Protection against nontypeable Haemophilus influenzae challenges by mucosal vaccination with a detoxified lipooligosaccharide conjugate in two chinchilla models

Abstract

Otitis media (OM) can occur following outset of upper respiratory tract infections. Inhibition of bacterial colonization in nasopharynx (NP) by mucosal vaccination may prevent OM by reducing bacterial invasion of the middle ears (MEs). In this study, 80 chinchillas were intranasally (i.n.) immunized with a detoxified lipooligosaccharide (dLOS)-tetanus toxoid conjugate vaccine of nontypeable Haemophilus influenzae (NTHi) mixed with cholera toxin (CT) or CT alone. All vaccinated animals responded with elevated levels of mucosal and serum anti-LOS antibodies. Two weeks after the last immunization, 40 chinchillas were challenged i.n. with NTHi to evaluate NP colonization and ME infection while the rest of the animals were challenged transbullarly (T.B.) to examine the development of OM. Compared to the control group, the vaccination inhibited not only bacterial colonization in NP and transmission to MEs in the i.n. challenge group but also bacterial colonization in NP and transmission to unchallenged ears in the T.B. challenge group. Though no difference was found in the challenged ears of either group right after the T.B. challenge, an early clearance of NTHi from NP and unchallenged ears as well as less severity of OM in the unchallenged ears were observed in vaccinated animals. Current results along with our previous data indicate that mucosal vaccination is capable of inhibiting NTHi NP colonization and preventing OM occurrence in chinchillas; the i.n. challenge model is preferable for testing the mucosal vaccines while the T.B. challenge model is superior for testing the systemic vaccines.

Fig. 1

Binding activities of mucosal antibodies elicited by dLOS-TT plus CT or CT alone to NTHi strains in the whole cell ELISA. Mucosal IgA and IgG (nasal lavages) bound to homologous strain 9274 and heterologous strains 1479, 3198, 5657 and 7502 but not to 2019. *: p <0.05; **: p <0.01.

Fig. 2

Time courses of bacterial counts (log 10) recovered from chinchilla lavage samples of nasopharynx (A) and middle ears (B) after an intranasal challenge; lavage samples of nasopharynx (C) and middle ears (D, E) after a transbullar challenge. Four chinchillas from vaccine or CT group were used at each time point and a total of 80 animals used for the entire course. Each CFU symbol represents an individual chinchilla. The ME counts (B) were bacterial CFUs from both ears of each animal. The bars indicated GM of each group. Chinchillas with culture-negative in bacterial counts were shown under the detecting level lines (CFU < 100/ml in the nasal lavages and CFU< 10/ml in the ME lavages). *: p <0.05; **: p <0.01.

Fig. 3

Indexes of otoscopic examination of chinchillas from different groups up to 28 days post transbulla challenge. The curve on each day is the mean index of each group, rated on a scale of 0 (normal ear) to 4 (severe otitis media with effusions), as determined by otoscopy (A, challenged middle ear; B, unchallenged middle ear). The mean index from both immunization groups, the dLOS-TT and the CT group, was obtained post challenge as follows: 20 chinchillas up to day 3, 16 up to day 7, 12 up to day 14, 8 up to day 21 and 4 up to day 28, were scored for each group, respectively.

Fig. 4

Tympanic membrane (TM) changes of a representative pair of chinchillas from a transbullar challenge. A double-blinded otoscopic examination was performed daily and the results were expressed as indexes (I: 0~4). Before challenge (day 0), all middle ears were rated as 0 for normal conditions. Overall, the vaccinated chinchillas showed less pathological TM changes in the challenged ears on days 21 and 28, and in the unchallenged ears during the entire course when compared with those of the control chinchillas.