Evaluation of an intranasal virosomal vaccine against respiratory syncytial virus in mice: effect of TLR2 and NOD2 ligands on induction of systemic and mucosal immune responses

Abstract

Introduction: RSV infection remains a serious threat to newborns and the elderly. Currently, there is no vaccine available to prevent RSV infection. A mucosal RSV vaccine would be attractive as it could induce mucosal as well as systemic antibodies, capable of protecting both the upper and lower respiratory tract. Previously, we reported on a virosomal RSV vaccine for intramuscular injection with intrinsic adjuvant properties mediated by an incorporated lipophilic Toll-like receptor 2 (TLR2) ligand. However, it has not been investigated whether this virosomal RSV vaccine candidate would be suitable for use in mucosal immunization strategies and if additional incorporation of other innate receptor ligands, like NOD2-ligand, could further enhance the immunogenicity and protective efficacy of the vaccine.

Objective: To explore if intranasal (IN) immunization with a virosomal RSV vaccine, supplemented with TLR2 and/or NOD2-ligands, is an effective strategy to induce RSV-specific immunity.

Methods: We produced RSV-virosomes carrying TLR2 (Pam3CSK4) and/or NOD2 (L18-MDP) ligands. We tested the immunopotentiating properties of these virosomes in vitro, using TLR2- and/or NOD2-ligand-responsive murine and human cell lines, and in vivo by assessing induction of protective antibody and cellular responses upon IN immunization of BALB/c mice.

Results: Incorporation of Pam3CSK4 and/or L18-MDP potentiates the capacity of virosomes to activate (antigen-presenting) cells in vitro, as demonstrated by NF-κB induction. In vivo, incorporation of Pam3CSK4 in virosomes boosted serum IgG antibody responses and mucosal antibody responses after IN immunization. While L18-MDP alone was ineffective, incorporation of L18-MDP in Pam3CSK4-carrying virosomes further boosted mucosal antibody responses. Finally, IN immunization with adjuvanted virosomes, particularly Pam3CSK4/L18-MDP-adjuvanted-virosomes, protected mice against infection with RSV, without priming for enhanced disease.

Conclusion: Mucosal immunization with RSV-virosomes, supplemented with incorporated TLR2- and/or NOD2-ligands, represents a promising approach to induce effective and safe RSV-specific immunity.

Figure 1. In vitro analysis of RSV virosomes and RSV virosomes adjuvanted with TLR2 and/or NOD2ligands.

RSV virosomes and RSV virosomes adjuvanted with TLR2 and/or NOD2 ligands were spun on an equilibrium density sucrose gradient. Subsequently, density, phospholipids and protein concentrations of each fraction was determined. Panel A shows a representative profile of a virosome purification gradient. Fractions (1,5,10; representing bottom, virosomal and top gradient fractions, respectively) were analyzed to determine their capacity to activate NF-κB in mouse macrophages (RAW-Blue cells; panel B) and human embryonic kidney cells (HEK-BlueTLR2 & HEK-Blue NOD2 cells; panel C). The level of NF-κB induced in RAW-blue cells was expressed as values relative to levels of NF-κB induced by CpG ODN, the positive control. To assess non-specific NF-κB activation by TLR2 and NOD2 ligand-carrying virosomes in HEK cells, control cells (HEK-Blue Null1 & HEK-Blue Null2 cells, respectively) were incubated with the same fractions and these values were subtracted from values obtained with HEK-BlueTLR2 & HEK-Blue NOD2 cells, respectively. As a control, HEK-Blue TLR2 and HEK-Blue NOD2 cells were stimulated with 100 ng/ml TNF-α. Bars represent the NF-κB activation relative to TNF-α control.

Figure 2. RSV-specific systemic IgG antibody responses after IN immunization of mice.

BALB/c mice were immunized IN with RSV virosomal vaccine formulations (5 µg of protein) or HNE. Control mouse groups were either immunized IM with FI-RSV or IN with live-RSV (L-RSV) on day 0 and 21. Six mice were used in each group. One week after the booster immunization, RSV-specific IgG responses in serum (A) and IgG-subtypes (IgG2a/IgG1) (B) were determined by ELISA. Panel A: Bars represent the geometric mean titer and standard deviation. Panel B: Bars represent the ratios of IgG2a/IgG1. The data shown are representative of at least 3 separate experiments. Data was analyzed by a Mann-Whitney U test and a p-value of≤0.05 was considered to represent a significant difference. * p≤0.05, ** p≤0.01.

Figure 3. RSV-specific mucosal IgA and IgG antibody responses in nasal washes and BAL after IN immunization of mice.

BALB/c mice were immunized IN with RSV virosomal vaccine formulations (5 µg of protein) or HNE. Control mouse groups were either immunized IM with FI-RSV or IN with L-RSV on day 0 and 21. Four days after challenge with live RSV (day 32), RSV-specific IgA responses in nasal washes (A), BAL (C) were determined by ELISA. RSV-specific IgG responses in nasal washes (B) and BAL (D) were also determined. For nasal washes, the data from 6 mice per group is shown and for BAL, data from 3 mice per group is shown. Panels A-D: Bars represent the mean absorbance (490 nm) and standard deviation. The data shown are representative data of at least 3 separate experiments. Data was analyzed by a Mann-Whitney U test and a p-value of ≤ 0.05 was considered to represent a significant difference. * p≤0.05, ** p≤0.01.

Figure 4. RSV-specific serum IgA and IgE antibody responses.

BALB/c mice were immunized IN with RSV-virosomal vaccine formulations (5 µg of protein) or HNE. Control mouse groups were either immunized IM with FI-RSV or IN with L-RSV on day 0 and 21. RSV-specific serum IgA (A) and IgE (B) were determined by ELISA. Data from 6 mice per group is shown. Panel A: Bars represent the geometric mean titer and standard deviation. Panel B: Bars represent the mean absorbance (490 nm) and standard deviation. The data shown are representative data of at least 2 separate experiments. Data was analyzed by a Mann-Whitney U test and a p-value of ≤0.05 was considered to represent a significant difference. * p≤0.05, ** p≤0.01.

Figure 5. Ex vivo cytokine production by splenocytes in response to stimulation with RSV.

IFN-γ (Panel A) and IL-5 (Panel B) production in splenocyte cultures after stimulation with inactivated RSV was determined by cytokine ELISA. Cytokines in the culture supernatants were assayed after 3 days of culturing. Data from 6 mice per group is shown. Bars and error bars represent means ± SD. The data shown are representative of at least 3 separate experiments. Data was analyzed by a Mann-Whitney U test and a p-value of ≤ 0.05 was considered to represent a significant difference. * p≤0.05, ** p≤0.01, *** p≤0.001.

Figure 6. Protection of mice from challenge with live RSV.

BALB/c mice were immunized IN with RSV-virosomal vaccine formulations (5 µg of protein) or HNE. Control mouse groups were either immunized IM with FI-RSV or IN with L-RSV on day 0 and 21. Mice were challenged with live-RSV on day 28 and four days after challenge (day 32), lung viral titers were determined. Data from 6 mice per group is shown. Viral titers are expressed as TCID₅₀. Bars and error bars represent means ± SD. The data shown are representative of at least 2 separate experiments. Data was analyzed by a Mann-Whitney U test and a p-value of ≤ 0.05 was considered to represent a significant difference. Asterisks indicate groups that had significantly lower viral titers compared to titers in the non-immune HNE group. Horizontal lines compares differences in titers in different immunized groups. * p≤0.05, ** p≤0.01.

Figure 7. Immunopathology after challenge.

BALB/c mice were immunized as described above. One week after the booster immunization, mice were challenged with live RSV (1*10⁶ TCID50). Four days after challenge, one lobe of lung was harvested, sliced and stained with H&E for pathology analysis using light microscopy. Panels are representative pictures of the lungs of mice immunized with (A) HNE, (B) L-RSV, (C) FI-RSV, (D) RSV virosomes, (E) RSV virosomes + TLR2-L, (F) RSV virosomes + NOD2-L and (G) RSV virosomes + TLR2-L + NOD2-L immunized mice. Black arrows indicate alveolitis, red arrows indicate peribronchiolitis and blue arrows indicate perivasculitis.