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

Titers of <i>M</i>. <i>hominis</i> in vaginal washes with “prophylactic” scheme of rAds inoculation.

<p>Vaginal washes were collected 5 days after <i>M</i>. <i>hominis</i> inoculation (6 days after rAds inoculation). Amount of <i>M</i>. <i>hominis</i> was evaluated with real-time PCR. The rAd5-CMV-PLAP-aMh-FcG2a group exhibited a significantly lower <i>M</i>. <i>hominis</i> titer (Student's t-test = 3.5; p<0.01). Ad-null n = 5, PBS n = 15, rAd5-CMV-PLAP-aMh-FcG2a n = 10, rAd5-CMV-PLAP-aMh-ILZ-HA n = 11. In the “prophylactic” scheme of rAds inoculation, the <i>M</i>. <i>hominis</i> titer was significantly decreased in the rAd5-CMV-PLAP-aMh-FcG2a group. The prophylactic scheme, however, is not practical for treating mycoplasma infection. In the “therapeutic” scheme, the number of animals diagnosed as positive at 7 days after inoculation with rAd5-CMV-PLAP-aMh-FcG2a (12 days after <i>M</i>. <i>hominis</i> inoculation) was significantly lower than that of the other groups. Nevertheless the mycoplasma titer was not significantly different among the infected animals in any of the groups and not informative due to the large variability in counts and the low number of infected animals in the rAd5-CMV-PLAP-aMh-FcG2a group (n = 2 at 3 days and n = 1 at 7 days).</p

rAd5-CMV-PLAP-aMh-FcG2a and control rAd5-CMV-PLAP-aMh-ILZ-HA constructions and purification.

<p>(A) Scheme of expression cassettes for rAd5-CMV constructions; (B) sizing of purified Ad5-CMV-PLAP-aMh-FcG2a corresponding to virions suspensions with a particle size of ~100 nM.</p