Ann Otol Rhinol Lary"l1ol 101:1992
INHIBITION OF NASOPHARYNGEAL COLONIZATION OF HEMOPHILUS INFLUENZAE BY ORAL IMMUNIZATION YUICHI KURONO, MD KOICHIRO SHIMAMURA, MD
GORO MOGI, MD OITA, JAPAN
Nontypeable Hemophilus influenzae organisms were inoculated into the nasopharynx of BALB/c mice immunized by oral administration offormalin-killed bacteria. Salivary antibodies and the colonization of H irfluenzae in the nasopharynx were investigated in order to clarify the effect of oral immunization. Salivary immunoglobulin A antibody titers against H influenzae were significantly increased by oral immunization, but salivary immunoglobulin G antibody titers were not. The bacteria inoculated into the nasopharynx were more rapidly eliminated in immunized mice than in control mice. The results suggest that oral immunization might be useful in preventing otitis media with effusion by inhibiting the colonization of the nasopharynx by pathogenic bacteria. KEY WORDS -
Hemophilus influenzae, nasopharyngeal colonization, oral immunization.
INTRODUCTION
still remains to be clarified whether nasopharyngeal bacterial colonization can be inhibited by oral immunization in vivo.
Nasopharyngeal adherence and colonization with pathogenic bacteria are known to be ofcrucial importance in the pathogenesis ofotitis media with effusion (OME).1-3 The bacteria colonizing the nasopharynx ascend into the middle ear through the eustachian tube and cause infection. Nontypeable Hemophilus influenzae (NTHi) and Streptococcus pneumoniae are frequently found in middle ear effusion and in the nasopharynx, and are considered the chief pathogens ofOME.4 Previously, we reported that the adherence of those bacterial strains to the nasopharyngeal mucosa is significantly higher in children with OME than in control children.I These findings suggest that bacterial adherence and colonization are prerequisites for the development of OME, and attempting to inhibit nasopharyngeal bacterial colonization might be effective in preventing OME. Mucociliary activity and external secretions containing antibodies and glycoproteins constitute barriers against bacterial invasion. Secretory immunoglobulin (Ig) A is the predominant immunoglobulin in external secretions and is known to be inhibitory to bacterial adherence.f The adherence of various species of Streptococcus to human buccal epithelial cells is specifically decreased by secretory IgA isolated from parotid fluid. 6 Specific secretory IgA antibody activity can, in many cases, be induced by peroral administration of antigen. 7 ,8 Murine salivary IgA antibody activity against S pneumoniae was enhanced by oral immunization with the bacterial preparation, and the adherence of S pneumoniae was reduced by pretreatment ofbacteria with the saliva.9 However, it
In the present study, we attempted to induce a specific mucosal immune response in mice by oral administration of NTHi. Nasopharyngeal colonization by NTHi following oral immunization was investigated in order to determine if it would be possible to use oral vaccination in preventing OME. MATERIALS AND METHODS
Animals. Conventional male BALB/c mice between 6 and 8 weeks of age were used. Bacteria. A strain ofNTHi isolated from the nasopharynx of a patient with OME was used for the experiment. The strain was stored in glycerol skim milk at -80°C. An aliquot of the stored sample was thawed and cultured on chocolate agar plate overnight at 37°C. The culture was transferred into tryptic soy broth containing 5% ofFildes enrichment (Difco, Detroit, Mich) and incubated for 6 hours at 37°C. The bacteria were harvested by centrifugation and suspended in phosphate-buffered saline solution (pBS, 0.01 mol/L, pH 7.4) to a concentration of approximately 109 colony-forming units per milliliter. In the preparation of the vaccine, NTHi organisms cultured on chocolate agar were collected and washed with PBS. The bacteria resuspended in PBS were killed by adding formalin to a concentration of 0.5% (vol/vol) and incubated for 24 hours at room temperature. The killing of the bacteria was confirmed by overnight culture on chocolate agar. The resulting suspension
From the Department of 0t01aryngology, Medical College of Oita, Oita, Japan. Presented at the conference on Immunologic Studies on the Ear, Oita, Japan, October 8-9,1991. REPRINTS - Goro Mogi, MD, Dept ofOto1aryngology, Medical College of Oita, I-I, Idaigaoka, Hazama-cho, Oita 879-56, Japan.
11
Kurono et al, Inhibition of Nasopharyngeal Colonization
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Fig 1. Salivary antibody activities against nontypeable Hemophilus influenzae after oral immunization. Mice were immunized daily for 14 days by oral administration of liposomes containing non typeable H influenzae and glucosaminyl-muramyl-peptide (GM) 53 (H influenzaei or GM 53 only (Control). Asterisk - p < .001.
was washed and resuspended in PBS to a concentration of approximately 109 organisms per milliliter. Vaccine. The bacterial antigen of NTHi was incorporated into liposomes and administered to mice as a vaccine. Preparation ofliposomes was accomplished by the method described by Ogawa et al. to Briefly, lecithin and cholesterol (both from Sigma, St Louis, Mo) were dissolved in chloroform and the mixture was evaporated. Aliquots of formalin-killed NTHi and adjuvant (glucosaminyl-muramyl-peptide 53, a gift from Dainippon Pharmaceutical Co, Osaka, Japan) were added, and the mixture was incubated for 30 minutes at 55°C. The contents were then sonicated for 3 minutes to obtain a homogeneous suspension of liposome vesicles. The liposomes given to control mice contained adjuvant alone. Immunization ofMice. Mice were given 0.25 mL ofliposomes containing NTHi antigen and adjuvant, or liposomes containing adjuvant alone as a control, daily for 14 days. All immunizations were given orally by using a bent animal feeding needle (Popper & Sons, New Hyde Park, NY). Samples of saliva were collected from the individual animals 24 hours after the final administration of antigen. Pilocarpine hydrochloride was injected subcutaneously (5 mg! kg) to stimulate salivary secretion. Blood was collected from the femoral artery of the mice at the time of the bacteriologic examination of the nasopharynx,
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Fig 2. Serum antibody activities against nontypeable Hemophilus influenzae after oral immunization. Mice were immunized daily for 14 days by oral administration of liposomes containing nontypeable H influenzae and glucosaminyl-muramyl-peptide (GM) 53 (H influenzae) or GM 53 only (Control). Asterisks - p < .001.
and serum was obtained by centrifugation. Saliva and serum specimens were stored at -80°C until use. Antibody Activities AgainstNTHi. The titer of antiNTHi IgA and IgG antibodies in the saliva and serum of mice was determined by enzyme-linked immunosorbent assay as previously reported'! with minor modification. One hundred microliters of formalinkilled NTHi suspension (l08 organisms per milliliter) in 0.1 mollL carbonate buffer (pH 9.6) was dispensed in each well of a polystyrene microtiter plate (Dynatech, Chantilly, Va) and adsorbed overnight at 4°C. The antigen-coated microplate was washed with PBS containing 0.05% Tween 20, and incubated with 200 !JLof 1% bovine serum albumin (Armour Pharmaceutical Co, Kankakee, TIl) for 1 hour at 37°C. After washing, l00!JL of each saliva or serum sample, appropriately diluted, was applied. The plate was incubated overnight at 4°C and washed again. Horseradish peroxidase-conjugated rabbit anti mouse IgA or IgG serum (Zymed, San Francisco, Calif), diluted 1to 1,000, was added and the plate was incubated for 1 hour at 37°C. After washing. citric acid phosphate buffer (pH 5.0) containing 0.4 mg!mL of o-phenylenediamine was added to each well. The color was allowed to develop at room temperature and the reaction was stopped with 2.5 mollL sulfuric acid. Absorbance was measured by spectrophotometry at 492 nm.
13
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Fig 3. Percentage of mice having nontypeable Hem~philus influenzae inoculated into nasopharynx after oral immunization with liposomes containing nontypeable H influenzae and glucosaminyl-muramyl-peptide (OM) 53 (H influenzae) or OM 53 only (Control).
Nasopharyngeal Inoculation ofNTHi. On day 15, after collection of saliva samples, 50 ~ of NTHi suspension (109 colony-forming units per milliliter) was inoculated into the nasopharynx ofmice through the nose by using a 26-gauge needle under anesthesia with diethyl ether. A group of five to six mice was painlessly killed on days 16, 17, 18, and 19 by decapitation following bloodletting. After the mandible of each mouse was removed, the surface of the palate and pharynx was disinfected with iodine solution to prevent bacterial contamination of the nasopharynx. The nasopharynx was opened by cutting the soft palate and was scraped with a sterile cottontipped swab. The swab was smeared and spread on chocolate agar and blood agar, respectively. Following overnight incubation at 37°C, the culture was examined. Colonies ofNTHi were identified by Gram stain and by their X and V growth factor requirements. Statistical Analysis. Student's t test was used to determine the statistical significance of the data. A difference at p < .05 was considered significant. RESULTS
Anti-NTHi Antibody Activities in Saliva and Serum. Salivary and serum anti-NTHi antibodies were enhanced by oral administration of liposomes containing NTHi and adjuvant (Figs 1 and 2). Specific anti-NTHi IgA antibodies were detected in 15 of 24 saliva samples from mice immunized with NTHi, and in 4 of23 saliva samples from control mice. Ten of 24 mice immunized with NTHi, and 7 of23 control mice were shown to have IgG antibody activity in their saliva. In the serum samples from control mice, specific IgA antibody activity was not detected, and IgG antibody activity was detected only in 2 of 23
o Day I
Day 2
Day 3
Day 4
Fig 4. Number of nontypeable Hemophilus influenzae organisms detected from nasopharynx of mice immunized with liposomes containing nontypeable H influenzae and glucosaminyl-muramyl-peptide (OM) 53 (H influenzae) or OM 53 only (Control) after nasopharyngeal inoculation of bacteria. Double asterisks - p < .05, CFU - colonyforming units.
samples. Eighteen and 22 of 24 serum samples from immunized mice were found to have specific IgA and IgG antibody activities, respectively. The mean values of salivary IgA, serum IgA, and serum IgG antibody titers were significantly greater in mice immunized with NTHi than in control mice (p < .001, respectively). There were no significant differences in the mean values of salivary IgG antibody titers between mice immunized with NTHi and control mice.
Nasopharyngeal Colonization ofNTHi. Colonization of NTHi inoculated into the nasopharynx after oral immunization is shown in Fig 3. The percentage of mice having NTHi in the nasopharynx decreased day by day after nasopharyngeal inoculation of bacteria. In the control group, NTHi was still cultured 4 days after inoculation. However, no NTHi was detected on the third day after inoculation in the group of mice immunized with NTHi. Figure 4 shows the number ofNTHi colonies on chocolate agar smeared with a nasopharyngeal swab. Although there was not much difference in the percentage of mice having NTHi in the nasopharynx 1 day after inoculation, the number of NTHi colonies was significantly smaller in mice immunized with NTHi than in control mice (p < .05). DISCUSSION
Adherence of bacteria to the surface ofthe mucosa is the initial step in colonization and infection. Although the mechanism of adherence of NTHi has not
14
Kurono et al, Inhibition ofNasopharyngeal Colonization
been demonstrated, the isolates are shown to be adherentto nasopharyngealepiith elial I ce11' s mviitrO. 5' 11 The strains are more adherent to nasopharyngeal epithelial cells from children with OME than to those from control children.' Stenfors and Raisanen 3 investigated the epithelial cells scraped from the nasopharynx of patients with OME and demonstrated that nasopharyngeal bacterial adherence is age-dependent in vivo. These findings suggest that nasopharyngeal colonization by NTHi plays an important role in the pathogenesis of OME in children. In the pre~ent study, we investigated the nasopharyngeal colonization by NTHi after the inoculation of bacteria into the nasopharynx. The methods used in this study could not offer conclusive proof as to whether NTHi isolates from the nasopharynx adhered to epithelial cells or whether they were merely harbored in the nasopharynx. However, the removal of NTHi from the nasopharynx after a short time, as shown in this study, indicates the presence of a dynamic system in the nasopharynx that prevents bacterial colonization. The system appears to consist of 1) the mucociliary function of the nasopharyngeal epithelium and 2) nasopharyngeal secretions containing specific antibodies and glycoproteins. Oral immunization is generally acknowledged to induce antigen-specific secretory IgA in external secretions.V In the present study, salivary anti-NTHi IgA antibody activity was significantly increased by oral administration of NTHi preparations. Salivary IgG antibody activity, however, was not. Although serum IgA and IgG antibody activities were also increased by oral immunization, no correlation between salivary and serum antibody activities was observed for either IgA or IgG antibodies. Ogawa et al lo reported similar responses of salivary and serum antibodies to oral immunization of Bacteroides gingivalis fimbriae. Bacterial fimbriae are known to be critical to the successful adherence ofmany pathogens to the host. Bakaletz et al l2,13 detected fimbriae in 100% of clinical isolates of NTHi and demonstrated that the antigenicity induced immunologic responses in chinchillas. Their findings suggest that
salivary IgA antibodies specific for NTHi are locally produced and might act against the fimbriae ofNTHi. The NTHi isolates inoculated into the nasopharynx were eliminated more rapidl~ in mice ~u nized with NTHi than in control truce. In a preVIOUS study, we showed that NTHi adherence to nasopharyngeal mucosal cells was inhibited by ~asoph~n geal secretions having secretory IgA ant~body ~CtlV ity against the bacteria. I I Although antl-N!Hi IgA antibody activity in nasopharyngeal secretions was not determined in the present study, specific IgA antibody activity in nasopharyngeal secretions may increase the inhibition of NTHi colonization in the nasopharynx. Since the number of NTHi colonies in the nasopharynx significantly decreased in an early phase, 1 day after inoculation, it see~~ that.the ~y~ temic immune response does not participate in elimination of bacteria from the nasopharynx. Williams and Gibbons" demonstrated that salivary secretory IgA has inhibitory activity against various species of Streptococcus, and the inhibitory activity of secretory IgA is correlated with the agglutinating activity. From these findings, it can be speculated that secretory IgA in nasopharyngeal secretions inhibits the adherence ofNTHi organisms and agglutinates them in order to enable the mucociliary function to eliminate them more easily from the nasopharynx. Recently, Yoshimura et all4 reported that intraduodenal immunization with S pneumoniae reduces the occurrenee of pneumococcal otitis media in guinea pigs. Pneumococcal colonization ofthe middle ear mucosa might also be prevented by enhanced mucosal immunity, as shown in the present study of the nasopharynx. In summary, in the present study anti-NTHi activity in saliva was enhanced by oral immunizati,on with NTHi incorporated into liposomes, and NTHI organisms inoculated into the nasopharynx were rapidly eliminated in immunized mice. The findings suggest that oral immunization activates the mucosal immunity and might be useful in preventing OME by inhibiting the colonization of the nasopharynx by pathogenic bacteria.
REFERENCES 1. Long SS, Henretig FM, Teter MJ. McCowan KL. Nasopharyngeal flora and acute otitis media. Infect Immun 1983;4: 987-91. 2, Serensen CH, Andersen LP, Tos M. Thomsen J, HolmJensen S. Nasopharyngeal bacteriology and secretory otitis media in young children. Acta Otolaryngol (Stockh) 1988;105:12631. 3. Stenfors L-E. Raisanen S. Age-dependent changes in bacterial adherence to epithelial cells of nasopharynx in vivo. Acta Otolaryngol (Stockh) 1990;110:292-9. 4. Kurono Y, Tomonaga K. Mogi G. Staphylococcus epi-
dermidis and Staphylococcus aureus in otitis media with effusion. Arch Otolaryngol Head Neck Surg 1988;114:1262-5. 5. Shimamura K, Shigemi H, Kurono Y, Mogi G. The role of bacterial adherence in otitis media with effusion, Arch Otolaryngol Head Neck Surg 1990;116:1143-6. 6. Williams RC, Gibbons RJ. Inhibition of bacterial adherence by secretory immunoglobulin A: a mechanism of antigen disposal. Science 1972;177:697-9. 7. Molinari JA, Ebersole JL, Platt D. Investigation of secretory immunoglobulin in saliva from germ free mice. Infect
Kuronoet al.Inhibition ofNasopharyngeal Colonization Immun 1974;10:1207-12. 8. Michalek SM. McGhee JR. Mesteeky J. Arnold RR. Bozzo L. Ingestion of Streptococcusmutans induces secretory IgA and carries immunity. Science 1976;192:1238-40. 9. Kurono Y. Bakaletz LO, Lim DJ. The effect of oral immunization on the adherence of Streptococcus pneumoniaein BALB/c mice. Abstracts of the Fourteenth Midwinter Research Meeting. St Petersburg Beach. Fla: Association for Research in Otolaryngology. 1991:56. 10. Ogawa T. Shimauchi H. HamadaS. Mucosal and systemic immune responses in BALB/c mice to Bacteroides gingivalis funbriae administered orally. Infect Immun 1989;57:3466-71. 11. Kurono Y. Shimamura K, Shigemi H. Mogi G. Inhibition
15
of bacterial adherence by nasopharyngeal secretions. Ann Otol Rhinol Laryngoll99I;I00:455-8. 12. Bakaletz LO. Tallan BM. Hoepf T. DeMaria TF, Birck HG.LimDJ.FrequencyoffunbriationofnontypeableHtU!mophiIus influenzae and its ability to adhere to chinchilla and human respiratory epithelium. Infect Immun 1988;56:331-5. 13. BakaletzW. TallanBM.Andrzejewski WJ.DeMariaTF. Lim DJ. Immunological responsiveness of chinchillas to outer membrane and isolated funbrial proteins of nontypeable HtU!mophilus influenzae. Infect Immun 1989;57:3226-9. 14. Yoshlmura H, WatanabeN.BundoJ. ShinodaM. MogiG. Oral vaccine therapy for pneumococcal otitis media in an animal model. Arch Otolaryngol Head Neck Surg 1991;117:889-94.
INHIBITION OF NASOPHARYNGEAL COLONIZATION OF HEMOPHILUS INFLUENZAE BY ORAL IMMUNIZATION YUICHI KURONO, MD KOICHIRO SHIMAMURA, MD
GORO MOGI, MD OITA, JAPAN
Nontypeable Hemophilus influenzae organisms were inoculated into the nasopharynx of BALB/c mice immunized by oral administration offormalin-killed bacteria. Salivary antibodies and the colonization of H irfluenzae in the nasopharynx were investigated in order to clarify the effect of oral immunization. Salivary immunoglobulin A antibody titers against H influenzae were significantly increased by oral immunization, but salivary immunoglobulin G antibody titers were not. The bacteria inoculated into the nasopharynx were more rapidly eliminated in immunized mice than in control mice. The results suggest that oral immunization might be useful in preventing otitis media with effusion by inhibiting the colonization of the nasopharynx by pathogenic bacteria. KEY WORDS -
Hemophilus influenzae, nasopharyngeal colonization, oral immunization.
INTRODUCTION
still remains to be clarified whether nasopharyngeal bacterial colonization can be inhibited by oral immunization in vivo.
Nasopharyngeal adherence and colonization with pathogenic bacteria are known to be ofcrucial importance in the pathogenesis ofotitis media with effusion (OME).1-3 The bacteria colonizing the nasopharynx ascend into the middle ear through the eustachian tube and cause infection. Nontypeable Hemophilus influenzae (NTHi) and Streptococcus pneumoniae are frequently found in middle ear effusion and in the nasopharynx, and are considered the chief pathogens ofOME.4 Previously, we reported that the adherence of those bacterial strains to the nasopharyngeal mucosa is significantly higher in children with OME than in control children.I These findings suggest that bacterial adherence and colonization are prerequisites for the development of OME, and attempting to inhibit nasopharyngeal bacterial colonization might be effective in preventing OME. Mucociliary activity and external secretions containing antibodies and glycoproteins constitute barriers against bacterial invasion. Secretory immunoglobulin (Ig) A is the predominant immunoglobulin in external secretions and is known to be inhibitory to bacterial adherence.f The adherence of various species of Streptococcus to human buccal epithelial cells is specifically decreased by secretory IgA isolated from parotid fluid. 6 Specific secretory IgA antibody activity can, in many cases, be induced by peroral administration of antigen. 7 ,8 Murine salivary IgA antibody activity against S pneumoniae was enhanced by oral immunization with the bacterial preparation, and the adherence of S pneumoniae was reduced by pretreatment ofbacteria with the saliva.9 However, it
In the present study, we attempted to induce a specific mucosal immune response in mice by oral administration of NTHi. Nasopharyngeal colonization by NTHi following oral immunization was investigated in order to determine if it would be possible to use oral vaccination in preventing OME. MATERIALS AND METHODS
Animals. Conventional male BALB/c mice between 6 and 8 weeks of age were used. Bacteria. A strain ofNTHi isolated from the nasopharynx of a patient with OME was used for the experiment. The strain was stored in glycerol skim milk at -80°C. An aliquot of the stored sample was thawed and cultured on chocolate agar plate overnight at 37°C. The culture was transferred into tryptic soy broth containing 5% ofFildes enrichment (Difco, Detroit, Mich) and incubated for 6 hours at 37°C. The bacteria were harvested by centrifugation and suspended in phosphate-buffered saline solution (pBS, 0.01 mol/L, pH 7.4) to a concentration of approximately 109 colony-forming units per milliliter. In the preparation of the vaccine, NTHi organisms cultured on chocolate agar were collected and washed with PBS. The bacteria resuspended in PBS were killed by adding formalin to a concentration of 0.5% (vol/vol) and incubated for 24 hours at room temperature. The killing of the bacteria was confirmed by overnight culture on chocolate agar. The resulting suspension
From the Department of 0t01aryngology, Medical College of Oita, Oita, Japan. Presented at the conference on Immunologic Studies on the Ear, Oita, Japan, October 8-9,1991. REPRINTS - Goro Mogi, MD, Dept ofOto1aryngology, Medical College of Oita, I-I, Idaigaoka, Hazama-cho, Oita 879-56, Japan.
11
Kurono et al, Inhibition of Nasopharyngeal Colonization
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Fig 1. Salivary antibody activities against nontypeable Hemophilus influenzae after oral immunization. Mice were immunized daily for 14 days by oral administration of liposomes containing non typeable H influenzae and glucosaminyl-muramyl-peptide (GM) 53 (H influenzaei or GM 53 only (Control). Asterisk - p < .001.
was washed and resuspended in PBS to a concentration of approximately 109 organisms per milliliter. Vaccine. The bacterial antigen of NTHi was incorporated into liposomes and administered to mice as a vaccine. Preparation ofliposomes was accomplished by the method described by Ogawa et al. to Briefly, lecithin and cholesterol (both from Sigma, St Louis, Mo) were dissolved in chloroform and the mixture was evaporated. Aliquots of formalin-killed NTHi and adjuvant (glucosaminyl-muramyl-peptide 53, a gift from Dainippon Pharmaceutical Co, Osaka, Japan) were added, and the mixture was incubated for 30 minutes at 55°C. The contents were then sonicated for 3 minutes to obtain a homogeneous suspension of liposome vesicles. The liposomes given to control mice contained adjuvant alone. Immunization ofMice. Mice were given 0.25 mL ofliposomes containing NTHi antigen and adjuvant, or liposomes containing adjuvant alone as a control, daily for 14 days. All immunizations were given orally by using a bent animal feeding needle (Popper & Sons, New Hyde Park, NY). Samples of saliva were collected from the individual animals 24 hours after the final administration of antigen. Pilocarpine hydrochloride was injected subcutaneously (5 mg! kg) to stimulate salivary secretion. Blood was collected from the femoral artery of the mice at the time of the bacteriologic examination of the nasopharynx,
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Fig 2. Serum antibody activities against nontypeable Hemophilus influenzae after oral immunization. Mice were immunized daily for 14 days by oral administration of liposomes containing nontypeable H influenzae and glucosaminyl-muramyl-peptide (GM) 53 (H influenzae) or GM 53 only (Control). Asterisks - p < .001.
and serum was obtained by centrifugation. Saliva and serum specimens were stored at -80°C until use. Antibody Activities AgainstNTHi. The titer of antiNTHi IgA and IgG antibodies in the saliva and serum of mice was determined by enzyme-linked immunosorbent assay as previously reported'! with minor modification. One hundred microliters of formalinkilled NTHi suspension (l08 organisms per milliliter) in 0.1 mollL carbonate buffer (pH 9.6) was dispensed in each well of a polystyrene microtiter plate (Dynatech, Chantilly, Va) and adsorbed overnight at 4°C. The antigen-coated microplate was washed with PBS containing 0.05% Tween 20, and incubated with 200 !JLof 1% bovine serum albumin (Armour Pharmaceutical Co, Kankakee, TIl) for 1 hour at 37°C. After washing, l00!JL of each saliva or serum sample, appropriately diluted, was applied. The plate was incubated overnight at 4°C and washed again. Horseradish peroxidase-conjugated rabbit anti mouse IgA or IgG serum (Zymed, San Francisco, Calif), diluted 1to 1,000, was added and the plate was incubated for 1 hour at 37°C. After washing. citric acid phosphate buffer (pH 5.0) containing 0.4 mg!mL of o-phenylenediamine was added to each well. The color was allowed to develop at room temperature and the reaction was stopped with 2.5 mollL sulfuric acid. Absorbance was measured by spectrophotometry at 492 nm.
13
Kurono et al, Inhibition of Nasopharyngeal Colonization % 100
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Fig 3. Percentage of mice having nontypeable Hem~philus influenzae inoculated into nasopharynx after oral immunization with liposomes containing nontypeable H influenzae and glucosaminyl-muramyl-peptide (OM) 53 (H influenzae) or OM 53 only (Control).
Nasopharyngeal Inoculation ofNTHi. On day 15, after collection of saliva samples, 50 ~ of NTHi suspension (109 colony-forming units per milliliter) was inoculated into the nasopharynx ofmice through the nose by using a 26-gauge needle under anesthesia with diethyl ether. A group of five to six mice was painlessly killed on days 16, 17, 18, and 19 by decapitation following bloodletting. After the mandible of each mouse was removed, the surface of the palate and pharynx was disinfected with iodine solution to prevent bacterial contamination of the nasopharynx. The nasopharynx was opened by cutting the soft palate and was scraped with a sterile cottontipped swab. The swab was smeared and spread on chocolate agar and blood agar, respectively. Following overnight incubation at 37°C, the culture was examined. Colonies ofNTHi were identified by Gram stain and by their X and V growth factor requirements. Statistical Analysis. Student's t test was used to determine the statistical significance of the data. A difference at p < .05 was considered significant. RESULTS
Anti-NTHi Antibody Activities in Saliva and Serum. Salivary and serum anti-NTHi antibodies were enhanced by oral administration of liposomes containing NTHi and adjuvant (Figs 1 and 2). Specific anti-NTHi IgA antibodies were detected in 15 of 24 saliva samples from mice immunized with NTHi, and in 4 of23 saliva samples from control mice. Ten of 24 mice immunized with NTHi, and 7 of23 control mice were shown to have IgG antibody activity in their saliva. In the serum samples from control mice, specific IgA antibody activity was not detected, and IgG antibody activity was detected only in 2 of 23
o Day I
Day 2
Day 3
Day 4
Fig 4. Number of nontypeable Hemophilus influenzae organisms detected from nasopharynx of mice immunized with liposomes containing nontypeable H influenzae and glucosaminyl-muramyl-peptide (OM) 53 (H influenzae) or OM 53 only (Control) after nasopharyngeal inoculation of bacteria. Double asterisks - p < .05, CFU - colonyforming units.
samples. Eighteen and 22 of 24 serum samples from immunized mice were found to have specific IgA and IgG antibody activities, respectively. The mean values of salivary IgA, serum IgA, and serum IgG antibody titers were significantly greater in mice immunized with NTHi than in control mice (p < .001, respectively). There were no significant differences in the mean values of salivary IgG antibody titers between mice immunized with NTHi and control mice.
Nasopharyngeal Colonization ofNTHi. Colonization of NTHi inoculated into the nasopharynx after oral immunization is shown in Fig 3. The percentage of mice having NTHi in the nasopharynx decreased day by day after nasopharyngeal inoculation of bacteria. In the control group, NTHi was still cultured 4 days after inoculation. However, no NTHi was detected on the third day after inoculation in the group of mice immunized with NTHi. Figure 4 shows the number ofNTHi colonies on chocolate agar smeared with a nasopharyngeal swab. Although there was not much difference in the percentage of mice having NTHi in the nasopharynx 1 day after inoculation, the number of NTHi colonies was significantly smaller in mice immunized with NTHi than in control mice (p < .05). DISCUSSION
Adherence of bacteria to the surface ofthe mucosa is the initial step in colonization and infection. Although the mechanism of adherence of NTHi has not
14
Kurono et al, Inhibition ofNasopharyngeal Colonization
been demonstrated, the isolates are shown to be adherentto nasopharyngealepiith elial I ce11' s mviitrO. 5' 11 The strains are more adherent to nasopharyngeal epithelial cells from children with OME than to those from control children.' Stenfors and Raisanen 3 investigated the epithelial cells scraped from the nasopharynx of patients with OME and demonstrated that nasopharyngeal bacterial adherence is age-dependent in vivo. These findings suggest that nasopharyngeal colonization by NTHi plays an important role in the pathogenesis of OME in children. In the pre~ent study, we investigated the nasopharyngeal colonization by NTHi after the inoculation of bacteria into the nasopharynx. The methods used in this study could not offer conclusive proof as to whether NTHi isolates from the nasopharynx adhered to epithelial cells or whether they were merely harbored in the nasopharynx. However, the removal of NTHi from the nasopharynx after a short time, as shown in this study, indicates the presence of a dynamic system in the nasopharynx that prevents bacterial colonization. The system appears to consist of 1) the mucociliary function of the nasopharyngeal epithelium and 2) nasopharyngeal secretions containing specific antibodies and glycoproteins. Oral immunization is generally acknowledged to induce antigen-specific secretory IgA in external secretions.V In the present study, salivary anti-NTHi IgA antibody activity was significantly increased by oral administration of NTHi preparations. Salivary IgG antibody activity, however, was not. Although serum IgA and IgG antibody activities were also increased by oral immunization, no correlation between salivary and serum antibody activities was observed for either IgA or IgG antibodies. Ogawa et al lo reported similar responses of salivary and serum antibodies to oral immunization of Bacteroides gingivalis fimbriae. Bacterial fimbriae are known to be critical to the successful adherence ofmany pathogens to the host. Bakaletz et al l2,13 detected fimbriae in 100% of clinical isolates of NTHi and demonstrated that the antigenicity induced immunologic responses in chinchillas. Their findings suggest that
salivary IgA antibodies specific for NTHi are locally produced and might act against the fimbriae ofNTHi. The NTHi isolates inoculated into the nasopharynx were eliminated more rapidl~ in mice ~u nized with NTHi than in control truce. In a preVIOUS study, we showed that NTHi adherence to nasopharyngeal mucosal cells was inhibited by ~asoph~n geal secretions having secretory IgA ant~body ~CtlV ity against the bacteria. I I Although antl-N!Hi IgA antibody activity in nasopharyngeal secretions was not determined in the present study, specific IgA antibody activity in nasopharyngeal secretions may increase the inhibition of NTHi colonization in the nasopharynx. Since the number of NTHi colonies in the nasopharynx significantly decreased in an early phase, 1 day after inoculation, it see~~ that.the ~y~ temic immune response does not participate in elimination of bacteria from the nasopharynx. Williams and Gibbons" demonstrated that salivary secretory IgA has inhibitory activity against various species of Streptococcus, and the inhibitory activity of secretory IgA is correlated with the agglutinating activity. From these findings, it can be speculated that secretory IgA in nasopharyngeal secretions inhibits the adherence ofNTHi organisms and agglutinates them in order to enable the mucociliary function to eliminate them more easily from the nasopharynx. Recently, Yoshimura et all4 reported that intraduodenal immunization with S pneumoniae reduces the occurrenee of pneumococcal otitis media in guinea pigs. Pneumococcal colonization ofthe middle ear mucosa might also be prevented by enhanced mucosal immunity, as shown in the present study of the nasopharynx. In summary, in the present study anti-NTHi activity in saliva was enhanced by oral immunizati,on with NTHi incorporated into liposomes, and NTHI organisms inoculated into the nasopharynx were rapidly eliminated in immunized mice. The findings suggest that oral immunization activates the mucosal immunity and might be useful in preventing OME by inhibiting the colonization of the nasopharynx by pathogenic bacteria.
REFERENCES 1. Long SS, Henretig FM, Teter MJ. McCowan KL. Nasopharyngeal flora and acute otitis media. Infect Immun 1983;4: 987-91. 2, Serensen CH, Andersen LP, Tos M. Thomsen J, HolmJensen S. Nasopharyngeal bacteriology and secretory otitis media in young children. Acta Otolaryngol (Stockh) 1988;105:12631. 3. Stenfors L-E. Raisanen S. Age-dependent changes in bacterial adherence to epithelial cells of nasopharynx in vivo. Acta Otolaryngol (Stockh) 1990;110:292-9. 4. Kurono Y, Tomonaga K. Mogi G. Staphylococcus epi-
dermidis and Staphylococcus aureus in otitis media with effusion. Arch Otolaryngol Head Neck Surg 1988;114:1262-5. 5. Shimamura K, Shigemi H, Kurono Y, Mogi G. The role of bacterial adherence in otitis media with effusion, Arch Otolaryngol Head Neck Surg 1990;116:1143-6. 6. Williams RC, Gibbons RJ. Inhibition of bacterial adherence by secretory immunoglobulin A: a mechanism of antigen disposal. Science 1972;177:697-9. 7. Molinari JA, Ebersole JL, Platt D. Investigation of secretory immunoglobulin in saliva from germ free mice. Infect
Kuronoet al.Inhibition ofNasopharyngeal Colonization Immun 1974;10:1207-12. 8. Michalek SM. McGhee JR. Mesteeky J. Arnold RR. Bozzo L. Ingestion of Streptococcusmutans induces secretory IgA and carries immunity. Science 1976;192:1238-40. 9. Kurono Y. Bakaletz LO, Lim DJ. The effect of oral immunization on the adherence of Streptococcus pneumoniaein BALB/c mice. Abstracts of the Fourteenth Midwinter Research Meeting. St Petersburg Beach. Fla: Association for Research in Otolaryngology. 1991:56. 10. Ogawa T. Shimauchi H. HamadaS. Mucosal and systemic immune responses in BALB/c mice to Bacteroides gingivalis funbriae administered orally. Infect Immun 1989;57:3466-71. 11. Kurono Y. Shimamura K, Shigemi H. Mogi G. Inhibition
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of bacterial adherence by nasopharyngeal secretions. Ann Otol Rhinol Laryngoll99I;I00:455-8. 12. Bakaletz LO. Tallan BM. Hoepf T. DeMaria TF, Birck HG.LimDJ.FrequencyoffunbriationofnontypeableHtU!mophiIus influenzae and its ability to adhere to chinchilla and human respiratory epithelium. Infect Immun 1988;56:331-5. 13. BakaletzW. TallanBM.Andrzejewski WJ.DeMariaTF. Lim DJ. Immunological responsiveness of chinchillas to outer membrane and isolated funbrial proteins of nontypeable HtU!mophilus influenzae. Infect Immun 1989;57:3226-9. 14. Yoshlmura H, WatanabeN.BundoJ. ShinodaM. MogiG. Oral vaccine therapy for pneumococcal otitis media in an animal model. Arch Otolaryngol Head Neck Surg 1991;117:889-94.
