Transformative vaccination: A pentavalent shield against COVID-19 and influenza with betulin-based adjuvant for enhanced immunity.

The development of an effective combined vaccine represents a crucial strategy for preventing outbreaks of infectious diseases and reducing the burden on healthcare resources. Developing a combined vaccine against both influenza and the coronavirus is a promising approach, but it is still in the early stages of development. This paper reports on a novel combined pentavalent candidate vaccine that has shown promising results in mice, with statistically significant differences in mean antibody titer against the coronavirus and the influenza antigens compared to placebo. We have shown that the coronavirus antigen is capable of inducing an immune response autonomously, regardless of the presence of the influenza antigens in a combined vaccine. On the other hand, the presence of the coronavirus antigen in a combined vaccine showed to enhance the immune response against some of the studied influenza antigens, suggesting that these antigens may act in synergy and elicit an enhanced immune response. The absence of dose-dependent difference in mean antibody titer within the same antigenic groups of vaccine preparations suggested that even small amounts of the coronavirus and the influenza antigens could induce an immune response just as good as high-dose vaccine preparations, which certainly has important safety and cost implications. The vaccine is soon to be ready for clinical trials and mass production.

Longitudinal Analysis of Neuraminidase and Hemagglutinin Antibodies to Influenza A Viruses after Immunization with Seasonal Inactivated Influenza Vaccines.

Neuraminidase (NA)-based immunity could reduce the harmful impact of novel antigenic variants of influenza viruses. The detection of neuraminidase-inhibiting (NI) antibodies in parallel with anti-hemagglutinin (HA) antibodies may enhance research on the immunogenicity and duration of antibody responses to influenza vaccines. To assess anti-NA antibodies after vaccination with seasonal inactivated influenza vaccines, we used the enzyme-linked lectin assay, and anti-HA antibodies were detected in the hemagglutination inhibition assay. The dynamics of the anti-NA antibody response differed depending on the virus subtype: antibodies to A/H3N2 virus neuraminidase increased later than antibodies to A/H1N1pdm09 subtype neuraminidase and persisted longer. In contrast to HA antibodies, the fold increase in antibody titers to NA after vaccination poorly depended on the preexisting level. At the same time, NA antibody levels after vaccination directly correlated with titers before vaccination. A difference was found in response to NA antigen between split and subunit-adjuvanted vaccines and in NA functional activity in the vaccine formulations.

IgGκ Signal Peptide Enhances the Efficacy of an Influenza Vector Vaccine against Respiratory Syncytial Virus Infection in Mice.

Intranasal vaccination using influenza vectors is a promising approach to developing vaccines against respiratory pathogens due to the activation of the mucosa-associated immune response. However, there is no clear evidence of a vector design that could be considered preferable. To find the optimal structure of an influenza vector with a modified NS genomic segment, we constructed four vector expressing identical transgene sequences inherited from the F protein of the respiratory syncytial virus (RSV). Two vectors were designed aiming at transgene accumulation in the cytosol. Another two were supplemented with an IgGκ signal peptide prior to the transgene for its extracellular delivery. Surprisingly, adding the IgGκ substantially enhanced the T-cell immune response to the CD8 epitope of the transgene. Moreover, this strategy allowed us to obtain a better protection of mice from the RSV challenge after a single intranasal immunization. Protection was achieved without antibodies, mediated by a balanced T-cell immune response including the formation of the RSV specific effector CD8+ IFNγ+/IL10+-producing cells and the accumulation of Treg cells preventing immunopathology in the lungs of infected mice. In addition to the presented method for optimizing the influenza vector, our results highlight the possibility of achieving protection against RSV through a respiratory-associated T-cell immune response alone.

Safety and Immunogenicity of Betuvax-CoV-2, an RBD-Fc-Based SARS-CoV-2 Recombinant Vaccine: Preliminary Results of the First-in-Human, Randomized, Double-Blind, Placebo-Controlled Phase I/II Clinical Trial.

COVID-19, being a life-threatening infection that evolves rapidly, remains a major public health concern calling for the development of vaccines with broad protection against different pathogenic strains and high immunogenicity. Aside from this, other concerns in mass immunization settings are also the scalability of production and relative affordability of the technology. In that regard, adjuvanted protein vaccines with particles mimicking the virus stand out among known vaccine technologies. The "Betuvax-CoV-2" vaccine, developed on the basis of a recombinant protein and an adjuvant, has already been tested in preclinical studies and has advanced to clinical evaluation. Open, double-blinded, placebo-controlled, randomized phase I/II clinical trial of the "Betuvax-CoV-2," recombinant protein subunit vaccine based on the S-protein RBD fused with the Fc-fragment of IgG, was conducted to evaluate safety and immunogenicity in response to the vaccination. METHODS: In the phase I/II clinical trial, 116 healthy adult men and women, ages 18-58, were enrolled: 20 in Stage I, and 96 in Stage II. In Stage I, 20 µg of the vaccine was administered intramuscularly on day 2, and either 5 µg (group 1) or 20 µg (group 2) on day 30. In Stage II, 20 µg of the vaccine was administered intramuscularly on day 2, and either 5 µg (group 3) or 20 µg (group 4) on day 30. In group 5, both injections were replaced with placebo. The primary outcome measures were safety (number of participants with adverse events throughout the study) and antigen-specific humoral immunity (SARS-CoV-2-specific antibodies measured by ELISA and CMIA). Antigen-specific cell-mediated immunity and changes in neutralizing antibodies (detected with a SARS-CoV-2 neutralization assay) were measured as a secondary outcome. The trial is registered with ClinicalTrials.gov (Study Identifier: NCT05270954). FINDINGS: Both vaccine formulations (20 µg + 5 µg and 20 µg + 20 µg) were safe and well tolerated. Most adverse events were mild, and no serious adverse events were detected. On day 51,anti-SARS-CoV-2 total and IgG antibody titers and anti-SARS-CoV-2 neutralizing antibodies were significantly higher in the vaccine groups (both formulations) than in the placebo. A more pronounced CD4+-mediated immune response was observed in the group of volunteers administered with the 20 + 20 µg vaccine formulation. INTERPRETATIONS: RBD-Fc-based COVID-19 "Betuvax-CoV-2" vaccine in doses (20 + 5 µg and 20 + 20 µg) demonstrated an excellent safety profile and induced a strong humoral response. Further research on the protective effectiveness of the "Betuvax-CoV-2" vaccine for the prevention of COVID-19 is on its way.

SARS-CoV-2 Subunit Virus-like Vaccine Demonstrates High Safety Profile and Protective Efficacy: Preclinical Study.

Public health threat coming from a rapidly developing COVID-19 pandemic calls for developing safe and effective vaccines with innovative designs. This paper presents preclinical trial results of "Betuvax-CoV-2", a vaccine developed as a subunit vaccine containing a recombinant RBD-Fc fusion protein and betulin-based spherical virus-like nanoparticles as an adjuvant ("Betuspheres"). The study aimed to demonstrate vaccine safety in mice, rats, and Chinchilla rabbits through acute, subchronic, and reproductive toxicity studies. Along with safety, the vaccine demonstrated protective efficacy through SARS-CoV-2-neutralizing antibody production in mice, rats, hamsters, rabbits, and primates (rhesus macaque), and lung damage and infection protection in hamsters and rhesus macaque model. Eventually, "Betuvax-CoV-2" was proved to confer superior efficacy and protection against the SARS-CoV-2 in preclinical studies. Based on the above results, the vaccine was enabled to enter clinical trials that are currently underway.

Hybrid Proteins with Short Conformational Epitopes of the Receptor-Binding Domain of SARS-CoV-2 Spike Protein Promote Production of Virus-Neutralizing Antibodies When Used for Immunization.

Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein-protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.

Design and Immunological Properties of the Novel Subunit Virus-like Vaccine against SARS-CoV-2.

The COVID-19 pandemic is ongoing, and the need for safe and effective vaccines to prevent infection and to control spread of the virus remains urgent. Here, we report the development of a SARS-CoV-2 subunit vaccine candidate (Betuvax-CoV-2) based on RBD and SD1 domains of the spike (S) protein fused to a human IgG1 Fc fragment. The antigen is adsorbed on betulin adjuvant, forming spherical particles with a size of 100-180 nm, mimicking the size of viral particles. Here we confirm the potent immunostimulatory activity of betulin adjuvant, and demonstrate that two immunizations of mice with Betuvax-CoV-2 elicited high titers of RBD-specific antibodies. The candidate vaccine was also effective in stimulating a neutralizing antibody response and T cell immunity. The results indicate that Betuvax-CoV-2 has good potential for further development as an effective vaccine against SARS-CoV-2.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Enhancement of the Local CD8+ T-Cellular Immune Response to Mycobacterium tuberculosis in BCG-Primed Mice after Intranasal Administration of Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens.

BCG is the only licensed vaccine against Mycobacterium tuberculosis (M.tb) infection. Due to its intramuscular administration route, BCG is unable to induce a local protective immune response in the respiratory system. Moreover, BCG has a diminished ability to induce long-lived memory T-cells which are indispensable for antituberculosis protection. Recently we described the protective efficacy of new mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing TB10.4 and HspX proteins of M.tb within an NS1 influenza protein open reading frame. In the present work, the innate and adaptive immune response to immunization with the Flu/THSP and the immunological properties of vaccine candidate in the BCG-prime → Flu/THSP vector boost vaccination scheme are studied in mice. It was shown that the mucosal administration of Flu/THSP induces the incoming of interstitial macrophages in the lung tissue and stimulates the expression of co-stimulatory CD86 and CD83 molecules on antigen-presenting cells. The T-cellular immune response to Flu/THSP vector was mediated predominantly by the IFNγ-producing CD8+ lymphocytes. BCG-prime → Flu/THSP vector boost immunization scheme was shown to protect mice from severe lung injury caused by M.tb infection due to the enhanced T-cellular immune response, mediated by antigen-specific effector and central memory CD4+ and CD8+ T-lymphocytes.

IFN-λ1 Displays Various Levels of Antiviral Activity In Vitro in a Select Panel of RNA Viruses.

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: 'preventive' (pretreatment); 'preventive/therapeutic' (pre/post); and 'therapeutic' (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the 'preventive' and 'preventive/therapeutic' regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.

Mucosal Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens Provides Protection against Mycobacterium tuberculosis in Mice and Guinea Pigs.

New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1-124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.

Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector.

Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors - B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials.

Antimycotic-antibiotic amphotericin B promotes influenza virus replication in cell culture.

In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.

Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1.

BACKGROUND: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH ≤ 5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. CONCLUSION/SIGNIFICANCE: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.