Effective induction of protective systemic immunity with nasally administered vaccines adjuvanted with IL-1

Abstract

IL-1α and IL-1β were evaluated for their ability to provide adjuvant activity for the induction of serum antibody responses when nasally administered with protein antigens in mice and rabbits. In mice, intranasal (i.n.) immunization with pneumococcal surface protein A (PspA) or tetanus toxoid (TT) combined with IL-1β induced protective immunity that was equivalent to that induced by parenteral immunization. Nasal immunization of awake (i.e., not anesthetized) rabbits with IL-1-adjuvanted vaccines induced highly variable serum antibody responses and was not as effective as parenteral immunization for the induction of antigen-specific serum IgG. However, i.n. immunization of deeply anesthetized rabbits with rPA+IL-1α consistently induced rPA-specific serum IgG ELISA titers that were not significantly different than those induced by intramuscular (IM) immunization with rPA+alum although lethal toxin-neutralizing titers induced by nasal immunization were lower than those induced by IM immunization. Gamma scintigraphy demonstrated that the enhanced immunogenicity of nasal immunization in anesthetized rabbits correlated with an increased nasal retention of i.n. delivered non-permeable radio-labeled colloidal particles. Our results demonstrate that, in mice, IL-1 is an effective adjuvant for nasally administered vaccines for the induction of protective systemic immunity and that in non-rodent species, effective induction of systemic immunity with nasally administered vaccines may require formulations that ensure adequate retention of the vaccine within the nasal cavity.

Figure 1. Intramuscular but not nasal delivery of IL-1β as a vaccine adjuvant induces increased core body temperature in rabbits

NZW female rabbits were immunized with 100 μg tetanus toxoid combined with rhIL-1β as adjuvant by the nasal or intramuscular route on day 0, 28 and 56. Rabbits were not sedated or anesthetized for performance of the immunizations. Body temperature was monitored immediately before (time 0) and at 2, 4, 6, 8 and 24 hours after immunization and temperatures were compared within each vaccine group using ANOVA followed by Dunnett’s test to compare the post-vaccination temperatures to the time 0 temperature. There were three rabbits per adjuvant group and temperature readings from each day (day 0, 28 and 56) were combined. Rabbits immunized by the intramuscular route with TT + IL-1β at 312.5 ng/kg had significantly increased core body temperatures at 2 and 4 hours after immunization (ANOVA, Dunnett’s test, p<0.05). Rabbits immunized with TT + IL-1β at 7,812.5 ng/kg had significantly increased core body temperatures at 2, 4, 6 and 8 hours after immunization (ANOVA, Dunnett’s, p<0.05). All other comparisons within groups between time 0 and post-vaccination body temperatures did not identify any significant change in core body temperature (ANOVA, Dunnett’s test).

Figure 2. Serum anti-TT IgG titers after nasal or intramuscular immunization with TT and IL-1β in rabbits

Female NZW rabbits were immunized by the nasal or intramuscular route while awake with 100 μg tetanus toxoid combined with rhIL-1β on day 0, 28 and 56. Rabbits were not sedated or anesthetized for performance of the immunizations. There were 3 rabbits in all groups. Serum was collected on day 21, 49 and 77 and tested for the presence of anti-TT IgG by ELISA (day 49 intranasal 7,812.5 ng/kg IL-1β and day 77 intranasal 12.5 ng/kg IL-1β had only two rabbits/group due to the inability to collect blood from one rabbit). The horizontal bars in each scatter plot represent the geometric mean titer (the solid bar for intramuscularly immunized rabbits and the dashed bar for intranasally immunized rabbits). No statistical comparisons were performed between groups due to the lack of power resulting from the low numbers of animals per group. Undetectable serum antibody responses are outside the range of the Y axis and are therefore not represented in the figure.

Figure 3. Nasal immunization of rabbits induces variable antigen-specific IgG responses that are significantly lower than IgG responses induced by intramuscular immunization

Female NZW rabbits (3 per group for each IL-1β adjuvant group) were immunized by the nasal or intramuscular route while awake with 100 μg tetanus toxoid combined with rhIL-1β (0.5, 12.5, 315.5 or 7812.5 ng/kg) on day 0, 28 and 56. Serum was collected on day 77 and tested for the presence of anti-TT IgG by ELISA. The serum anti-TT IgG titer in nasally immunized rabbits was 1:21,081 (geometric mean titer) while the serum anti-TT IgG titer in intramuscularly immunized rabbits was 1:4,194,304 (geometric mean titer; p=0.0001 vs nasal immunization, Mann Whitney).

Figure 4. The state of anesthesia influences the efficacy of nasal immunization in rabbits

Female NZW rabbits were nasally immunized with 20 μg rPA combined with rhIL-1α (5–15 μg) on day 0, 21 and 42. There were 4 - 8 rabbits per group Control rabbits were immunized intramuscularly with 10 μg rPA + alum on day 0, 21 and 42. Nasally immunized rabbits were awake, sedated with acepromazine/butorphanol (sedated but upright and mobile in their cage), sedated with acepromazine and anesthetized with isoflurane immediately before immunization (sedated with anesthesia lasting less than 5 minutes after vaccination) or anesthetized with ketamine/xylazine before immunization (deeply anesthetized for at least 30 minutes following nasal immunization). Serum was collected on day 42 and 56 and tested for the presence of anti-PA IgG by ELISA. Serum anti-PA IgG were compared between groups using a one way ANOVA and Tukey’s Multiple Comparison Test (PRISM). a. serum anti-PA IgG titers significantly greater than in intranasally immunized awake rabbits, p<0.05. b. serum anti-PA IgG titers significantly greater than in intranasally immunized rabbits treated with acepromazine/butorphanol p<0.05. c. serum anti-PA IgG titers significantly greater than in intranasally immunized rabbits treated with acepromazine/isoflurane p<0.05. d. serum anti-PA IgG titers significantly greater than in intranasally immunized rabbits treated with ketamine/xylazine p<0.05.

Figure 5. Anesthetic state influences nasal retention in rabbits

Female NZW rabbits (3–4 per group) were used to monitor the nasal retention of non-permeable Tc^99m-sulfur colloid (Tc^99m-SC) after delivery to nasal mucosal surfaces in acepromazine/butorphanol-sedated, or ketamine/xylazine-anesthetized, rabbits. A. Typical scintigraphy image of Tc^99m-SC acquired from the lateral aspect immediately after delivery with activity deposited within the nasal cavity (rabbit outline drawn for orientation purposes only). B. Efficacy of delivery to rabbits. Total radioactivity delivered to rabbits at time zero (includes nasal cavity, lungs, and swallowed activity within the digestive tract) as percentage of expected delivery. No difference between groups; p=0.8571, Mann Whitney. C. Efficacy of nasal cavity delivery. Radioactivity deposited regionally within the nasal cavity as percentage of total activity administered to rabbits. No difference between groups; p=0.6286, Mann Whitney. D. One of seven rabbits aspirated the Tc^99m-sulfur colloid at the time of delivery resulting in immediate accumulation of activity in the lungs and digestive tract (the ketamine/xylazine-anesthetized rabbit with a low percentage of nasal cavity delivery in Figure 5C). E. Retention of radioactivity initially deposited within nasal cavity. Percent of delivered activity remaining in nasal cavity region of interest, compared between groups at each time point using two-tailed unpaired Student’s T-test; *: p=0.0245. F. Nasal retention: area under the retention versus time curve. The retention data of Figure 5E were used to calculate the total area under the curve for the acepromazine/butorphanol and ketamine/xylazine groups, respectively:* p =0.063 two-tailed unpaired Student’s T-test. G. Typical scintigraphy image of Tc^99m-sulfur colloid 180 minutes after delivery with regional retention of radioactivity within the nasal cavity as well as clearance to the stomach.