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.

In silico design of influenza a virus artificial epitope-based T-cell antigens and the evaluation of their immunogenicity in mice.

The polyepitope strategy is promising approach for successfully creating a broadly protective flu vaccine, which targets T-lymphocytes (both CD4+ and CD8+) to recognise the most conserved epitopes of viral proteins. In this study, we employed a computer-aided approach to develop several artificial antigens potentially capable of evoking immune responses to different virus subtypes. These antigens included conservative T-cell epitopes of different influenza A virus proteins. To design epitope-based antigens we used experimentally verified information regarding influenza virus T-cell epitopes from the Immune Epitope Database (IEDB) (http://www.iedb.org). We constructed two "human" and two "murine" variants of polyepitope antigens. Amino acid sequences of target polyepitope antigens were designed using our original TEpredict/PolyCTLDesigner software. Immunogenic and protective features of DNA constructs encoding "murine" target T-cell immunogens were studied in BALB/c mice. We showed that mice groups immunised with a combination of computer-generated "murine" DNA immunogens had a 37.5% survival rate after receiving a lethal dose of either A/California/4/2009 (H1N1) virus or A/Aichi/2/68 (H3N2) virus, while immunisation with live flu H1N1 and H3N2 vaccine strains provided protection against homologous viruses and failed to protect against heterologous viruses. These results demonstrate that mechanisms of cross-protective immunity may be associated with the stimulation of specific T-cell responses. This study demonstrates that our computer-aided approach may be successfully used for rational designing artificial polyepitope antigens capable of inducing virus-specific T-lymphocyte responses and providing partial protection against two different influenza virus subtypes.Communicated by Ramaswamy H. Sarma.

Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens.

Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A-H1N1 and H3N2-and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity. We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0-M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.

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.

In silico Designed Ebola Virus T-Cell Multi-Epitope DNA Vaccine Constructions Are Immunogenic in Mice.

Background: The lack of effective vaccines against Ebola virus initiates a search for new approaches to overcoming this problem. The aim of the study was to design artificial polyepitope T-cell immunogens⁻⁻candidate DNA vaccines against Ebola virus and to evaluate their capacity to induce a specific immune response in a laboratory animal model. Method: Design of two artificial polyepitope T-cell immunogens, one of which (EV.CTL) includes cytotoxic and the other (EV.Th)⁻⁻T-helper epitopes of Ebola virus proteins was carried out using original TEpredict/PolyCTLDesigner software. Synthesized genes were cloned in pcDNA3.1 plasmid vector. Target gene expression was estimated by synthesis of specific mRNAs and proteins in cells transfected with recombinant plasmids. Immunogenicity of obtained DNA vaccine constructs was evaluated according to their capacity to induce T-cell response in BALB/c mice using IFNγ ELISpot and ICS. Results: We show that recombinant plasmids pEV.CTL and pEV.Th encoding artificial antigens provide synthesis of corresponding mRNAs and proteins in transfected cells, as well as induce specific responses both to CD4+ and CD8+ T-lymphocytes in immunized animals. Conclusions: The obtained recombinant plasmids can be regarded as promising DNA vaccine candidates in future studies of their capacity to induce cytotoxic and protective responses against Ebola virus.

Evaluation of Influenza Vaccination Efficacy: A Universal Epidemic Model.

By means of a designed epidemic model, we evaluated the influence of seasonal vaccination coverage as well as a potential universal vaccine with differing efficacy on the aftermath of seasonal and pandemic influenza. The results of the modeling enabled us to conclude that, to control a seasonal influenza epidemic with a reproduction coefficient R0 ≤ 1.5, a 35% vaccination coverage with the current seasonal influenza vaccine formulation is sufficient, provided that other epidemiology measures are regularly implemented. Increasing R0 level of pandemic strains will obviously require stronger intervention. In addition, seasonal influenza vaccines fail to confer protection against antigenically distinct pandemic influenza strains. Therefore, the necessity of a universal influenza vaccine is clear. The model predicts that a potential universal vaccine will be able to provide sufficient reliable (90%) protection against pandemic influenza only if its efficacy is comparable with the effectiveness of modern vaccines against seasonal influenza strains (70%-80%); given that at least 40% of the population has been vaccinated in advance, ill individuals have been isolated (observed), and a quarantine has been introduced. If other antiepidemic measures are absent, a vaccination coverage of at least 80% is required.

Novel approaches in polyepitope T-cell vaccine development against HIV-1.

RV144 clinical trial was modestly effective in preventing HIV infection. New alternative approaches are needed to design improved HIV-1 vaccines and their delivery strategies. One of these approaches is construction of synthetic polyepitope HIV-1 immunogen using protective T- and B-cell epitopes that can induce broadly neutralizing antibodies and responses of cytotoxic (CD8(+) CTL) and helpers (CD4(+) Th) T-lymphocytes. This approach seems to be promising for designing of new generation of vaccines against HIV-1, enables in theory to cope with HIV-1 antigenic variability, focuses immune responses on protective determinants and enables to exclude from the vaccine compound that can induce autoantibodies or antibodies enhancing HIV-1 infectivity. Herein, the authors will focus on construction and rational design of polyepitope T-cell HIV-1 immunogens and their delivery, including: advantages and disadvantages of existing T-cell epitope prediction methods; features of organization of polyepitope immunogens, which can generate high-level CD8(+) and CD4(+) T-lymphocyte responses; the strategies to optimize efficient processing, presentation and immunogenicity of polyepitope constructs; original software to design polyepitope immunogens; and delivery vectors as well as mucosal strategies of vaccination. This new knowledge may bring us a one step closer to developing an effective T-cell vaccine against HIV-1, other chronic viral infections and cancer.

Mathematical model of the Tat-Rev regulation of HIV-1 replication in an activated cell predicts the existence of oscillatory dynamics in the synthesis of viral components.

BACKGROUND: The life cycle of human immunodeficiency virus type-1 (HIV-1) makes possible the realization of regulatory strategies that can lead to complex dynamical behavior of the system. We analyze the strategy which is based on two feedback mechanisms, one mediating a positive regulation of the virus replication by Tat protein via the antitermination of the genomic RNAs transcription on TAR (transactivation responsive) element of the proviral DNA and the second mechanism providing a negative regulation of the splicing of the full-length (9 kb) RNAs and incompletely spliced (4 kb) RNAs via their transport from the nucleus to the cytoplasm. Although the existence of these two regulatory feedback loops has been considered in other mathematical models, none of them examined the conditions for the emergence of complex oscillatory patterns in the intracellular dynamics of viral components. RESULTS: We developed a mechanistic mathematical model for the Tat-Rev mediated regulation of HIV-1 replication, which considers the activation of proviral DNA transcription, the Tat-specific antitermination of transcription on TAR-element, resulting in the synthesis of the full-length 9 kb RNA, the splicing of the 9 kb RNA down to the 4 kb RNA and the 4 kb RNA to 2 kb RNA, the transport of 2 kb mRNAs from the nucleus to the cytoplasm by the intracellular mechanisms, the multiple binding of the Rev protein to RRE (Rev Response Element) sites on 9 kb and 4 kb RNA resulting in their export to the cytoplasm and the synthesis of Tat and Rev proteins in the cytoplasm followed by their transport into the nucleus. The degradation of all viral proteins and RNAs both in the cytoplasm and the nucleus is described. The model parameters values were derived from the published literature data. The model was used to examine the dynamics of the synthesis of the viral proteins Tat and Rev, the mRNAs under the intracellular conditions specific for activated HIV-1 infected macrophages. In addition, we analyzed alternative hypotheses for the re-cycling of the Rev proteins both in the cytoplasm and the nuclear pore complex. CONCLUSIONS: The quantitative mathematical model of the Tat-Rev regulation of HIV-1 replication predicts the existence of oscillatory dynamics which depends on the efficacy of the Tat and TAR interaction as well as on the Rev-mediated transport processes. The biological relevance of the oscillatory regimes for the HIV-1 life cycle is discussed.

PolyCTLDesigner: a computational tool for constructing polyepitope T-cell antigens.

BACKGROUND: Construction of artificial polyepitope antigens is one of the most promising strategies for developing more efficient and safer vaccines evoking T-cell immune responses. Epitope rearrangements and utilization of certain spacer sequences have been proven to greatly influence the immunogenicity of polyepitope constructs. However, despite numerous efforts towards constructing and evaluating artificial polyepitope immunogens as well as despite numerous computational methods elaborated to date for predicting T-cell epitopes, peptides binding to TAP and for antigen processing prediction, only a few computational tools were currently developed for rational design of polyepitope antigens. FINDINGS: Here we present a PolyCTLDesigner program that is intended for constructing polyepitope immunogens. Given a set of either known or predicted T-cell epitopes the program selects N-terminal flanking sequences for each epitope to optimize its binding to TAP (if necessary) and joins resulting oligopeptides into a polyepitope in a way providing efficient liberation of potential epitopes by proteasomal and/or immunoproteasomal processing. And it also tries to minimize the number of non-target junctional epitopes resulting from artificial juxtaposition of target epitopes within the polyepitope. For constructing polyepitopes, PolyCTLDesigner utilizes known amino acid patterns of TAP-binding and proteasomal/immunoproteasomal cleavage specificity together with genetic algorithm and graph theory approaches. The program was implemented using Python programming language and it can be used either interactively or through scripting, which allows users familiar with Python to create custom pipelines. CONCLUSIONS: The developed software realizes a rational approach to designing poly-CTL-epitope antigens and can be used to develop new candidate polyepitope vaccines. The current version of PolyCTLDesigner is integrated with our TEpredict program for predicting T-cell epitopes, and thus it can be used not only for constructing the polyepitope antigens based on preselected sets of T-cell epitopes, but also for predicting cytotoxic and helper T-cell epitopes within selected protein antigens. PolyCTLDesigner is freely available from the project's web site: http://tepredict.sourceforge.net/PolyCTLDesigner.html.

Attenuated Salmonella enteritidis E23 as a vehicle for the rectal delivery of DNA vaccine coding for HIV-1 polyepitope CTL immunogen.

This study is focusing on elucidation of the capacity of attenuated Salmonella enteritidis E23 (cya, crp) to serve as a vehicle for the rectal delivery of the DNA vaccine. Earlier for creation HIV-1 candidate DNA vaccine we have designed the polyepitope protein TCI (T-cell immunogen), which comprises over 80 CTL epitopes from subtype A, B and C HIV-1 proteins. The gene coding for TCI protein was used to construct the eukaryotic expression plasmid pcDNA-TCI. The attenuated S. enteritidis E23 was transformed by electroporation with recombinant plasmid pcDNA-TCI and the expression of the TCI gene was determined in vitro and in vivo. BALB/c mice were rectally immunized with S. enteritidis E23/pcDNA-TCI (108 cfu) twice at 4 week interval. Bacteria were not pathogenic for mice and spontaneously eliminated from mice spleen and liver to 60 days post the immunization. Detectable antibodies were generated in 2 weeks after immunization and their level increased after second immunization. The results of INF-γ ELISpot show that mice immunized with S. enteritidis E23/pcDNA-TCI elicited HIV-specific cellular immune response. This study demonstrates that attenuated S. enteritidis E23 is an effective live vector for rectal delivery of the DNA vaccine pcDNA-TCI to generate humoral and T-cellular responses against HIV-1.

A synergistic effect of a combined bivalent DNA-protein anti-HIV-1 vaccine containing multiple T- and B-cell epitopes of HIV-1 proteins.

Immunogenic properties of the combined vaccine CombiHIVvac, comprising polyepitope HIV-1 immunogens, one being the artificial polyepitope protein TBI, containing the T- and B-cell epitopes from Env and Gag proteins, and the DNA vaccine construct pcDNA-TCI coding for the artificial protein TCI, carrying over 80 T-cell epitopes (both CD4+ CTL and CD8+ Th) from Env, Gag, Pol, and Nef proteins, are studied in this work. The data reported demonstrate clearly that a combination of two B- and T-cell immunogens (TBI and TCI) in one construct results in a synergistic increase in the antibody response to both TBI protein and the proteins from HIV-1 lysate. The level of antibodies induced by immunization with the constructs containing either immunogen alone (TBI protein or the plasmid pcDNA-TCI) was significantly lower as compared to that induced by the combined vaccine. The analysis performed suggests that the presence of CD4+ T-helper epitopes, which can be presented by MHC class II, in the protein TCI may be the main reason underlying the increased synthesis of antibodies to TBI protein due to a CD4-mediated stimulation of B-cell proliferation and differentiation.

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.

Exclusion of HIV epitopes shared with human proteins is prerequisite for designing safer AIDS vaccines.

BACKGROUND: The safety of a potential AIDS vaccine is an issue that will become critical at later stages of product development and needs to be addressed before it is too late. OBJECTIVE: In order to design safer vaccine, the HIV antigens, to be deployed in it, should be free of regions that are either present in human proteins or exhibit pronounced structural similarity to proteins responsible for important physiological functions. STUDY DESIGN: The approach is based on the use of an original matrix predicting the antigenic similarity of amino acids. This mathematical approach developed by us was applied for identification of fragments with similarity to human proteins within potentially immunodominant regions of HIV proteins. A potential self-sensitization by viral quasispecies with variants of hypervariable V3 region, generated as a result of immune pressure on the immunodominant region of envelope, was considered in detail. RESULTS: Viral fragments occurring in normal human proteins as well as regions exhibiting high similarity to proteins responsible for physiological homeostasis were identified in every HIV protein at a frequency higher than expected. Most such regions contained either T-cell (CD8(+) CTL or CD4(+) Helper) or B-cell epitopes, or both of them simultaneously. The gained knowledge was applied in designing a synthetic immunogen containing multiple CTL epitopes. The synthesis of series of chimeric peptides representing hypervariable region of V3 loop of HIV envelope, to be used as a multi-epitope or mixotope vaccine candidate, has been achieved. Such a vaccine could theoretically pre-empt any escape mutant borrowing from antigenic diversity of hypervariable region of V3 loop of HIV envelope. CONCLUSIONS: The epitopes shared by HIV and its host are likely to be implicated in the immunopathogenesis of AIDS through induction of cross-reacting effectors of the immune system. The prospect that 'house-keeping' immune mechanism can be foiled by molecular mimicry of HIV with physiologically important human proteins should be taken into consideration in safer vaccine design.

Designing and engineering of DNA-vaccine construction encoding multiple CTL-epitopes of major HIV-1 antigens.

A synthetic T cell immunogen (TCI) has been designed as a candidate DNA-based vaccine against Human immunodeficiency virus (HIV)-1 using cytotoxic T lymphocytes (CD8(+) CTL) and T-helper lymphocytes (CD4(+) Th) epitopes retrieved from the Los Alamos HIV Molecular Immunology Database. The protein 392 amino acids in length contains about eighty CTL-epitopes, many of which are overlapping and are totally restricted by ten different HLA class I molecules. To be able to detect CTL responses induced by a DNA vaccine in experimental animals, additional epitopes, restricted by mouse and Macaque rhesus major histocompatibility complex (MHC) class I molecules, were included in the target immunogen. The gene encoding the TCI protein was assembled, cloned into vector plasmids and expressed in a prokaryotic and a eukaryotic system. The presence of HIV-1 protein fragments in the immunogen structure was ascertained by ELISA and immunoblotting using panels of HIV-1-positive sera and monoclonal antibodies to p24. It has been demonstrated that DNA vaccine can induce both specific T cell responses (CTL and blast transformation) and specific antibodies in mice immunized with pcDNA-TCI.

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.