In the present study, we have shown that the incidence of AOM was sequentially lowest in breast, followed by breast/formula and the formula fed infants. The results support other studies demonstrating that breast-feeding has a protective effect against acute and prolonged infections, including otitis media (15
). The protective effect of breast-feeding against respiratory diseases, including otitis media, has been ascribed to passively transfer maternal antibodies, mainly secretory IgA, (1
). A novel aspect of our work involved examination of another possible mechanism whereby breast-feeding might be protective against AOM – stimulation of higher serum antibody levels against a major otopathogen, NTHi. Indeed, we found that serum antibody responses to NTHi and an OMP vaccine candidate of NTHi, P6, were enhanced in breast compared to breast/formula and formula fed infants. This observation supports the notion that breast-feeding facilitates immune responses in infants and suggests that feeding status may be an important co-variate in studies that compare antibody levels among AOM groups.
Others have reported that children with AOM or NP colonization by NTHi develop an immune response to this pathogen (17
). The NP is considered a reservoir for NTHi, and NP flora becomes established during the first year of life. In group 1 of our study were some children without prior AOM at 2 and 6 months of age, yet they had an increase in serum antibody to whole NTHi and P6 suggesting that NP colonization by NTHi was an immunizing event.
We measured the serum antibody levels to a well-characterized NTHi strain. Several studies have reported that NP colonization causes children to develop antibody to homologous and heterologous NTHi strains (18
). More recent studies demonstrated that all NTHi strains share antigenically conserved OMP P6 (3
), and an antibody response to P6 may confer protection against NTHi, rendering infants less susceptible to AOM (2
). The increase in levels of specific serum antibodies to whole-cell NTHi and to P6 in our study is consistent with these prior results.
We demonstrated that children develop bactericidal antibodies to homologous NTHi strains isolated from ME fluid during AOM, similar to the findings by others (2
). Two lines of evidence suggest that IgG antibody directed against highly conserved OMP P6 likely accounts for some bactericidal activity against NTHi. There was a reduction in bactericidal activity after removal of P6 antibodies; and, P6-specific IgG antibodies and bactericidal titers correlated. Variable anti-P6 IgG responses among children with AOM caused by NTHi suggest that children also vary in their ability to mount protective responses to highly conserved OMP P6.
Specific serum antibodies are likely to protect against NP carriage and AOM provided that the antibody can reach the mucosal surface (21
). If breast-feeding does enhance the serum antibody responses to NTHi, a protective effect would occur in support of this hypothesis. In group 2b, we observed decreased NP carriage in breast compared to non-breast fed infants with AOM. Thus, serum antibodies that are transudated to the NP during an upper respiratory infection could provide protection against NP carriage by NTHi, more so in breast fed children. In the absence of inflammation, serum antibody transudation would be less and NP colonization would be impacted less. This proposal is supported by the observation that carriage of NTHi does not typically result in inflammation in the nasal passage (22
Bernstein et al (17
) reported that passively acquired serum antibody in the newborn did not play a role in the prevention of NTHi NP colonization. However, during episodes of respiratory illness, there is inflammation in the upper respiratory tract mucosa (23
) and an increase in NP flora (9
). AOM is known to be accompanied by inflammation in the NP and ME, and this facilitates the transfer of circulating IgG antibodies into the NP and ME in the peak of the inflammatory response (21
). Therefore, our observations are not inconsistent with earlier studies.
Increased naturally occurring NTHi-specific serum IgG responses may not completely prevent AOM in certain infants, as evidenced by episodes of AOM even in the breast fed infants; however, a protective role of specific antibodies might be demonstrated in future studies and vaccine-induced antibody levels may be much higher than achievable by colonization and/or AOM. Importantly, among children with medium or high frequency of AOM, the levels of serum IgG antibody to whole-cell NTHi were significantly higher in the breast fed group. Although NTHi-specific serum IgG responses did not prevent AOM in otitis-prone children, it is possible that these antibodies could facilitate faster resolution from AOM.
Others speculate the mechanism of protection against respiratory infections afforded by breast milk might be anti-idiotypic antibodies as well as T and B lymphocytes, which are transferred via milk (25
). Breast milk also contains numerous anti-inflammatory factors, nucleotides, cytokines, growth factors, macrophages, and granulocytes in the milk that might stimulate the immune system of the infant (26
). Increased responses of serum antibodies after vaccination with Haemophilus influenzae
type b (Hib) conjugate vaccines, tetanus and diphtheria toxoids, BCG, live oral poliovirus, and pneumococcal vaccines have been shown in breast fed infants (10
). Nucleotides present in breast milk have been added to many infant formulas, but were not present in the infant formulas used in 1990-1991 when our group 1 healthy and AOM children's sera were collected. Nucleotide supplementation has been shown to influence immune responses to vaccines (10
It is likely that repeated contact with NTHi on the mucosal surface could stimulate the production of systemic IgG as well as local IgA responses to this pathogen. However, the presence of specific serum IgG antibodies in infants less than 6 months of age could be due to maternal antibody passage. Specific IgG antibody usually decline by 6 months of age, and in the present study the levels of these antibodies increased from 2 months to 6 months. These findings provide evidence that we were measuring an active infant's immune response in breast fed children.
Our study has limitations. We did not confirm NP colonization by NTHi through NP cultures in group 1; therefore, we could not compare the specific antibody titers in children who were proven culture-positive vs. culture-negative for NTHi. We made an assumption that enough infants were colonized with NTHi at least once during the sampling times based on NP carriage studies that have reported colonization rates ranging to > 50% (9
). To supplement this assumption, we did examine the overall rate of NTHi NP colonization in the group 2 population and found colonization rates among all enrolled infants to be 25.3% (33 of 130) as confirmed by culture. The rate of NTHi NP colonization most likely was higher, as evidenced by the presence of NTHi in 32% (47 of 147) of our culture-negative NP samples using a sensitive multiplex PCR technique (unpublished observation).
In conclusion, our findings suggest that breast-feeding plays a significant role in modulating serum antibody levels to NTHi and OMP P6 during the first 6 months after birth. Transudation of serum antibody to NTHi and OMP P6 might protect against AOM during upper respiratory infections. This finding has implications for studies seeking a population-based serum correlate of protection against NTHi infection. The addition of nucleotides to a commercially available formula might produce the same effect as breastfeeding and deserves study.