Intranasal Immunization with Killed Unencapsulated Whole Cells Prevents Colonization and Invasive Disease by Capsulated Pneumococci

Vaccine preparation.Strain RX1 is a capsule-negative mutant derived from a pneumococcus capsular serotype 2 (22). To allow for growth to high concentrations, an autolysin (lytA)-negative mutant of Rx1 (Rx1AL⁻) was used to prepare the killed vaccine preparations. This strain carries an erythromycin resistance gene (1). For vaccine production, RX1AL⁻ was grown at 37°C in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) and 0.3 μg of erythromycin/ml to about 10⁹cells/ml. The cells were washed and suspended in saline at 10% of the original volume. Samples were mixed 3:7 (volume/volume) with ethanol, washed and resuspended in saline, and then frozen in small aliquots. When the killed vaccine preparation was cultured on blood agar, no viable bacteria were detected (lower limit of detection, 1 CFU/0.1 ml). The final vaccine mixture also contained CT (List Biological Laboratories, Campbell, Calif.) at 1 μg of CT per dose of vaccine. Control mice or rats were immunized with 1 μg of CT in saline.

Bacteria for animal challenge. S. pneumoniae strains GA03212, SF07348, and CT80231 are of capsular serotype 6B, 10F, and 14, respectively; all clinical isolates were originally from the Active Bacterial Core Surveillance (ABCs) of the Centers for Disease Control and Prevention. Strain WU2 is a type 3 pneumococcus that has been previously described (4). WU2r was passaged intraperitoneally in rats to increase its virulence (21). These strains were stored at −70°C in either skim milk or THY with 10% glycerol. For challenge in mice, frozen suspensions of S. pneumoniae type 6B were thawed and diluted in saline to a concentration of 10⁶CFU/10 μl. For intrathoracic challenge in rats, WU2r was grown overnight on blood agar plates and then grown in THY on the morning of the experiment. In early log phase it was diluted to an estimated concentration of 8 × 10⁵/ml in 0.5% low-melting-point agarose to increase its virulence (21); the actual colony count was determined on blood agar. For passive-protection experiments in infant rats, frozen suspensions of WU2r were thawed, diluted in sera, and incubated at 4°C for 90 min. Following incubation, the bacterial preparations were further diluted in 0.5% low-gelling-point agarose to the desired concentration.

Animal Models. (i) Mouse colonization model.C57BL/6J mice, 4 to 6 weeks old, were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed four per cage. They were randomized by cage to receive vaccine or control preparations. Immunization was delivered by gently restraining the unanesthetized mice and applying 10 μl atraumatically to the nostrils. Live pneumococcal preparations (at a concentration of 10⁶ CFU/inoculation), killed Rx1AL⁻ without or with CT, CT alone, or saline was given three times at weekly intervals. One week following the last immunization, animals in all groups received 1 mg of rifampin in 0.25 ml subcutaneously (a dose which effectively eradicates pneumococcal colonization and does not prevent subsequent colonization [data not shown]). At 1 or 7 weeks later, the mice were challenged with 10⁶ CFU of S. pneumoniae type 6B applied as in the immunizations. At 1 week after challenge, the mice were euthanized by CO₂ inhalation; the trachea was exposed and transected by careful dissection. An upper respiratory wash was done by instilling sterile, nonbacteriostatic saline retrograde through the transected trachea and collecting the first 6 drops (about 0.1 ml) from the nostrils. An animal was considered to be nasally colonized if ≥1 CFU/50 μl of wash fluid was detected on blood agar containing 2.5 μg of gentamicin/ml. In addition, quantitative cultures of these upper respiratory washes were performed; the lower limit of detection of these cultures was 1 CFU per 50 μl.

(ii) Young-rat sepsis model.Outbred virus-free Sprague-Dawley 3-week-old male rats were obtained from Charles River Laboratories (Wilmington, Mass.) and housed four per cage. They were randomized by cage to receive vaccine or control immunizations, which were administered as for the mice except that volumes of 100 μl (for the first two immunizations) and 30 μl (for the third, if applicable) were applied to the left nostril only. In pilot studies using methylene blue-containing solutions, delivery of up to 100 μl did not result in aspiration of material into the lungs (data not shown). Two weeks following the last immunization, intrathoracic challenge with S. pneumoniae type 3 was performed. The right chest was prepared with an alcohol swab, and a 0.05-ml inoculum was injected transthoracically into the right mid-lung via a 29-gauge needle on an insulin syringe. The depth of the intrathoracic injection (5 mm) was controlled by a small hemostat clipped at the base of the needle. Clinical appearance was monitored daily by an investigator blinded to immunization assignment, using a 5-point scoring system (0, well; 1, ruffled fur; 2, ruffled fur and decreased activity; 3, ruffled fur and inactivity; 4, hunched and gaunt). An animal receiving a score of ≥2 on any day of the experiment was considered to have become ill. Any animal receiving a score of 3 or above was immediately euthanized, since such a score is a reliable predictor of mortality in our experience (data not shown). Clinical appearance and mortality were assessed for 10 days after inoculation, after which time the experiment was concluded.

(iii) Passive-protection studies: infant-rat model.For passive-protection studies, we used the infant-rat sepsis model as published previously (21). Briefly, timed-pregnant Sprague-Dawley dams were allowed to deliver in our animal-housing facilities. On day 3 of life, infant rats were randomly redistributed across dams, so that each cage had approximately 12 infant rats. On day 4 of life, infant rats were randomized by cage to receive an intrathoracic injection of S. pneumoniae type 3 that had been previously incubated in various sera. The sera of main interest were pools from the young rats immunized (in experiment 2 of Table 2) three times with Rx1AL⁻ plus CT or with CT alone. Additional controls were normal rat serum (NRS) and a positive control consisting of bacterial polysaccharide immune globulin (BPIG; hyperimmune serum obtained from healthy adult volunteers immunized with 23-valent pneumococcal PS vaccine [gift of the Massachusetts Biological Laboratories, Jamaica Plain, Mass.]). Low and high inocula (corresponding to a final challenge of ca 5 and 50 CFU, respectively) were added to each serum or BPIG sample. Following incubation for 90 min at 4°C, bacteria were diluted to the desired concentration in 0.5% low-gelling-point agarose, as described above. Bacterial challenge via the intrathoracic route was performed as described above, except that the volume of the injection was 25 μl. Blood cultures of specimens from all infant rats were performed 24 h after challenge. For this purpose, the tail vein of each infant rat was swabbed with 70% ethanol and punctured aseptically with a sterile lancet. Approximately 10 μl of blood was plated on a blood agar plate containing 2.5 mg of gentamicin per liter (to suppress growth of the normal skin flora) and incubated overnight at 37°C with 5% CO₂. Bacteremia was defined as the presence of ≥1 CFU ofS. pneumoniae. The lower limit of detection of bacteremia was 100 CFU/ml. The end point of these experiments was mortality by day 7 after challenge.

Statistical Analysis.Fisher's exact test was used to compare the frequency of nasopharyngeal colonization following challenge in mice immunized with various preparations. To compare the density of colonization in mice, log-transformed counts of CFU of pneumococci were compared using Student's t test. For these purposes, the density of colonization in noncolonized animals was assigned a value equal to half the lower limit of detection, or 0.5 CFU per 50 μl. Fisher's test was used to compare the mortality rates following intrathoracic challenge in the rats that were immunized with Rx1AL⁻ plus CT versus CT alone as well as in the passive-protection experiments.