Mucosal Immunization with DTaP Confers Protection against Bordetella pertussis Infection and Cough in Sprague-Dawley Rats

Abstract

Pertussis is a respiratory disease caused by the Gram-negative pathogen, Bordetella pertussis. The transition from a whole-cell pertussis vaccine (wP and DTP) to an acellular pertussis vaccine (aP, DTaP, and Tdap) correlates with an increase in pertussis cases, despite widespread vaccine implementation and coverage, and it is now appreciated that the protection provided by aP rapidly wanes. To recapitulate the localized immunity observed from natural infection, mucosal vaccination with aP was explored using the coughing rat model of pertussis. Overall, our goal was to evaluate the route of vaccination in the coughing rat model of pertussis. Immunity induced by both oral gavage and intranasal vaccination of aP in B. pertussis challenged rats over a 9-day infection was compared to intramuscular wP (IM-wP)- and IM-aP-immunized rats that were used as positive controls. Our data demonstrate that mucosal immunization of aP resulted in the production of anti-B. pertussis IgG antibody titers similar to IM-wP- and IM-aP-vaccinated controls postchallenge. IN-aP also induced anti-B. pertussis IgA antibodies in the nasal cavity. Immunization with IM-wP, IM-aP, IN-aP, and OG-aP immunization protected against B. pertussis-induced cough, whereas OG-aP immunization did not protect against respiratory distress. Mucosal immunization by both intranasal and oral gavage administration protected against acute inflammation and decreased bacterial burden in the lung compared to mock-vaccinated challenge rats. The data presented in this study suggest that mucosal vaccination with aP can induce a mucosal immune response and provide protection against B. pertussis challenge. This study highlights the potential benefits and uses of the coughing rat model of pertussis; however, further questions regarding waning immunity still require additional investigation.

Keywords: Bordetella pertussis; DTaP; immunization; mucosal; pertussis; plethysmography; rats.

FIG 1

i.n. booster vaccination induces systemic anti-B. pertussis (anti-Bp) and anti-PT antibody titers. At 1 and 2 weeks after prime immunization and at 1 week after boost blood was collected via the saphenous vein, and anti-Bp and anti PT IgM (A and C)- and IgG (B and D)-specific antibodies were measured. The results are shown on a log scale and as means ± the standard errors of the mean (SEM; n = 3 to 8). The dotted line represents the limit of detection. *, P < 0.05. (n = 4 to 8). P values were determined by two-way ANOVA with Tukey’s post hoc test compared between groups. Asterisks (*) under each graph annotate the significance between labeled groups under the y axis and the group under the corresponding bar. Grayed-out boxes indicate that no stats were calculated.

FIG 2

Intranasal and oral vaccination of acellular pertussis vaccine decreases cough in B. pertussis-infected rats. Coughs were counted every day of the 9-day infection using WBP. Coughs were counted for mock-vaccinated challenge rats (A) and for IM-wP (B)-, IM-aP (C)-, IN-aP (D)-, and OG-aP (E)-vaccinated and challenged rats. (F) To assess any potential differences between vaccine groups over the entire course of infection, the average total numbers of coughs for each rat per group were compared. The results are shown as means ± the SEM (n = 3 to 4). P values were determined by two-way ANOVA with Dunnett’s post hoc test and one-way ANOVA with Dunnett post hoc test for total cough count (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [compared to the mock-vaccinated challenge control group]).

FIG 3

Intranasal vaccination decreases the pulmonary restriction of B. pertussis-infected rats. Bronchiole restriction was measured over the course of infection by WBP. Bronchiole restriction was determined by the factor Penh for mock-vaccinated challenge rats (A) and for IM-wP (B)-, IM-aP (C)-, IN-aP (D)-, and OG-aP (E)-vaccinated and challenged rats. The results are shown as means ± the SEM (n = 3 to 4). (F) Differences observed between groups at days 5 to 7 postchallenge. P values were determined by two-way ANOVA with Dunnett’s post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001 [compared to the mock-vaccinated challenge group]).

FIG 4

Mucosal vaccination induces the production of anti-B. pertussis IgG, whereas i.n. immunization also induces both anti-PT IgM and IgG antibodies. ELISAs were used to determine and compare the induced serological responses from vaccinated and challenge rats in the serum. Both IgM (A and C) and IgG (B and D) serum antibody titers from immunized and challenged rats were measured postchallenge. The dotted line represents the limit of detection. The results are shown on a log scale as means ± the SEM (*, P < 0.05; n = 4). P values were determined by two-way ANOVA with Tukey’s post hoc test compared between groups. Asterisks (*) under each graph annotate the significance between labeled group under the y axis and the group under the corresponding bar. Grayed-out boxes indicate that no stats were calculated.

FIG 5

Intranasal immunization elicits the production of anti-B. pertussis IgA in the respiratory tract. ELISA was used to analyze antibodies in the lung (A and B) and nasal cavity (C and D) from lung homogenate supernatant and PBS flushed through the nasal cavity from vaccinated and challenge rats at days 1 and 9 postchallenge. IgA titers were determined against pertussis toxin and B. pertussis. The dotted line represents the limit of detection. The results are shown on a log scale as a means ± the SEM (n = 3 to 4; **, P < 0.01; ****, P < 0.0001). P values were determined by the Kruskal-Wallis test, using Dunn’s post hoc test to compare between groups.

FIG 6

Mucosal vaccination protects against acute and total inflammation in the lung of B. pertussis-infected Sprague-Dawley rats. After euthanasia, the left lobe of the lung was excised, sectioned, and stained with H&E. Lung samples scores are based on standard qualitative toxicologic scoring criteria: 0, none; 1, minimal (rare); 2, mild (slight); 3, moderate; 4, marked; and 5, severe. (A) Representative image of acute inflammation of rat lung showing increased numbers of neutrophils and edema surrounding blood vessel (asterisk). (B) Representative image of chronic inflammation of the rat lung showing increased numbers of mononuclear cells surrounding bronchioles (asterisk). Inflammatory cells are also present in the lamina propria (arrow) and epithelium (arrowhead) of bronchioles. (C) Average acute inflammation scores of the lung are detailed by the presence of neutrophils in the parenchyma, blood vessels, and the airways. (D) Average chronic inflammation scores are distinguished by mononuclear infiltrates in the parenchyma, blood vessels, and airway of the lung. All scoring assessments were determined with no knowledge of the groups. The results are shown as means ± the SEM (n = 3 to 4). P values were determined by two-way ANOVA, followed by Dunnett’s comparison test (*, P < 0.05; **, P < 0.01 [compared to mock challenge]).

FIG 7

Oral and intranasal immunization decreased the B. pertussis bacterial burden in the respiratory tract. Bacteria were quantified by serially diluted CFU after vaccination and intranasal challenge. CFU counts were determined from lung homogenate (A), trachea (B), and nasal lavage (C) 1.5 h, 1 day, and 9 days after B. pertussis challenge. The results are shown as means ± the SEM (n = 2 to 4). P values were determined by one-way ANOVA with Dunnett’s post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [compared to the mock-vaccinated challenge group]).

FIG 8

Systemic and mucosal anti-B. pertussis and anti-PT antibodies correlate with observed protection. Correlograms were generated using the observed data for IM-wP (A and B), IM-aP (C and D), IN-aP (E and F), and OG-aP (G and H) immunizations. Program R was used to make correlation graphs from raw data for both day 1 and day 9 postchallenge. R² values were generated when generating the correlograms. Positive correlations are annotated by blue circles, while the negative correlations are annotated by red circles. The size of the circle indicates the strength of the correlation.