Immunization with the Haemophilus ducreyi trimeric autotransporter adhesin DsrA with alum, CpG or imiquimod generates a persistent humoral immune response that recognizes the bacterial surface

Abstract

The Ducreyi serum resistance A (DsrA) protein of Haemophilus ducreyi belongs to a large family of multifunctional outer membrane proteins termed trimeric autotransporter adhesins responsible for resistance to the bactericidal activity of human complement (serum resistance), agglutination and adhesion. The ability of DsrA to confer serum resistance and bind extracellular matrix proteins lies in its N-terminal passenger domain. We have previously reported that immunization with a recombinant form of the passenger domain of DsrA, rNT-DsrA, in complete/incomplete Freund's adjuvant, protects against a homologous challenge in swine. We present herein the results of an immunogenicity study in mice aimed at investigating the persistence, type of immune response, and the effect of immunization route and adjuvants on surrogates of protection. Our results indicate that a 20 μg dose of rNT-DsrA administered with alum elicited antisera with comparable bacterial surface reactivity to that obtained with complete/incomplete Freund's adjuvant. At that dose, high titers and bacterial surface reactivity persisted for 211 days after the first immunization. Administration of rNT-DsrA with CpG or imiquimod as adjuvants elicited a humoral response with similar quantity and quality of antibodies (Abs) as seen with Freund's adjuvant. Furthermore, intramuscular administration of rNT-DsrA elicited high-titer Abs with significantly higher reactivity to the bacterial surface than those obtained with subcutaneous immunization. All rNT-DsrA/adjuvant combinations tested, save CpG, elicited a Th2-type response. Taken together, these findings show that a 20 μg dose of rNT-DsrA administered with the adjuvants alum, CpG or imiquimod elicits high-quality Abs with reactivity to the bacterial surface that could protect against an H. ducreyi infection.

Keywords: DsrA; Haemophilus ducreyi; Trimeric autotransporter adhesin; Vaccine.

Fig. 1.. The recombinant immunogen rNT-DsrA_I is devoid of contaminants.

A. Preparations of rNT-DsrA_I were subjected to SDS-PAGE and Coomassie Blue staining to determine the presence of foreign proteins. Bovine Serum Albumin (BSA) standards were run concurrently with the purified immunogen to confirm protein concentration previously measured using a commercial reagent. B. rNT-DsrA_I preparations were subjected to Western blotting with antibodies to rFL-DsrA_I [17].

Fig. 2.. Quantity and quality of the humoral immune response is enhanced in animals receiving the rNT-DsrA vaccine administered with alum or Freund’s adjuvants.

A. Log endpoint titers (means ± standard deviations) of individual rNT-DsrA antisera from mice receiving 0.04, 4, or 20 µg of rNT-DsrA_I either alone, in alum or Freund’s adjuvant. B. Reactivity (means ± standard deviations of 3 independent experiments) of pooled rNT-DsrA_I antisera (day 56) to the surface of viable homologous H. ducreyi. *, p<0.05; **, p<0.01 using an unpaired t-test.

Fig. 3.. rNT-DsrA elicits a persistent humoral immune response.

Antisera from mice immunized subcutaneously three times, three weeks apart with 4 (A) or 20 µg (B) of rNT-DsrA_I administered alone or in conjunction with alum or Freund’s adjuvant were tested for reactivity to purified rNT-DsrA_I (top) or to the surface of viable homologous H. ducreyi (bottom). Antisera were collected on days 0, 21, 42, 56, 122, 146 and 211, where day 0 indicates the first immunization. Shown are means ± standard deviations of individual sera (5 mice/group) for ELISA (top) or pooled sera (whole-cell binding ELISA, bottom) tested on three consecutive days. *, p<0.05 using an unpaired t-test.

Fig. 4.. The humoral immune response to rNT-DsrA_I is similar when administered with Freund’s, MPL, CpG or imiquimod adjuvant, and improved with intramuscular compared to subcutaneous immunization route.

Groups of 5 mice were immunized either with 4 (A) or 20 (B) µg of rNT-DsrA_I in the absence or presence of 5 different adjuvants (Alum, Freund’s, MPL, CpG and Imiquimod) following subcutaneous (SQ, top) or intramuscular (IM, middle) routes. Mice were immunized and bled three times, three weeks apart (days 0, 21, and 42), and bled at day 56. Top and middle, means ± standard deviations of endpoint titers of individual antisera after SQ or IM immunization, respectively. Bottom, reactivity of individual antisera from day 56 to viable, homologous H. ducreyi strain 35000HP (white column, SQ route; black column, IM route). * indicates p<0.05 as compared to the response obtained with “no adjuvant” at that specific dose/route for the entire time of the trial (56 days); for whole cell binding ELISAs (bottom), “*p=“ compares SQ and IM routes for pairs of doses and adjuvants using an unpaired t-test.

Fig. 5.. Intramuscular administration of rNT-DsrA_I using the human-approved adjuvants CpG and Imiquimod elicits a humoral immune response that recognizes equally well DsrA at the surface of both homologous and heterologous H. ducreyi strains.

Reactivity of pooled rNT-DsrA_I antisera to the surface of viable homologous (35000HP) and heterologous (HMC50) H. ducreyi strains. Shown are means ± standard deviations of three experiments conducted on three consecutive days. (A) Data from immunization trial 1 (20 µg dose, subcutaneous route only) are shown according to time point (days) and adjuvant. (B) Reactivity of antisera (day 56) from mice receiving a 20 µg dose of rNT-DsrA_I alone or in combination with 5 different adjuvants, using the subcutaneous (SQ) or intramuscular (IM) route. *, p<0.05 using an unpaired t-test.

Fig. 6.. Adjuvant, dose and route of immunization affect isotype switching of the humoral immune response to rNT-DsrA_I.

Log₂ IgG1/IgG2a ratios of pooled (A) and individual (B) antisera from animals receiving 0.04, 4 and 20 µg doses administered alone, or in combination with one of 5 different adjuvants. A. Log₂ IgG1/IgG2a ratios for different doses of rNT-DsrA_I administered subcutaneously either alone, with Alum or Freund’s adjuvant were measured for the length of the study, up to 211 days. B. Log₂ IgG1/IgG2a ratios of day 56 antisera from mice receiving the rNT-DsrA_I vaccine with several different human-approved adjuvants administered subcutaneously (SQ) or intramuscularly (IM). *, p<0.05 compared to the “no adjuvant” control for each dose/route tested.