated with PAMAM 4G. In the group of animals immunized with complexes of the DNA vaccines with PAMAM 4G, no statistical differences were found in the ability to induce T-cell responses, as compared with the group of mice immunized with the combination of naked DNAs. CONCLUSIONS: The PG conjugate can be considered as a promising and safe means to deliver DNA-based vaccines. The use of PAMAM requires further optimization.

The action of stress hormones on the structure and function of erythrocyte membrane.

The action of a mixture of hormones (cortisol and adrenaline) on erythrocyte membrane during their binding was investigated. Changes in the membrane structure were elucidated by atomic force microscopy; microviscosity of the lipid bilayer and changes in the activity of Na(+),K(+)-ATPase at different concentrations of the hormones in erythrocyte suspension were estimated by the fluorescence method. Cortisol and adrenaline were shown to compete for the binding sites. A hormone that managed to bind nonspecifically to the membrane hindered the binding of another hormone. In a mixture of these hormones, cortisol won a competition for the binding sites; therewith, microviscosity of the membranes increased by 25%, which corresponds to a change in microviscosity produced by the action of cortisol alone. The competitive relationships affected also the Na(+),K(+)-ATPase activity, which was indicated by appearance of the second maximum of enzyme activity. It is assumed that an increase in microviscosity of erythrocyte membrane first raises the Na(+),K(+)-ATPase activity due to a growth of the maximum energy of membrane phonons, and then decreases the activity due to hindering of conformational transitions in the enzyme molecule.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVß3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process.

Immunochemical and single molecule force spectroscopy studies of specific interaction between the laminin binding protein and the West Nile virus surface glycoprotein E domain II.

ELISA and Western blot immunochemical data attest an effective and highly specific interaction of the surface glycoprotein E domain II (DII) of the tick born encephalitis and Dengue viruses with the laminin binding protein (LBP). Based on a highly conservative structure of the DII in different flaviviruses we propose a similarly effective interaction between the LBP and the DII of the surface glycoprotein E of the West Nile virus. We report the results of studies of this interaction by immunochemical and single molecule force spectroscopy methods. The specific binding between these species is confirmed by both methods.

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.

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.