Intradermal immunization improves protective efficacy of a novel TB vaccine candidate

Abstract

We have developed the Mycobacterium tuberculosis (Mtb) fusion protein (ID83), which contains the three Mtb proteins Rv1813, Rv3620 and Rv2608. We evaluated the immunogenicity and protective efficacy of ID83 in combination with several emulsion-formulated toll-like receptor agonists. The ID83 subunit vaccines containing synthetic TLR4 or TLR9 agonists generated a T helper-1 immune response and protected mice against challenge with Mtb regardless of route. The ID83 vaccine formulated with gardiquimod (a TLR7 agonist) also resulted in a protective response when administered intradermally, whereas the same vaccine given subcutaneously failed to provide protection. This highlights the need to explore different routes of immunization based on the adjuvant formulations used.

Fig. 1

ID83-specific IgG1 and IgG2c endpoint antibody titers and IgG2c:IgG1 ratios from mice immunized via the s.c. route (A, C) and the i.d. route (B, D). Serum was collected 1 wk following the third immunization. Mean reciprocal dilutions are represented as the endpoint titer (Log₁₀) ± SD. Asterisks represent statistical significance between IgG2c and IgG1 within each vaccine group, p<0.05.

Fig. 2

IFN-γ production from splenocytes following either (A) s.c. or (B) i.d. immunization. Mice were immunized three times, 3 wk apart and spleens were removed 4 wk after the last immunization. Splenocytes isolated from immunized animals were plated in duplicate at 2 × 10⁵ cells/well and cultured with medium, PPD, ConA (3 µg/ml), or the recombinant fusion protein ID83 (10 µg/ml) for 48 hours. Frequencies of IFN-γ-secreting cells were determined by ELISPOT (e-Bioscience). Results are represented as the mean SFU (spot forming units) per 10⁶ cells ± SD.

Fig. 3

Cytokine production from ID83-specific CD4 T cells in immunized mice was measured by flow cytometry. Splenocytes from vaccinated mice were stimulated with ID83 or peptides for 12 hr in the presence of GolgiStop. ID83-stimulated or peptide-stimulated splenocytes were identified by ICS based on CD3 and CD4 expression and were further gated as CD44high (antigen experienced). The percent frequency of cells expressing either IFN-γ or TNF was determined from either s.c. immunized mice (A–B) or from i.d. immunized mice (C–D). The values from pooled splenocytes (n=3 mice/group) are shown.

Fig. 4

IFN-γ production from ID83-specific CD8 T cells in immunized mice was measured by flow cytometry. Splenocytes from vaccinated mice were stimulated with ID83 or peptides for 12 hr in the presence of GolgiStop. ID83-stimulated or peptide-stimulated splenocytes were identified by ICS based on CD3 and CD8 expression and were further gated as CD44high (antigen experienced). The percent frequency of cells expressing IFN-γ was determined from either s.c. immunized mice (A–B) or from i.d. immunized mice (C–D). The values from pooled splenocytes (n=3 mice/group) are shown.