Differential Biodistribution of Adenoviral-Vectored Vaccine Following Intranasal and Endotracheal Deliveries Leads to Different Immune Outcomes

Abstract

Infectious diseases of the respiratory tract are one of the top causes of global morbidity and mortality with lower respiratory tract infections being the fourth leading cause of death. The respiratory mucosal (RM) route of vaccine delivery represents a promising strategy against respiratory infections. Although both intranasal and inhaled aerosol methods have been established for human application, there is a considerable knowledge gap in the relationship of vaccine biodistribution to immune efficacy in the lung. Here, by using a murine model and an adenovirus-vectored model vaccine, we have compared the intranasal and endotracheal delivery methods in their biodistribution, immunogenicity and protective efficacy. We find that compared to intranasal delivery, the deepened and widened biodistribution in the lung following endotracheal delivery is associated with much improved vaccine-mediated immunogenicity and protection against the target pathogen. Our findings thus support further development of inhaled aerosol delivery of vaccines over intranasal delivery for human application.

Keywords: Adenovirus-vectored vaccine; T cells; Tuberculosis; biodistribution; endotracheal; intranasal; mucosal immunity; respiratory mucosal immunization.

Figure 1

Step-wise illustration of endotracheal delivery method (I.T). Endotracheal intubation was carried out in a C57BL/6 mouse anesthetized with 2% isoflurane and oxygen at a flow rate of 2 liters/min using an illuminated otoscope, a 22G blunt-tip intravenous catheter, a 45⁰ C-angled intubation stand, a P200 pipette with P200 tip and a P200 pipette with extended length gel loading tip (A). Unconscious mouse breathing at a respiration rate of 30 breaths per min was placed by hooking its upper incisor over a string attached to the intubation board. An otoscope was placed into the mouth and the vocal cord was visualized (B). A 22G blunt-tip intravenous catheter attached to a syringe was then inserted into the trachea (C). The syringe was then replaced with a P200 pipette attached to a p200 tip containing water and the movement of water in the tip during breathing was confirmed to affirm the insertion of catheter into the trachea (D). Next, P200 pipette attached to an extended length gel-loading tip loaded with 50 μL AdHu5Luc or vaccine was inserted into the catheter and allow to inhale by the mouse (E).

Figure 2

Biodistribution of vaccine surrogate in the lung following intranasal or endotracheal inoculation. (A) Experimental schema. Mice were inoculated intranasally (I.N.) or endotracheally (I.T) with adenovirus-vector expressing luciferase (AdHu5Luc) or PBS. Biodistribution was visualized as a factor of light emission upon intranasal administration of luciferin. Images were obtained using an IVIS Spectrum and presented as pseudocolour images of bioluminescence in PBS (B) or AdHu5Luc inoculated animals (C). Red represents the most intense areas of biodistribution while the blue corresponds to the weakest areas of biodistribution. Mice were imaged with an integration time of 30 sec. Three mice per treatment group is shown. (D) Bar graph shows corrected total fluorescence intensity measured in relative fluorescence units (RFU) and quantified using ImageJ in either right or left lung. Data is from 3 mice/group and RFU are presented as mean ± SEM. **p < 0.01.

Figure 3

Vaccine-specific T cell responses in the airways following endotracheal immunization compared to intranasal immunization. Experimental schema (A). Mice immunized intranasally (I.N.) or endotracheally (I.T.) with AdHu5Ag85A were scarified 4 weeks post-immunization and mononuclear cells from airways were examined for vaccine-specific responses. (B) Bar graphs comparing total number of mononuclear cells in bronchoalveolar lavage (BAL) fluid. (C) Representative flow cytometric dotplots showing frequencies of Ag85A-specific CD8 T cells (CD8+tet+) determined by tetramer staining, and frequencies of IFNγ+ CD8+ and CD4+ T cells determined by intracellular cytokine staining of cells stimulated with Ag85A CD8 or CD4 T-cell specific peptides in BAL. Top row dotplots (Control) show the defining gates for tetramer population gated out of total CD8 T cells from unimmunized animal and the gates for CD8+IFNγ+ and CD4+IFNγ+ T cells out of total unstimulated CD8 and CD4 T cells from BAL of immunized mice. Numerical indicated in the dotplots represent the mean frequency of parent (CD4 or CD8 T cells) ± SEM. (D) Bar graphs comparing absolute number of CD8+tet+, CD8+IFNγ+, and CD4+IFNγ+ T cells in BAL of intranasal- and endotracheal-immunized mice. Absolute numbers of CD8+tetramer+, CD8+ IFN-γ+ and CD4+ IFN-γ+ shown in bar graphs were calculated based on frequency of CD3+live cells gated out of total events to exclude all non-immune cells. Data is from 3 mice/group, representative of two independent experiments and presented as mean ± SEM.*p < 0.05.

Figure 4

Vaccine-specific T cell responses in the left and right lung tissues following endotracheal immunization compared to intranasal immunization. Experimental schema (A). Mice immunized intranasally (I.N.) or endotracheally (I.T.) with AdHu5Ag85A were scarified 4 weeks post-immunization and mononuclear cells from left and right lung homogenates were examined for vaccine-specific responses separately. (B) Bar graphs comparing total numbers of mononuclear cells in left and right lung tissues. (C–E) Representative flow cytometric dotplots showing frequencies of Ag85A-specific CD8 T cells (CD8+tet+) determined by tetramer staining, and frequencies of IFNγ+ CD8+ and CD4+ T cells determined by intracellular cytokine staining of cells stimulated with Ag85A CD8 or CD4 T-cell specific peptides in left and right lung tissues. Top row dotplots (Control) show the defining gates for tetramer population gated out of total CD8 T cells from unimmunized animal and the gates for CD8+IFNγ+ and CD4+IFNγ+ T cells out of total unstimulated CD8 and CD4 T cells from left and right lung tissues of immunized mice. Numericals indicated in the dotplots represent the mean frequency of parent (CD4 or CD8 T cells) ± SEM. (F–H) Bar graphs comparing absolute numbers of CD8+tet+, CD8+IFNγ+, and CD4+IFNγ+ T cells in left and right lung tissue of intranasal and endotracheal-immunized mice. Absolute numbers of CD8+tetramer+, CD8+ IFN-γ+ and CD4+ IFN-γ+ cells were calculated based on frequencies of CD3+live cells gated out of total events to exclude all non-immune cells. Data is from 3 mice/group, representative of two independent experiments and presented as mean ± SEM.*p < 0.05, **p < 0.01.

Figure 5

Immune protection against pulmonary tuberculosis following endotracheal immunization compared to intranasal immunization. Experimental schema (A). Mice immunized intranasally (I.N.) or endotracheally (I.T.) with AdHu5Ag85A were infected with virulent M. tuberculosis (Mtb) and sacrificed 4 weeks post-infection. Unimmunized mice were included as controls (Naïve). Right lung homogenates were serially diluted and plated for the assessment of mycobacterial burden (colony forming unit -CFU). (B) Bar graph comparing Log₁₀ CFU/lung in unimmunized (naïve), or I.N. or I.T. immunized mice. Data is from n = 6 mice/group and presented as mean ± SEM. *p < 0.05; **p <0.01; ****p < 0.0001. (C–H) Following M.tb infection, lungs were processed for H&E staining and examined for immunopathological changes. Representative low-power micrographs showing overall lung architectural changes and higher-power micrographs showing granuloma, areas of pneumonitis and peribronchial/perivascular infiltrates. (I–K) Bar graphs comparing the semi-quantitative scoring of the extent of lung granuloma, pneumonitis and infiltration of cells in naïve, I.N. and I.T. immunized mice. Scoring was carried out on a scale of 1 to 10 and independently verified by another researcher blinded to the experimental groups. Data is from n = 6 mice/group. Data in bar graphs are presented as mean ± SEM. ***p <0.001; ****p < 0.0001.