Sublingual vaccination induces mucosal and systemic adaptive immunity for protection against lung tumor challenge

Abstract

Sublingual route offers a safer and more practical approach for delivering vaccines relative to other systemic and mucosal immunization strategies. Here we present evidence demonstrating protection against ovalbumin expressing B16 (B16-OVA) metastatic melanoma lung tumor formation by sublingual vaccination with the model tumor antigen OVA plus synthetic glycolipid alpha-galactosylceramide (aGalCer) for harnessing the adjuvant potential of natural killer T (NKT) cells, which effectively bridge innate and adaptive arms of the immune system. The protective efficacy of immunization with OVA plus aGalCer was antigen-specific as immunized mice challenged with parental B16 tumors lacking OVA expression were not protected. Multiple sublingual immunizations in the presence, but not in the absence of aGalCer, resulted in repeated activation of NKT cells in the draining lymph nodes, spleens, and lungs of immunized animals concurrent with progressively increasing OVA-specific CD8+ T cell responses as well as serum IgG and vaginal IgA levels. Furthermore, sublingual administration of the antigen only in the presence of the aGalCer adjuvant effectively boosted the OVA-specific immune responses. These results support potential clinical utility of sublingual route of vaccination with aGalCer-for prevention of pulmonary metastases.

Figure 1. Multiple rounds of sublingual immunization employing the aGalCer adjuvant induce progressively increasing antigen-specific cellular and humoral responses.

Effector responses were determined in mice immunized by sublingual route three times at 7 day intervals and boosting on day 41 (i.e. 27 days post last immunization) with OVA or OVA+aGalCer. Vertical arrows at different time points indicate the time of immunization. The kinetics of the development of adaptive immune responses was determined at 7 days post each immunization. (A) Antigen-specific CD8⁺ T lymphocytes were detected in the PBMC by staining with fluorescently labeled OVA/K^b tetramer and antibodies to CD44 and CD8, and representative flow plots for OVA/K^b tetramer⁺ cells expressed as a percentage of CD8⁺ T lymphocytes from each time point are presented. (B) Cumulative data for percentages of OVA/K^b tetramer⁺, CD8⁺ T lymphocytes in PBMC at different time points in mice immunized with OVA or OVA+aGalCer. (C) Single cell suspensions from spleen and CLN were analyzed for antigen-specific IFN-γ production in response to stimulation with the CD8 T cell epitope peptide SIINFEKL from OVA using a standard IFN-γ ELISpot assay. Data are shown as IFN-γ spot forming cells (SFC) per million input cells and OVA-specific responses were adjusted to background medium control and expressed as mean ± S.D. (D) Splenocytes isolated from immunized mice were also analyzed for antigen-specific cytolytic activity by the standard chromium-release assay employing the syngeneic EL-4 target cells pulsed with the OVA peptide, at 100∶1 effector to target cell ratio. Data were adjusted for background by subtracting control values (target cells not pulsed with the OVA peptide) and expressed as mean ±S.D. (E and F) Antigen specific antibody response after each dose was determined by ELISA. The log10 titers of serum IgG and vaginal IgA respectively at different time points in mice immunized with OVA or OVA+ aGalCer were calculated by adjusting to background pre-immune values. Data are expressed as mean ± S.D. and representative of two separate experiments. The statistical significance (p≤0.05), between same number of immunizations with OVA alone and OVA+aGalCer is shown as * and between each additional immunization with either OVA alone or admixed with aGalCer is shown as **.

Figure 2. Repeated activation of NKT and dendritic cells with each immunization employing the aGalCer adjuvant delivered by the sublingual route.

(A) Mice were immunized by sublingual route with one or two doses of OVA or OVA+aGalCer at 7 day intervals and sacrificed at different time points as shown to determine activation of NKT cells and DC. (B) Gating strategy for staining NKT cells isolated from the spleens, CLNs and lungs with fluorescently labeled NKT tetramer, antibodies to CD3 and IFN-γ, and Aqua live/dead stain. (C) The total number of NKT cells (CD1d tetramer⁺ CD3⁺) at different times post immunization in each tissue. (D) The total number of activated of NKT cells in each tissue were determined at different times post immunization by intracellular staining for IFN-γ. (E) Gating tree and representative histograms for CD86 expression on CD11c⁺ cells (activated DC) from mice immunized with OVA+aGalCer (black) in comparison to that from mice immunized with OVA alone (gray) after one or two immunizations (1× and 2×, respectively) are shown. (F) Cumulative data for activated DC from spleens, CLNs and lungs of mice immunized with OVA+aGalCer (black) compared to animals immunized with OVA alone (white) was evaluated by measuring the MFI of CD86 expression on CD11c⁺ cells at day 3 after either 1^st or 2^nd immunization (i.e. day 3 and day 10 respectively). Data are representative of two separate experiments and expressed as mean ± S.D. The statistical significance (p≤0.05) between groups of mice that were immunized with OVA alone and OVA+aGalCer after 1^st and 2^nd immunization at different time points is shown as *.

Figure 3. Efficacy of antigen-specific immune responses induced by sublingual immunization employing the aGalCer adjuvant against lung tumor challenge.

(A) Mice were immunized three times by sublingual route with either OVA admixed with aGalCer, OVA alone, aGalCer alone or PBS on days 0, 7 and 14. Seven days after final immunization, the mice were challenged by the intravenous route with 5×10⁴ control or OVA-transgenic B16 tumor cells (B16 and B16-OVA, respectively) and lungs were harvested 14 days post challenge to determine the number of tumor foci. (B) Numbers of tumor foci/lung were shown as mean ± S.D. for each of the different groups of mice. Statistical analyses between different groups were performed using student t-test between different treatments and the different levels of significance are shown as * (p≤0.05) and ** (p≤0.001). (C) Representative lungs corresponding to the different groups of mice in panel B. (D) In a separate group of similarly immunized mice, single cell suspensions from the lungs were analyzed 7 days post each immunization including at the time of tumor challenge (day 21) for antigen-specific IFN-γ production in response to stimulation with the CD8 T cell epitope peptide SIINFEKL from OVA using a standard IFN-γ ELISpot assay. Vertical arrows represent the time of immunization and data are shown as IFN-γ spot forming cells (SFC) per million input cells and OVA specific responses were adjusted to background medium control and expressed as mean ± S.D. The statistical significance (p≤0.05) between groups of mice that were immunized with OVA alone and OVA+aGalCer at different time points is shown as *. (E) Representative dot plots showing antigen specific effector CD8 T lymphocyte population (OVA/K^b tetramer⁺, CD44^hi cells) in the lungs at the time of tumor challenge (day 21) for mice immunized with OVA alone and OVA admixed with aGalCer.