Protection against development of otitis media induced by nontypeable Haemophilus influenzae by both active and passive immunization in a chinchilla model of virus-bacterium superinfection

Abstract

Three separate studies, two involving active-immunization regimens and one involving a passive-transfer protocol, were conducted to initially screen and ultimately more fully assess several nontypeable Haemophilus influenzae outer membrane proteins or their derivatives for their relative protective efficacy in chinchilla models of otitis media. Initial screening of these antigens (P5-fimbrin, lipoprotein D, and P6), delivered singly or in combination with either Freund's adjuvant or alum, indicated that augmented bacterial clearance from the nasopharynx, the middle ears, or both anatomical sites could be induced by parenteral immunization with P5-fimbrin combined with lipoprotein D, lipoprotein D alone, or the synthetic chimeric peptide LB1 (derived from P5-fimbrin), respectively. Data from a second study, wherein chinchillas were immunized with LB1 or lipoprotein D, each delivered with alum, again indicated that clearance of nontypeable H. influenzae could be augmented by immunization with either of these immunogens; however, when this adjuvant was used, both antibody titers in serum and efficacy were reduced. A third study was performed to investigate passive delivery of antisera directed against either LB1, lipoprotein D, nonacylated lipoprotein D, or a unique recombinant peptide designated LPD-LB1(f)2,1,3. The last three antiserum pools were generated by using the combined adjuvant of alum plus monophosphoryl lipid A. Passive transfer of sera specific for LB1 or LPD-LB1(f)2,1,3 to adenovirus-compromised chinchillas, prior to intranasal challenge with nontypeable H. influenzae, significantly reduced the severity of signs and incidence of otitis media which developed (P </= 0.001). Collectively, these data indicate the continued merit of further developing LB1 and LPD-LB1(f)2,1,3 as components of vaccines for otitis media.

FIG. 1

Silver stain of the immunogens used in this study. Lanes: 1, P5-fimbrin; 2, P6; 3, LPD; 4, LB1; 5, LB2; 6, PDm; 7, LPD-LB1(f)_2,1,3; 8, prestained molecular mass standards.

FIG. 2

Three-dimensional bar graph depicting the percentage of ears per cohort which were culture positive for NTHI in middle ear fluids over time.

FIG. 3

(A) Dynamics of middle ear colonization in CFU of NTHI per milliliter of fluids recovered from adenovirus-infected chinchilla cohorts immunized as indicated and challenged t.b. and i.n. with NTHI 86-028NP. Only four cohorts which demonstrated moderate to significantly augmented clearance plus the naive and adjuvant-only control cohorts are included. (B) NP colonization dynamics in CFU of NTHI per milliliter of lavage fluid of adenovirus-infected chinchilla cohorts immunized as indicated and challenged with NTHI 86-028NP. Only three cohorts which demonstrated moderate to significantly augmented clearance plus naive and adjuvant-only cohorts are included. The asterisk at day 0 indicates the inoculum of NTHI instilled (10⁸ CFU). Results are means and standard deviations (number of determinations were six per cohort for days 1 to 10 and five per cohort for days 14 to 35).

FIG. 4

Three-dimensional bar graph depicting the percentage of animals per cohort which were culture positive for NTHI in NP lavage fluids over time.

FIG. 5

CFU of NTHI per milliliter of recovered middle ear fluids (A) or NP lavage fluids (B) in chinchillas immunized as indicated in the figure and challenged i.n. and t.b. with NTHI 86-028NP. Results are means and standard deviations.

FIG. 6

Amino acid sequence of LPD-LB1(f)_2,1,3. Capital letters represent the LPD sequence; bold capital letters represent the sequences of the three LB1(f) peptides; underlined capital letters represent the histidine tag; lowercase letters represent linker regions. The box indicates the signal peptide.

FIG. 7

Western blot composite of the pooled chinchilla sera that were passively transferred to adenovirus-infected chinchillas prior to i.n. challenge with NTHI 86-028NP. Lanes: 1, whole NTHI 86-028NP outer membrane protein; 2, LB1; 3, LPD; 4, PDm; 5, LPD-LB1(f)_2,1,3. The serum pools used were anti-LB1 (A), anti-LPD (B), anti-PDm (C), and anti-LPD-LB1(f)_2,1,3 (D).

FIG. 8

Percentage of total abnormal ears based on otoscopy and tympanometry in the five adenovirus-compromised chinchilla cohorts which received either sterile diluent or a 1:5 dilution of anti-LB1, anti-LPD, anti-PDm, or anti-LPD-LB1(f)_2,1,3 by passive transfer prior to i.n. challenge with NTHI 86-028NP.

FIG. 9

TEM composite of NTHI 86-028NP indirectly immunogold labeled with chinchilla antisera followed by either gold-conjugated protein A or gold-conjugated goat anti-mouse Igs. NTHI was incubated with chinchilla anti-LPD (A), MAb 2C7 (B), chinchilla anti-LB1 (C), or chinchilla anti-LPD-LB1(f)_2,1,3 (D). Magnification, ca. ×65,000 (A and B) and ×54,000 (C and D).