A single sublingual dose of an adenovirus-based vaccine protects against lethal Ebola challenge in mice and guinea pigs

Abstract

Sublingual (SL) delivery, a noninvasive immunization method that bypasses the intestinal tract for direct entry into the circulation, was evaluated with an adenovirus (Ad5)-based vaccine for Ebola. Mice and guinea pigs were immunized via the intramuscular (IM), nasal (IN), oral (PO) and SL routes. SL immunization elicited strong transgene expression in and attracted CD11c(+) antigen presenting cells to the mucosa. A SL dose of 1 × 10⁸ infectious particles induced Ebola Zaire glycoprotein (ZGP)-specific IFN-γ⁺ T cells in spleen, bronchoalveolar lavage, mesenteric lymph nodes and submandibular lymph nodes (SMLN) of naive mice in a manner similar to the same dose given IN. Ex vivo CFSE and in vivo cytotoxic T lymphocyte (CTL) assays confirmed that SL immunization elicits a notable population of effector memory CD8+ T cells and strong CTL responses in spleen and SMLN. SL immunization induced significant ZGP-specific Th1 and Th2 type responses unaffected by pre-existing immunity (PEI) that protected mice and guinea pigs from lethal challenge. SL delivery protected more mice with PEI to Ad5 than IM injection. SL immunization also reduced systemic anti-Ad5 T and B cell responses in naive mice and those with PEI, suggesting that secondary immunizations could be highly effective for both populations.

Figure 1. Adenovirus Serotype 5 Efficiently Transduces TR146 Cells and the Murine Oral Mucosa after Sublingual Administration and Stimulates Migration of CD11c(+) and CD11b(+) Antigen Presenting Cells to the Delivery Site

A) Uninfected TR146 cells. B) TR146 cells infected with first generation adenovirus 5 expressing beta-galactosidase (AdlacZ, moi 30) for 24 hours prior to histochemical staining for transgene expression. C) Representative longitudinal section of murine sublingual mucosa obtained from an animal 2 hours after administration of saline (Vehicle) stained for endogenous beta-galactosidase expression. D) Section of sublingual mucosa obtained 2 hours after sublingual administration of 1 × 10⁸ infectious particles of AdlacZ stained for beta-galactosidase expression. E) Beta-galactosidase expression in submandibular glands 24 hours after treatment (black arrows). F) Cryosection from mouse given saline and stained for cells expressing MHC class II surface antigens (brown dots). G) Cryosection taken 2 hours after administration of adenovirus and stained for the presence of MHC class II+ cells. A significant number of cells were visualized at the delivery site (arrows). H) Spotted pattern of MHC class II+ cells in the oral mucosa 24 hours after sublingual administration of adenovirus. Additional staining revealed that cells positive for MHC II antigens were also positive for CD11b (Panels i–k) and CD11c surface molecules (Panels l–n). Magnification: Panels a–f, i and l 200×; Panel g, h, j, k, m, and n 100×.

Figure 2. Pre-Existing Immunity to Adenovirus Strengthens the CD8⁺ T cell Response against Ebola Zaire Glycoprotein (ZGP) in Mice Immunized by the Sublingual Route in Local Compartments

B10.Br mice (10/group) were given 1 × 10⁸ infectious particles of recombinant adenovirus expressing ZGP by various routes. A subset of mice from each group (n=5) were given 2.5 × 10¹¹ particles of adenovirus containing the beta-galactosidase transgene by intramuscular injection 28 days prior to vaccination to establish pre-existing immunity (PEI). Mice treated in this manner had an average neutralizing antibody titer of 1:165 ± 22 prior to vaccination. Panel A. Systemic CD8+ T Cell Response in Naïve Mice and Those with Pre-Existing Immunity. Ten days after vaccination, splenocytes were harvested, pooled according to treatment, stimulated with a ZGP-specific peptide (TELRTFSI) and stained with antibodies against CD8 surface proteins and intracellular interferon gamma (IFN-γ). Positive cells were identified by flow cytometry. Numbers in each box represent the proportion of each cell population that was activated by the antigen-specific peptide. Panel B. Mucosal Response. Mononuclear cells were harvested from various compartments of individual mice and analyzed for production of IFN-γ in response to the antigen-specific peptide by ELISPOT 10 days after immunization. Results are reported as the mean ± the standard error of the mean. *, p<0.05, **, p<0.01, ***, p<0.001, one-way ANOVA, Bonferroni/Dunn post-hoc analysis. I.M. – intramuscular, I.N. – intranasal, S.L. – sublingual, P.O. – oral, PEI – pre-existing immunity, BAL - bronchioalveolar lavage fluid, SMLN – submandibular lymph nodes, MLN – mesenteric lymph nodes.

Figure 3. Sublingual Immunization Induces Higher Levels of Ebola Zaire-Specific IgG and IgM Antibodies in Serum and Mucosal Secretions than Intramuscular Immunization that are not Compromised by Pre-Existing Immunity

Serum (Panels A and B) and bronchoalveolar lavage fluid (BAL, Panels C and D) were collected from naïve mice and those with pre-existing immunity 42 days after immunization with 1 × 10⁸ infectious particles of a recombinant adenovirus expressing ZGP by various routes. Samples from individual mice were evaluated for the presence of ZGP-specific IgG subclasses, IgM and IgA by ELISA. End point titers are expressed as the reciprocal log₂ titer of the last dilution giving an OD at 450 nm of 0.1 units higher than background. Results are expressed as average values ± the standard error of the means and are representative of two separate experiments each containing 4 mice per immunization route. *, p<0.05, **, p<0.01, ***, p<0.001, one-way ANOVA, Bonferroni/Dunn post-hoc analysis. I.M. – intramuscular, I.N. – intranasal, S.L. – sublingual, P.O. – oral, PEI – pre-existing immunity, BAL - bronchioalveolar lavage fluid.

Figure 4. Sublingual Immunization Can Induce Long-Lasting Antigen-Specific T Cell-Mediated Immune Responses

The presence of immunological memory to Ebola Zaire GP was assessed in mice immunized with recombinant adenovirus by various routes 42 days after treatment by two separate assays. Panel A. Scatter Dot Plot Illustrating the CD8 Effector Memory T Cell Response to an Ebola Glycoprotein-Specific Peptide as Determined In Vitro. Splenocytes were stained with CFSE and stimulated with the TELRTFSI peptide for 5 days. Cells positive for CD8⁺, CD44^HI and CD62L^LOW were then evaluated for CFSE by four-color flow cytometry. A decrease in CFSE staining denotes cell division/expansion. Panel B. Quantitative Analysis of the Effector Memory T Cell Response: CFSE Assay. Data was generated from scatter plots shown in Panel A and represent the average values obtained from two separate experiments containing 4 mice per treatment. Error bars reflect the standard error of the data. Panel C. Representative Histograms Illustrating the In Vivo Ebola Glycoprotein-Specific Cytolytic T Cell Response. An equal mixture of TELRTFSI peptide pulsed CFSE^HI and unpulsed CFSE^LOW splenocytes (2 × 10⁷ cells total) were adoptively transferred to immunized mice by tail vein injection. Twenty-four hours later, splenocytes and mononuclear cells from submandibular lymph nodes (SMLN) were harvested and analyzed using flow cytometry. The number above each peak in the histogram plot denotes the percentage of gated CFSE^HI (right peak) and CFSE^LOW (left peak) cells for each subpopulation. Panel D. Quantitative Analysis of the Effector Cytotoxic T Cell Response: In Vivo CTL Assay. Data represent the average values obtained from two separate experiments each containing 4 mice per treatment. Error bars reflect the standard error of the data. I.M. – intramuscular, I.N. – intranasal, S.L. – sublingual, P.O. – oral

Figure 5. Sublingual Immunization Significantly Reduces Production of IL-6 in Response to the Adenovirus Vector and Minimizes Toxicity Associated with Adenovirus-Based Vaccines

Serum IL-6 (A), IL-12 (B) and TNF-α (C) were assessed from samples taken from B10.Br mice 6 hours after administration of a single dose of 1 × 10⁸ infectious particles of a first generation adenovirus expressing Ebola Zaire glycoprotein. (D) Serum alanine (ALT) and aspartate (AST) aminotransferase levels were evaluated 4 days after immunization. Data reflect average values ± the standard error of the mean for four mice from each group. *, p<0.05, **, p<0.01, ***, p<0.001, one-way ANOVA, Bonferroni/Dunn post-hoc analysis. I.M. – intramuscular, I.N. – intranasal, S.L. – sublingual, P.O. – oral

Figure 6. Sublingual Vaccination Performs in a Manner Similar to that of Traditional Intramuscular Vaccination with Respect to Survival after Lethal Challenge in Mice and Guinea Pigs

Naïve mice and those with prior exposure to adenovirus serotype 5 (indicated by PEI, n=10) were challenged with a lethal dose of 1,000 pfu mouse-adapted Ebola Zaire (30,000 × LD₅₀) by intraperitoneal injection 28 days after immunization with a single dose of 1 ×10⁸ infectious particles of vaccine. A separate group of naïve mice was given 1×10⁷ infectious particles of the vaccine (SL (low)). Two groups of mice were given 2.5 ×10¹¹ particles of adenovirus to establish pre-existing immunity (indicated by PEI) while another was given 5×10¹⁰ particles (indicated by PEI**) A) Mouse Kaplan-Meier survival curve. *indicates a significant difference with respect to the PEI/IM treatment group. B) Mouse Body Weight Profile After Challenge. No significant changes in body weight were noted in animals that survived challenge. C) Serum alanine (ALT) and aspartate (AST) aminotransferase levels Post-Challenge (mouse). Samples from non-survivors were taken at time of death. Samples from survivors were taken 14 days post-challenge. D) Serum Cytokines Post-Challenge. For panels c and d, data reflect average values ± the standard error of the mean for five mice per group. *indicates a significant difference between values from survivors and those that did not survive challenge (fatalities). E) Kaplan-Meier survival curve (Guinea Pig). Naïve Guinea pigs were challenged with a uniformly lethal dose of Guinea pig-adapted Ebola Zaire (1,000 pfu, ~1,000 × LD₅₀) by intraperitoneal injection 28 days after immunization. *indicates a significant difference with respect to saline controls (PBS). F) Change in Body Weight After Challenge (Guinea Pig). No significant changes in body weight were noted in animals that survived challenge. G) Serum alanine (ALT) and H) aspartate (AST) aminotransferase levels Post-Challenge (Guinea Pig). Samples were taken from survivors at days 0, 5, 7 and 14 post-challenge. Samples were taken from non-survivors at the described times and at time of death. In all panels, * p<0.05, ** p<0.01, ***p<0.001, one-way ANOVA, Bonferroni/Dunn post-hoc analysis.

Figure 7. Sublingual Immunization Does Not Facilitate Preferential Expansion of Anti-Adenovirus CD4+ Memory T Cells and Curtails Production of Anti-Adenovirus Neutralizing Antibodies in Mice with Pre-Existing Immunity

Pre-existing immunity was established by a single intramuscular injection of 2.5 × 10¹¹ particles of adenovirus containing the beta-galactosidase transgene 28 days prior to vaccination by various routes. a) Adenovirus-Specific Memory T Cell Proliferation. Splenocytes were harvested, labeled with CFSE and co-cultured with a recombinant adenovirus serotype 5 vector without a transgene cassette (AdNull) for 5 days. The number in each dot plot denotes the percentage of gated CFSE-negative cells for each subpopulation. Numbers and gates in red indicate the CD4+ population. Numbers and gates in blue indicate the CD8+ population. Dot plots were generated with samples pooled from 5 mice/treatment. b) Anti-Adenovirus Neutralizing Antibodies Produced after Immunization of Mice with Pre-Existing Immunity. Serum was collected from mice prior to immunization and 28 days after treatment. Neutralization was assessed by serial dilution of each sample, incubation with a set amount of adenovirus expressing beta-galactosidase and assessment of transduction efficiency on HeLa cells as described in the Materials and Methods section. Data reflect average values ± the standard error of the mean for samples collected from eight mice per group over 2 separate experiments. *indicates a significant difference with respect to immunization route. ***p<0.001, one-way ANOVA, Bonferroni/Dunn post-hoc analysis. Naïve – samples obtained from mice given saline (negative control).