Immunity to Haemophilus influenzae type b in young adults: correlation of bactericidal and opsonizing activity of serum with antibody to polyribosylribitol phosphate and lipooligosaccharide before and after vaccination.

Naturally acquired humoral immunity is thought to protect adults against serious infections due to Haemophilus influenzae type b (Hib). Antibody to the polyribosylribitol phosphate (PRP) capsule is generally considered protective; antibody to lipooligosaccharide (LOS) or outer membrane protein (OMP) may also play a role. Serum from 23 of 50 healthy young adults had no bactericidal effect (BE) against Hib yet opsonized these organisms for approximately 30% uptake by polymorphonuclear leukocytes. The degree of bactericidal and opsonizing activity in serum from the other 27 subjects generally correlated with the level of antibody to PRP but not to LOS or OMP. However, serum from some individuals had levels of antibody to PRP as high as 4.9 micrograms/ml without BE, and seven of 27 subjects with BE had antibody levels of less than 1 microgram/ml. After vaccination with 20 micrograms of conjugated PRP, the level of antibody to PRP was greater than 5 micrograms/ml in all 50 subjects. BE appeared in 22 of those who originally lacked it, and opsonization increased to approximately 50%.

Natural and vaccine-related immunity to Streptococcus pneumoniae.

To investigate the protective effects of pneumococcal vaccine, we assayed serum from healthy adults and from elderly bronchitics for antibody and opsonic activity against nine serotypes of S. pneumoniae. Before vaccination, there was no relation between opsonization and the level of antibody measured by RIA. Some serotypes were well opsonized in the absence of detectable antibody to capsular polysaccharide; others were not, despite modest levels of antibody. These in vitro studies did not support the concept that a certain level of antibody (e.g., greater than or equal to 250 ng of antibody nitrogen/ml) was specifically associated with the capacity to opsonize pneumococci. Nearly all postvaccination sera had increased antibody and opsonic activity against all serotypes, but the lack of correlation in any individual serum persisted. RIA showed that pre- and postvaccination levels of antibody in elderly adults with chronic lung disease were similar to those of younger adults. In elderly bronchitics, opsonizing activity for six of the nine serotypes was lower after vaccination, a result of suggesting a possible explanation for the failure of pneumococcal vaccine to be fully protective in these subjects. Elderly subjects had higher levels of antibody to phosphocholine, but when isolated, this antibody did not opsonize any of the vaccine strains of pneumococci. These results suggest that alternative strategies are needed to maximize the protective effect of pneumococcal vaccine in the population at greatest risk.

Emergence of bactericidal and opsonizing antibody to Vibrio vulnificus following bacterial infection.

Virulent isolates of Vibrio vulnificus resist the bactericidal and opsonizing effects of normal human serum, in contrast to environmental isolates, which are highly serum susceptible. Immune responses to bacteremic V. vulnificus infections in human subjects have not been characterized. Serum from a patient who survived sepsis caused by V. vulnificus had substantial bactericidal and opsonizing immunoglobulin G (IgG) for his own bloodstream isolate. Killing was mediated by the classical complement pathway, whereas opsonization was effected by either the classical or the alternative pathway. IgG that reacted strongly with 55-, 58-, and 68-kilodalton outer membrane proteins was present in the patient's convalescent-phase serum but was absent from normal human serum. These findings suggest that humoral immunity to V. vulnificus, mediated by bactericidal and opsonizing antibody, emerges during infection and may be due, in part, to IgG directed against identifiable outer membrane proteins.

Human alveolar lining material and antibacterial defenses.

To investigate the possible antibacterial properties of human alveolar lining material (ALM), we obtained ALM and pulmonary alveolar macrophages (PAM) by bronchoalveolar lavage of healthy nonsmokers. Alveolar lining material was isolated by centrifugation or micropore filtration; electron microscopy revealed lamellar bodies, and lipid analysis showed that 98% of the lipid fraction was phospholipid. No free fatty acids were detected. Streptococcus pneumoniae and non-typable Haemophilus influenzae (NTHI) died spontaneously in PBS at a mean rate of log10 0.75 and 0.95 in 90 min, respectively; the addition of ALM appeared to exert a slight protective effect, and at higher concentrations supported replication of NTHI. There was no difference in the uptake of the bacteria by PAM when ALM was present. Phagocytosed NTHI were killed rapidly and completely within 60 min by PAM with or without ALM. A greater proportion of S. aureus were killed by PAM alone than in the presence of ALM. Alveolar lining material from healthy humans thus appears to have no demonstrable effect on host defense against these bacteria. The differences between our results and those of earlier studies using ALM from rats may relate to interspecies differences in the composition of ALM.

Phagocytosis and killing of common bacterial pathogens of the lung by human alveolar macrophages.

To investigate factors that determine susceptibility of the lungs to infection with common respiratory pathogens, we studied phagocytosis and killing of nontypable Haemophilus influenzae, H. influenzae type b, Streptococcus pneumoniae types III, VI, and XIV, an unencapsulated variant of S. pneumoniae type III, and Staphylococcus aureus Cowan I, by using human alveolar macrophages obtained by bronchoalveolar lavage of healthy nonsmokers. After opsonization with 10% pooled human serum, mean uptake (+/- standard deviation) of nontypable H. influenzae (67.5% +/- 15.0%), unencapsulated S. pneumoniae type III (71.2% +/- 4.8%) and S. aureus (79.1% +/- 10.2%) was significantly greater (P less than .01) than that of H. influenzae type b (40.1% +/- 15.0%), and S. pneumoniae types III (4.4% +/- 3.1%), VI (11.8% +/- 9.6%), or XIV (8.7% +/- 7.0%). Nontypable H. influenzae was ingested after opsonization with much less pooled human serum than was H. influenzae type b, and uptake of encapsulated S. pneumoniae was not enhanced by as much as 80% pooled human serum. Intracellular killing of unencapsulated S. pneumoniae type III and nontypable H. influenzae was rapid and complete and corresponded to the degree of phagocytosis, but despite a high uptake, S. aureus were killed slowly and incompletely. The virulence of S. pneumoniae and H. influenzae as lung pathogens is thus determined jointly by encapsulation and the inadequate opsonizing effect of normal human serum, whereas that of S. aureus may be related to the organism's relative resistance to intracellular killing by alveolar macrophages.

Resistance of Vibrio vulnificus to serum bactericidal and opsonizing factors: relation to virulence in suckling mice and humans.

Vibrio vulnificus causes soft-tissue infections, gastrointestinal disease, and severe sepsis in humans. Bacterial and host factors in virulence have remained poorly defined. We found that blood culture isolates of V. vulnificus were completely resistant to the bactericidal effects of 10% normal human serum, in contrast to soft-tissue and environmental isolates that showed a mean 2.6 log10 decline during 120 min of incubation. Following opsonization by 10% normal human serum, mean uptake of blood isolates by normal human polymorphonuclear leukocytes during 20 min of incubation in vitro was 45.8% compared with 83.2% for isolates from other sites. Blood isolates were lethal for suckling mice (mean LD50, 1.3 X 10(6)) in contrast with isolates from other sites, which were less virulent (mean LD50, 1 X 10(9)); lethality correlated well with bacteremia at 6 hr. These studies show a close correlation between bacterial virulence for humans and suckling mice and suggest that resistance to the bactericidal and opsonizing effects of normal human serum may be important factors governing that virulence.

Immunoglobulin A from bronchopulmonary secretions blocks bactericidal and opsonizing effects of antibody to nontypable Haemophilus influenzae.

Patients with chronic bronchitis are colonized by and may develop acute bronchopulmonary infection due to nontypable Haemophilus influenzae (NTHI) despite the presence of bactericidal and opsonizing antibody to the infecting organism. To test the hypothesis that secretory immunoglobulin A (IgA) interferes with host defense mechanisms, we extracted secretory IgA from bronchopulmonary secretions of five patients with NTHI pneumonia. NTHI was incubated with IgA before or during incubation with each patient's own serum or normal human serum. IgA from four of these individuals blocked the bactericidal and opsonizing effects of normal human serum and/or their own serum against their own and/or other NTHI. IgA from bronchopulmonary secretions of patients not infected with NTHI or from the serum of a patient with an IgA myeloma had no such effect. Blocking appeared to result from a direct interaction between IgA and the bacteria. The presumed mechanism is an interaction with bacterial surface antigens, although it is not known whether this occurs at antigenic sites responsible for bactericidal and opsonizing activity or whether interaction with adjacent antigenic sites and subsequent steric interference is responsible. This blocking effect of IgA may be one mechanism that allows for the development of NTHI colonization or pneumonia in an individual who already has seemingly adequate antibody against the infecting organism.

Nontypable Haemophilus influenzae are unencapsulated both in vivo and in vitro.

Some investigators have suggested that nontypable Haemophilus influenzae isolated from sputum of adults with pneumonia are variant forms of typable H influenzae that have lost their capsule during passage in vitro. We examined colonies of both typable and nontypable H influenzae after they had been grown in vitro, as well as bronchopulmonary secretions from patients with pneumonia or acute, purulent tracheobronchitis due to H influenzae; electron microscopy combined with ruthenium-red staining was used to detect the presence of capsular glycocalyx. H influenzae types a, b, and e', whether grown in vitro or observed directly in bronchopulmonary secretions, had readily detectable capsular glycocalyx external to the cell membrane. In contrast, non-typable H influenzae appeared to be unencapsulated after cultivation in vitro or when directly visualized in bronchopulmonary secretions of infected patients.