Safety and immunogenicity of rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine against SARS-CoV-2 in healthy adolescents: an open-label, non-randomized, multicenter, phase 1/2, dose-escalation study

To protect young individuals against SARS-CoV-2 infection, we conducted an open-label, prospective, non-randomised dose-escalation Phase 1/2 clinical trial to evaluate the immunogenicity and safety of the prime-boost “Sputnik V” vaccine administered at 1/10 and 1/5 doses to adolescents aged 12–17 years. The study began with the vaccination of the older cohort (15-to-17-year-old participants) with the lower (1/10) dose of vaccine and then expanded to the whole group (12-to-17-year-old participants). Next, 1/5 dose was used according to the same scheme. Both doses were well tolerated by all age groups. No serious or severe adverse events were detected. Most of the solicited adverse reactions were mild. No significant differences in total frequencies of adverse events were registered between low and high doses in age-pooled groups (69.6% versus 66.7%). In contrast, the 1/5 dose induced significantly higher humoral and T cell-mediated immune responses than the 1/10 dose. The 1/5 vaccine dose elicited higher antigen-binding (both S and RBD-specific) as well as virus-neutralising antibody titres at the maximum of response (day 42), also resulting in a statistically significant difference at a distanced timepoint (day 180) compared to the 1/10 vaccine dose. Higher dose resulted in increased cross-neutralization of Delta and Omicron variants.;Clinical Trial RegistrationClinicalTrials.gov, NCT04954092, LP-007632

Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa

Induction of a robust and long-lived mucosal immune response during vaccination is critical to achieve protection against numerous pathogens. However, traditional injected vaccines are generally poor inducers of mucosal immunity. One of the effective strategies to improve vaccine efficacy is incorporation of adjuvant molecules that enhance and polarize adaptive immune reactions. Effects of Syk-coupled lectin receptor agonists as adjuvants to induce mucosal immune reactions during parenteral immunization are not fully studied. We now report that the agonists trehalose-6,6-dibehenate (TDB), curdlan, and furfurman, which stimulate Dectin-1, Dectin-2, and Mincle, respectively, activate transcription factors (NF-κB, NFAT, and AP-1) to various extents in murine RAW 264.7 macrophages, even though similar pathways are activated. The agonists also elicit differential expression of maturation markers in bone marrow-derived dendritic cells, as well as differential cytokine secretion from these cells and from splenic mononuclear cells. In vivo assays also show that agonists of Dectin-1 and Dectin-2, but not Mincle, induce heavy IgA secretion in intestinal mucosa even when delivered parenterally. Strikingly, this effect appears to be formulation-independent. Collectively, the data suggest that adjuvants based on Dectin-1 and Dectin-2 agonists may significantly improve the efficacy of parenteral vaccines by inducing robust local immune reactions in intestinal mucosa

Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which bind to the stem domain of hemagglutinin and efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 80-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc antibodies showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection

Powerful Complex Immunoadjuvant Based on Synergistic Effect of Combined TLR4 and NOD2 Activation Significantly Enhances Magnitude of Humoral and Cellular Adaptive Immune Responses

<div><p>Binding of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) activates innate immune responses and contributes to development of adaptive immunity. Simultaneous stimulation of different types of PRRs can have synergistic immunostimulatory effects resulting in enhanced production of molecules that mediate innate immunity such as inflammatory cytokines, antimicrobial peptides, etc. Here, we evaluated the impact of combined stimulation of PRRs from different families on adaptive immunity by generating alum-based vaccine formulations with ovalbumin as a model antigen and the Toll-like receptor 4 (TLR4) agonist MPLA and the Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) agonist MDP adsorbed individually or together on the alum-ovalbumin particles. Multiple <i>in vitro</i> and <i>in vivo</i> readouts of immune system activation all showed that while individual PRR agonists increased the immunogenicity of vaccines compared to alum alone, the combination of both PRR agonists was significantly more effective. Combined stimulation of TLR4 and NOD2 results in a stronger and broader transcriptional response in THP-1 cells compared to individual PRR stimulation. Immunostimulatory composition containing both PRR agonists (MPLA and MDP) in the context of the alum-based ovalbumin vaccine also enhanced uptake of vaccine particles by bone marrow derived dendritic cells (BMDCs) and promoted maturation (up-regulation of expression of CD80, CD86, MHCII) and activation (production of cytokines) of BMDCs. Finally, immunization of mice with vaccine particles containing both PRR agonists resulted in enhanced cellular immunity as indicated by increased proliferation and activation (IFN-γ production) of splenic CD4+ and CD8+ T cells following <i>in vitro</i> restimulation with ovalbumin and enhanced humoral immunity as indicated by higher titers of ovalbumin-specific IgG antibodies. These results indicate that combined stimulation of TLR4 and NOD2 receptors dramatically enhances activation of both the humoral and cellular branches of adaptive immunity and suggests that inclusion of agonists of these receptors in standard alum-based adjuvants could be used to improve the effectiveness of vaccination.</p></div

Vaccine formulations containing a combination of TLR4 and NOD2 agonists significantly enhance maturation of BMDC compared to formulations containing individual PRR agonists.

<p>BMDCs were harvested on day 8 of culture and incubated in 24-well plates for 24 hours with vaccine formulations. Expression of the maturation markers, CD80 <b>(A)</b>, CD86 <b>(B)</b>, and major histocompatibility complex (MHC) class II <b>(C)</b> was assessed by flow cytometric analysis of 5x10⁴ CD11c+ cells. Mean MFI values (indicating expression level) ± SEM from two independent experiments with 5 replicates each are shown. * indicates significant difference (P≤0.05) between formulations containing MDP or MPLA individually and the formulation without PRR agonists. # indicates significant difference (P≤0.05) between Alum+OVA+MDP+MPLA treatment and Alum+OVA+MPLA or Alum+OVA+MDP (Student’s t-test). <b>(D)</b> Cytokine levels were measured in cell-free culture supernatants collected 24 hours after addition of vaccine formulations to BMDCs (4x10⁴ cells/well) using bead-based immunoassay. Data represent mean ± SD. * indicates significant difference (P≤0.05) between formulations containing MDP or MPLA individually and the formulation without PRR agonists. # indicates significant difference (P≤0.05) between Alum+OVA+MDP+MPLA treatment and Alum+OVA+MPLA or Alum+OVA+MDP (Student’s t-test).</p

Composition of prepared model vaccine formulations.

<p>Composition of prepared model vaccine formulations.</p

Vaccine formulations containing a combination of TLR4 and NOD2 agonists lead to a stronger ovalbumin-specific antibody response in mice than formulations containing individual PRR agonists.

<p>Mice (n = 5/group) were immunized s.c. twice with alum-based vaccine formulations: Alum+OVA, Alum+OVA+MDP, Alum+OVA+MPLA, and Alum+OVA+MDP+MPLA. Controls included naıve (untreated) mice and mice immunized with soluble OVA (no Alum or PRR agonist). Blood was collected from mice 14 days after the last immunization and serum levels of ovalbumin-specific total IgG (A), IgG1 (B), IgG2a (C), IgG2b (D) and IgG2c (E) antibodies were detected by ELISA. The mean for 5 mice/group ± SEM is shown. Experiment was repeated three times with analogous results.</p

Vaccine formulations containing a combination of TLR4 and NOD2 agonists enhance NF-κB/AP-1 activation in THP-1 cells compared to formulations containing individual agonists.

<p><b>(A)</b> Addition of the vaccine formulation containing both TLR4 and NOD2 agonists to THP1-XBlue^™-CD14 cells leads to enhanced NF-κB/AP-1-dependent SEAP activity compared to formulations with individual PRR agonists. SEAP activity was measured in cell-free culture supernatants 18 h after vaccine formulation addition. Results are expressed as the fold-increase in SEAP activity relative to untreated (intact) cells; mean values ± SD from three independent experiments, each performed in duplicate. * indicates significant difference (P≤0.05) between formulations containing MDP or MPLA individually and the formulation without PRR agonists. # indicates significant difference (P≤0.05) between Alum+OVA+MDP+MPLA treatment and Alum+OVA+MPLA or Alum+OVA+MDP (Student’s t-test). <b>(B)</b> Addition of the vaccine formulation containing both TLR4 and NOD2 agonists to THP1 cells leads to enhanced cytokine production in comparison to vaccine formulations with individual PRR agonists. Cells were left untreated or treated with Alum+OVA, Alum+OVA+MDP, Alum+OVA+MPLA, or Alum+OVA+MDP+MPLA formulations for 18 hrs. Cell-free supernatants were prepared and analyzed by multiplex-bead ELISA Bio-Plex Pro kit (BioRad, USA) for production of IL-1β, TNF-α, and IL-8. Results are representative of two separate experiments, each performed in triplicate. Mean ± SD is shown for triplicate samples. * and # indicate significant differences as described for (A).</p

Vaccine formulations containing a combination of TLR4 and NOD2 agonists induce stronger antigen-specific CD4 and CD8 T-cell responses in mice than formulations containing individual PRR agonists.

<p>Mice (n = 5/group) were immunized s.c. twice with alum-based vaccine formulations: Alum+OVA, Alum+OVA+MDP, Alum+OVA+MPLA, and Alum+OVA+MDP+MPLA. Controls included naıve (untreated) mice and mice immunized with soluble OVA (no Alum or PRR agonist). Splenocytes were harvested from mice 14 days after the last immunization. <b>(A-B)</b> T-cell proliferation in response to ovalbumin restimulation. Splenocytes were CFSE-labeled, restimulated with whole OVA protein (1μg/ml) for 72h, stained with fluorescently-tagged antibodies against CD3, CD4 and CD8 and analyzed by flow cytometry. A. Gating strategy used for determination of antigen-specific CD4 and CD8 T-cell responses. B. Representative dot plots for the naïve (intact) group and each immunized group showing CFSE fluorescence (x-axis) of CD4 and CD8 T cells (distinguished by CD8 expression shown on the y-axis). The percentage of proliferating CD4+ and CD8+ T cells in response to antigen represent mean from two independent experiments with 5 mice/group each. <b>(C-D)</b> T-cell activation indicated by IFN-γ production in response to ovalbumin restimulation. Splenocytes were restimulated for 18 hours with whole OVA antigen at 1μg/ml in the presence of BD GolgiPlug solution (BD biosciences), stained with fluorescently-tagged antibodies against CD3, CD4 and CD8, permeabilized, stained with a fluorescently-tagged antibody against IFN-γ and then analyzed by flow cytometry. The percentage of CD4 (C) or CD8 (D) T cells expressing IFN-γ is shown as the mean ± SD for 5 mice per group. Two additional experiments yielded similar results * indicates significant difference (P≤0.05) between formulations containing MDP or MPLA individually and the formulation without PRR agonists (Alum+OVA). # indicates significant difference (P≤0.05) between Alum+OVA+MDP+MPLA treatment and Alum+OVA+MPLA or Alum+OVA+MDP (Student’s t-test).</p