Antigen Targeting to Human HLA Class II Molecules Increases Efficacy of DNA Vaccination

Abstract

It has been difficult to translate promising results from DNA vaccination in mice to larger animals and humans. Previously, DNA vaccines encoding proteins that target Ag to MHC class II (MHC-II) molecules on APCs have been shown to induce rapid, enhanced, and long-lasting Ag-specific Ab titers in mice. In this study, we describe two novel DNA vaccines that as proteins target HLA class II (HLA-II) molecules. These vaccine proteins cross-react with MHC-II molecules in several species of larger mammals. When tested in ferrets and pigs, a single DNA delivery with low doses of the HLA-II-targeted vaccines resulted in rapid and increased Ab responses. Importantly, painless intradermal jet delivery of DNA was as effective as delivery by needle injection followed by electroporation. As an indication that the vaccines could also be useful for human application, HLA-II-targeted vaccine proteins were found to increase human CD4⁺ T cell responses by a factor of ×10³ in vitro. Thus, targeting of Ag to MHC-II molecules may represent an attractive strategy for increasing efficacy of DNA vaccines in larger animals and humans.

FIGURE 1.

Characterization of vaccine proteins. (A) Schematic structure of a DNA-delivered dimeric vaccine protein. The vaccine protein consists of two N-terminal HLA-II–specific targeting units in an scFv format that are linked to an Ig-based dimerization unit and two C-terminal antigenic units. (B) Presence of vaccine proteins in supernatants of transiently transfected 293E cells detected by sandwich ELISA. (C) Western blot of affinity-purified vaccine proteins detected with mAb against human C_H3 dimerization unit. The vaccine proteins are indicated below lanes; molecular mass is indicated by arrows. The vertical line indicates joining of two parts from the same blot. (D) FACS analyses of human PBMCs stained with the indicated vaccine proteins. Binding to gated CD19⁺, CD11c⁺, and CD11b⁺ cells is shown.

FIGURE 2.

Specificity of vaccine proteins for HLA-II molecules. (A) The indicated vaccine proteins and HKB1 IgM mAb (donor of scFv for αpHLAII vaccine proteins) were assayed for binding to 91 beads coated with either HLA-DR (36 different types), HLA-DQ (29 types), or HLA-DP (26 types) molecules. Shown is the percentage of binding to each series of HLA-II molecules. αNIP-CκCκ did not bind any of the HLA-II molecules tested. Information on specific beads employed, and binding, is provided in Supplemental Fig. 1. (B) Sequence alignment of selected vaccine-interacting and noninteracting β₁ domains with the critical residue 58 boxed. The relevant HLA sequences were downloaded from the IMGT/HLA database, aligned using ClustalX, and annotated using GenDoc. HLA-DP and HLA-DQ sequences are provided in Supplemental Fig. 2. (C) Structural and topological comparison of the postulated HKB1 epitope centered on the critical residue position 58 of the β₁ chain (side view onto the β₁ domain). Position 59 is also solvent exposed and is likely to represent an anchor residue for binding to the postulated epitope. The solvent-exposed surface electrostatic potentials were generated using APBS and contoured onto the molecular surfaces using PyMOL. Positively charged amino acids are colored in blue, whereas negatively charged amino acids are colored in red. Protein Data Bank ID codes: 1V9S (DQB1*02:01), 2NNA (DQB1*03:02), 2IAN (DRB1*01:01), and 3LQZ (DPB1*02:01).

FIGURE 3.

HLA-II–specific vaccine proteins enhance proliferation of Ag-specific CD4⁺ T cells. Irradiated human PBMCs (HLA-DR4⁺ [DRA1, B1*0401]) were incubated with cloned mouse Cκ-specific DR4-restricted CD4⁺ T cells in the presence of titrated amounts of various affinity-purified vaccine proteins expressing mouse Cκ Ag in a CκCκ scFv format. The cultures were pulsed with [³H]thymidine, and incorporation into DNA was assessed as an indicator of T cell proliferation.

FIGURE 4.

DNA vaccination with αpHLAII-HA confers protection against influenza challenge in DQ2-transgenic mice. (A) Supernatants from 293E cells transiently transfected with DNA expressing the indicated vaccine proteins were examined for binding to mAb against the C_H3 dimerization unit (MCA878) in sandwich ELISA, followed by detection with biotinylated mAb (H36-4-52) directed against PR8 HA in the antigenic unit. (B) Western blot of unreduced supernatants from transfected 293E cells, detected with anti-HA mAb specific for PR8 HA expressed in the vaccine proteins. Constructs are indicated below lanes; molecular mass is indicated by arrows. (C) Binding of indicated vaccine proteins to splenocytes from DQ2-transgenic mice on a BALB/c background. (D–G) Mice were vaccinated once with 25 μg of the indicated DNA plasmids i.d. combined with electroporation. Sera obtained on days 7, 14, and 21 were tested for presence of (D) IgG, (E) IgG1, and (F) IgG2a anti-HA Abs in ELISA. (G) At day 22, mice were challenged i.n. with a lethal dose of PR8 virus and monitored for weight. In (D), (E), and (G), mean ± SEM is given (n = 6 per group). *p < 0.05 for αpHLAII-HA compared with all other controls (two-way ANOVA and Bonferroni posttest).

FIGURE 5.

αpHLAII-HA DNA vaccine delivered by injection and electroporation increases immune responses in ferrets and pigs. (A and B) The indicated vaccine proteins were examined for specific binding to PBMCs from ferrets (A) and pigs (B). Selection gates for live cells were used on ferret PBMCs, whereas an additional gate for CD11R⁻ cells was used for pig PBMCs. Representative images are shown (n = 3 for both species). (C) Ferrets were immunized once on day 0 (↑) with 100 μg of DNA i.d./electroporation. Vaccine constructs are indicated; HA was from A/California/07/2009 (H1N1) (Cal07). Serum samples were tested for IgG against inactivated Cal07 influenza in ELISA (mean ± SEM, n = 6 per group) (*p < 0.05, two-way ANOVA). (D) Norwegian farm pigs were immunized on day 0 (↑) and boosted on day 21 (↑) with 400 μg of DNA i.d./electroporation. Vaccine constructs are indicated, with plasmids encoding HA from Cal07. Sera were assayed for IgG in ELISA (mean ± SEM, n = 4 per group) (*p < 0.05, two-way ANOVA). (E) Ferret sera from animals with negative prevaccination titers (<2³) were collected at day 29 after vaccination and tested in an HI assay (mean ± SEM, n = 6 per group). (F) Pig sera from animals with negative prevaccination titers (<2³) were collected at day 21 after vaccination and tested in an HI assay (mean ± SEM, n = 4 per group). (G) Sera harvested at day 21 after the first immunization of pigs were assayed in a microneutralization assay against influenza Cal07 (n = 4 per group). Individual values are depicted, and the dotted line represents the 50% neutralization threshold used for positive scoring (*p < 0.05, two-way ANOVA).

FIGURE 6.

Painless jet delivery of DNA vaccine in pigs with maintenance of efficiency. (A) Norwegian farm pigs were DNA immunized twice (days 0 and 28, ↑) with titrated amounts of αpHLAII-HA (encoding HA from influenza virus PR8). Plasmids were delivered i.d. either with needle injection/electroporation (EP) (n = 6 per group) or by needle-free jet delivery (Jet) (n = 5 per group). Sera were harvested at the indicated time points and assayed for IgG against inactivated influenza PR8 by ELISA (mean ± SEM). (B and C) Sera collected either 21 d after the first vaccination (B) or 1 wk after the second vaccination (C) were assayed in microneutralization assays against influenza PR8. Individual values are given, and the dotted lines indicate the threshold for positive neutralization.

FIGURE 7.

Painless jet delivery of DNA vaccine in pigs with maintenance of targeting effect. (A) Norwegian farm pigs (n = 6 per group) were immunized twice (days 0 and 28, ↑) with 75 μg of DNA i.d. by jet delivery. The indicated constructs expressed HA from PR8 influenza. Sera obtained at the indicated time points were tested for IgG Abs binding recombinant HA (PR8) (mean ± SEM). (B and C) Sera of (A) were collected either 28 d after a single vaccination (B) or 1 wk after the second vaccination (C) and assayed in microneutralization assays against influenza PR8. Individual values are given, and the dotted lines indicate threshold for positive neutralization. *p < 0.05 compared with αNIP-HA, HA, and NaCl by two-way ANOVA.