The anti-influenza M2e antibody response is promoted by XCR1 targeting in pig skin

Abstract

XCR1 is selectively expressed on a conventional dendritic cell subset, the cDC1 subset, through phylogenetically distant species. The outcome of antigen-targeting to XCR1 may therefore be similar across species, permitting the translation of results from experimental models to human and veterinary applications. Here we evaluated in pigs the immunogenicity of bivalent protein structures made of XCL1 fused to the external portion of the influenza virus M2 proton pump, which is conserved through strains and a candidate for universal influenza vaccines. Pigs represent a relevant target of such universal vaccines as pigs can be infected by swine, human and avian strains. We found that cDC1 were the only cell type labeled by XCR1-targeted mCherry upon intradermal injection in pig skin. XCR1-targeted M2e induced higher IgG responses in seronegative and seropositive pigs as compared to non-targeted M2e. The IgG response was less significantly enhanced by CpG than by XCR1 targeting, and CpG did not further increase the response elicited by XCR1 targeting. Monophosphoryl lipid A with neutral liposomes did not have significant effect. Thus altogether M2e-targeting to XCR1 shows promises for a trans-species universal influenza vaccine strategy, possibly avoiding the use of classical adjuvants.

Figure 1

Selective targeting of pig skin cDC1 subset with poXCL1-mCherry upon intradermal injection with different adjuvant formulations. NIP-mCherry and poXCL1-mCherry (60 µg) were injected intradermally in different adjuvant formulations, skin cells were collected after 24 h by enzymatic dissociation and labeled for MHC class II and CD172A detection. FSC^high and MHC class II^high cells (cDC) were gated as shown in A (left panel) and their percentage among total extracted skin cells is indicated. The FSC^high and MHC class II^high gated cells were analyzed for CD172A and mCherry expression (in A (right panel) to E) and the percentage of each quadrant among gated cells is indicated. The injections are: in A (right panel), PBS; in B, plain VB; in C, VB formulated with SWE adjuvant; in D, VB formulated with CL + MPL; in E, VB formulated with NL + MPL.

Figure 2

Characterization of poXCL1-M2e and NIP-M2e VB. In (A) the M2e VB consist of a targeting unit (poXCL1 or NIP), a dimerization domain with hinge and CH₃ from human IgG3 and an antigenic unit with three repeats of the M2e sequence. In (B) purified poXCL1-M2e (predicted size 33 kDa) and NIP-M2e (predicted size 48 kDa) were analyzed by SDS-PAGE under reducing (+DTT) and non-reducing (-DTT) conditions. In (C) normalized concentrations of purified poXCL1-M2e and NIP-M2e preparations were evaluated by ELISA, and detected using an antibody specific for the VB dimerization domain.

Figure 3

Effect of XCR1 targeting and MPL adjuvant on the anti-M2e IgG response in seronegative pigs. Pigs were inoculated twice (D0 and D28) intradermally with 0.25 or 2.5 µg of poXCL1-M2e or NIP-M2e VB with or without CL + MPL. In (A and B), the anti-M2e IgG titers obtained in the different groups at D28 (A) and at D56 (B) are shown. Signals with sera from D0 were all at the level of background. In (C), as neither the dose nor the formulated MPL were confounding factors, the NIP-M2e and the poXCL1-M2e vaccinated groups were combined to increase the statistical power. In (D and E), the IgG1 and IgG2 anti-M2e titers at D56 are shown for the different groups. In all panels, each dot corresponds to individual pig values. Arithmetic means and standard errors of the mean are shown. Statistical significance between 2 groups was calculated with unpaired t-test (p values are reported when > 0.05, * < 0.05; ** < 0.01).

Figure 4

Effect of XCR1 targeting and CpG adjuvant on the anti-M2e IgG response in seropositive pigs. Pigs with pre-existing antibodies against M2e were inoculated 3 times intradermally (D0, 28, 55) with 8 µg poXCL1-M2e or NIP-M2e VB with or without CpG. In (A), the anti-M2e IgG titers obtained in the different groups at D0, D55 and D85 are shown. In (B), the D0 versus D85 anti-M2e IgG titers from poXCL1-M2e vaccinated pigs (with and without CpG) were plotted and correlation was estimated with a two-tailed Pearson test. In (C and D), the IgG1 and IgG2 anti-M2e titers at D85 are shown for the different groups. In (E) the IgG2 titer: IgG1 titer ratios are depicted. In all panels, each dot corresponds to individual pig values. Arithmetic means and standard errors of the mean are shown. Statistical significance between 2 groups was calculated with unpaired t-test (p values = * < 0.05; ** < 0.01).