Mucosal immunization against respiratory bacterial pathogens

Bacterial respiratory diseases remain a major cause of morbidity and mortality throughout the world. The young and the elderly are particularly susceptible to the pathogens that cause these diseases. Therapeutic approaches remain dependent upon antibiotics contributing to the persistent increases in antibiotic resistance. The main causes of respiratory disease discussed in this review are Mycobacterium tuberculosis, Corynebacterium diphtheriae, Bordatella pertussis, Streptococcus pneumoniae, non-typeable Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa. All these organisms initiate disease at the mucosal surface of the respiratory tract and thus the efficacy of the host's response to infection needs to be optimal at this site. Vaccines available for diseases caused by many of these pathogens have limitations in accessibility or efficacy, highlighting the need for improvements in approaches and products. The most significant challenges in both therapy and prevention of disease induced by bacteria in the respiratory tract remain the development of non-injectable vaccines and delivery systems/immunization regimens that improve mucosal immunity.     

Specific immune response in the human respiratory tract following oral immunization with live typhoid vaccine

Specific antibody responses in the lower respiratory tract of human subjects to orally administered Salmonella typhi Ty21a are reported. These responses, predominantly of the immunoglobulin G class, were determined to be a transudate from serum. These results were supported by the similarity in responses to parenteral administration of heat-killed typhoid vaccine. Specific immunoglobulin A antibody was a poor contributor to the respiratory antibody response to either vaccine.     

Immune responsiveness and oral immunization.

The effects on the immune response of daily feeding of 30 mg of human serum albumin to rats have been studied. Feeding for periods of 17-20 days consistently resulted in a specific systemic hyporesponsiveness evident on subsequent parenteral immunogen challenge. Local secretory sites such as the major salivary glands were not made hyporesponsive as evidenced by the salivary antibody titres and the enumeration of glandular plaque-forming cells. The levels and classes of antibodies present in the secretions and sera were identified by passive haemagglutination, and a sensitive red-cell-linked antigen-antiglobulin reaction. By the use of radioimmunoassay and haemagglutination inhibition assays it was possible to quantitate the amount of antigen appearing in the circulation at varying times after feeding. It was shown in the use of oesophagectomised rats that absorption of small amounts of intact protein occurs from undefined sites in the oral cavity. Attempts were made to transfer the specific systemic hyporesponsiveness to syngeneic animals using spleen cell and serum transfers.     

Cell-mediated immunity after oral immunization with ribonucleic acid-protein fractions of Vibrio cholerae L-form lysates.

Oral administration of a single dose of ribonucleic acid-protein fraction of lysates of Vibrio cholerae subtype Ogawa L-forms induced an increase in cell-mediated immunity in rabbits. This was demonstrated by an increase in leukocyte migration inhibition in peripheral blood leukocytes, in macrophages migration inhibition, and in microbicidal activity against Listeria monocytogenes in peritoneal macrophages obtained from orally immunized rabbits. Increased cell-mediated immunity was induced mainly with V. cholerae Ogawa and ribonucleic acid-protein fractions. It appeared in 20 days and persisted for at least 90 days.     

Decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection

Virus respiratory infections often precede bacterial pneumonia in healthy individuals. In order to determine the potential role of respiratory syncytial virus (RSV) in bacterial secondary infections, a mouse sequential pulmonary infection model was developed. Mice were exposed to RSV then challenged with Streptococcus pneumoniae (StPn). Exposure of BALB/c mice to 10(6)-10(7) plaque forming units (pfu) of virus of RSV significantly decreased StPn clearance 1-7 days following RSV exposure. This finding was not restricted to StPn alone: exposure to RSV followed by Staphylococcus aureus (SA) or Pseudomonas aeruginosa(PA) resulted in similar decreases in bacterial clearance. Both bronchoalveolar lavage (BAL) cell counts and pulmonary histopathology demonstrated that RSV-StPn exposed mice had increased lung cellular inflammation compared to mice receiving StPn or RSV alone. The effect of RSV infection on bacterial clearance was dependent on the mouse genetic background: C57BL/6J mice (relatively resistant to RSV infection) demonstrated a modest change in StPn clearance following RSV exposure, whereas FVBN/J mice (similar to the BALB/cJ mice in RSV susceptibility) demonstrated a similar degree of RSV-associated decrease in StPn clearance 7 days following RSV exposure. Neutrophils from the RSV-StPn sequentially exposed BALB/cJ mice were functionally altered-produced greater levels of peroxide production but less myeloperoxidase (MPO) compared to mice receiving StPn alone. These data demonstrate that RSV infection decreases bacterial clearance, potentially predisposing to secondary bacterial pneumonia despite increased lung cellular inflammation, and suggest that functional changes occur in the recruited neutrophils that may contribute to the decreased bacterial clearance.     

Age-dependent antibody response in mice and humans following oral influenza immunization

In order to compare the antibody response in serum and secretions from healthy young subjects and the elderly (>60 years), volunteers were immunized with the commercial inactivated influenza virus vaccine, by the usual (parenteral) route or orally. Also, young and old mice (mean age, 20 months) were orally immunized with live influenza virus. The older mice responded with a very slight rise in their serum and respiratory tract antibody levels compared with the young mice but showed no diminution in protection against lethal viral challenge. Elderly volunteers showed only slight serum antibody responses after parenteral immunization compared with the young. Neither group demonstrated a rise in serum antibody following oral immunization. With respect to the secretory IgA (SIgA) antibody response, certain differences were noted between the young and the elderly: the preimmunization levels of antibody to influenza virus were significantly greater in nasal secretions and saliva in the elderly as compared to the young volunteers, and the salivary antibody response was diminished in the elderly. This lack of a salivary antibody response in the elderly was explicable by the inverse relationship between the preimmunization SIgA antibody titers and the response to immunization. Oral immunization led to no more side effects than observed in the placebo control group.     

Specific immune response in the respiratory tract after administration of an oral polyvalent bacterial vaccine.

An oral killed polyvalent bacterial vaccine was assessed in a double-blind trial involving healthy volunteers. Three courses of oral vaccine were given over a 2-month period; each course contained 10(10) Haemophilus influenzae and 7 X 10(9) Staphylococcus aureus organisms. Immunity was assessed by monitoring antibody in saliva and serum over a 3-month period. No evidence of a nonspecific effect on immune parameters (immunoglobulin levels and Escherichia coli antibody) was detected in saliva or serum. An increase in H. influenzae antibody in saliva was detected in 55% of subjects receiving the vaccine compared with 6.7% of the placebo group. Antibody was associated with immunoglobulin A (IgA), IgG, and IgM, but the greatest increases over preimmunization levels were detected in the IgA class. No increase in serum antibody levels was detected. Subjects with higher preimmunization levels of salivary antibody to H. influenzae were less likely to respond to the oral bacterial vaccine. No increase in S. aureus antibody was detected in saliva or serum.     

Effect of oral immunization with Pseudomonas aeruginosa on the development of specific antibacterial immunity in the lungs.

Oral administration of Pseudomonas aeruginosa to rats elicited a systemic and mucosal antibody response in the intestinal and respiratory tracts. The antibody response did not influence the course of the disease when immunized animals were subsequently challenged by the introduction of viable bacteria into the lungs.     

Intranasal immunization with a colloid-formulated bacterial extract induces an acute inflammatory response in the lungs and elicits specific immune responses

Nonspecific stimulation of lung defenses by repeated oral administration of immunomodulators, such as bacterial extracts, has shown potential for the prevention of respiratory tract infections. Here, we show that intranasal (i.n.) immunization with a bacterial extract formulated as a colloid induces an acute inflammatory response in the lungs characterized by increased production of CCL and CXCL chemokines and a major influx of dendritic cells (DCs) and neutrophils, with a higher proportion of DCs showing an activated phenotype (high CD80/CD86 expression). Cytokine levels measured in bronchoalveolar-lavage samples showed a small increase in the production of tumor necrosis factor alpha and similar levels of the other cytokines measured (interleukin 10 [IL-10], IL-12, and gamma interferon [IFN-gamma]) in immunized mice compared with control mice. However, the recall response of primed animals after antigenic challenge induced increased expression of IL-12 and IFN-gamma mRNAs in lung homogenates. Overall, all these effects were not due to the lipopolysaccharide content in the bacterial extract. Furthermore, we found that three i.n. doses administered 2 to 3 weeks apart were enough to elicit long-lasting specific serum immunoglobulin G (IgG) and secretory IgA antibody responses. Assessment of IgG subclasses showed a balanced pattern of IgG1-IgG2a responses. The serum total IgE concentrations were also elevated in immunized mice 2 weeks after the third dose, but they significantly decreased soon afterwards. Our results suggest that simple formulations of bacterial extracts administered i.n. are highly immunogenic, eliciting local and systemic immune responses, and may serve as the basis for cost-effective immunotherapies for the prevention and treatment of respiratory infections.     

Immune response in oral and rectal immunization by the attenuated strain of salmonella carrying the HIV DNA-vaccine

The recombinant strain of Salmonella typhimurium SL7207/pBMC-env, carrying a plasmid containing the gene of protein HIV-1 gp-160, was obtained under the monitoring by CMV-promoter. The above strain was used in the rectal and oral immunization of BALB/c mice. HIV-specific antibodies were detected in serum after a one-time immunization; such antibodies were able to inhibit the viral replication in vitro. Furthermore, the shaping-up of the specific cytotoxic and of proliferative responses was registered. Finally, the rectal immunization by cells of the Salmonella recombinant strain can be regarded as a promising delivery system of DNA-vaccine (pBMC-env), and it is more effective versus the oral immunization variant.     

Prevalence of bacterial respiratory pathogens in the nasopharynx in breast-fed versus formula-fed infants.

In several studies, breast-feeding has been associated with decreased frequency or duration of otitis media episodes. If a causal relationship exists, the mechanism of protection of breast-feeding has not been established. We hypothesized that infants who are breast-fed, compared with infants who are formula-fed, have a lower prevalence of nasopharyngeal colonization with the bacterial respiratory pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes) commonly isolated from the middle ear effusions of children with acute otitis media. In two private pediatric practices, we obtained specimens from the nasopharynx for culture from 211 infants at 1 month of age and from 173 of these infants at 2 months of age. A swab was left in place in the nasopharynx for 45 s and was then immediately transferred onto appropriate culture media. Exclusively breast-fed (n = 84) and exclusively formula-fed (n = 76) infants were similar regarding the number of persons in the household, the number of children in the household, the number of siblings in day care, and the proportion with a recent upper respiratory tract infection. The two groups did not differ significantly in the proportions found to have one or more respiratory pathogens at 1 month of age (10.7 versus 18.4%; P = 0.12) or 2 months of age (34.8 versus 35.1%; P = 0.57). We conclude that during the first 2 months after birth, the exclusive receipt of breast milk appears not to substantially influence the prevalence of nasopharyngeal colonization with common bacterial respiratory pathogens.     

Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis.

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.     

Protective secretory immunoglobulin A antibodies in humans following oral immunization with Streptococcus mutans.

Ingestion of a vaccine containing killed Streptococcus mutans, originally isolated from each volunteer, daily for 10 consecutive days induced increased levels of specific secretory immunoglobulin A (sIgA) antibodies to S. mutans cells and two cell surface proteins, glucosyltransferase and surface antigen I/II, in parotid saliva and tears of four healthy males and in parotid saliva, tears, colostrum, and milk of a pregnant woman. In addition, these antibodies inhibited glucosyltransferase activity. Both IgA1 and IgA2 antibodies were induced. The levels of IgA antibodies in all secretions remained significantly above preimmunization levels for more than 50 days after oral administration of antigen. A second series of immunizations for 7 consecutive days resulted in even higher levels of sIgA antibodies, which peaked earlier and persisted longer than those observed after the primary immunizations. No increase in levels of antibodies in serum were detected in any subject. Antibodies reactive with human heart and kidney antigens could not be detected in saliva, tears, colostrum, milk, or serum samples collected at any time during the immunization regimen. The numbers of viable S. mutans organisms in dental plaque and whole saliva decreased after each series of immunizations, which correlated with increased levels of IgA antibodies in saliva, suggesting that IgA antibodies in saliva were responsible for the reduced adherence of this bacterium. These results indicate that ingested S. mutans antigen induces secretion of specific IgA1 and IgA2 antibodies in saliva, tears, colostrum, and milk, providing further evidence for the existence of a common mucosal immune system.     

Phagocytosis of bacterial pathogens: implications in the host response

Phagocytosis of bacterial pathogens is at the heart of the pathogenesis of infections. Pathogens have evolved a large array of strategies to escape the deleterious effect of phagocytosis by professional phagocytes among which avoiding phagocytosis, killing the phagocytes or surviving inside them are the most 'popular' solutions. Bacterial pathogens are also using induction of phagocytic entry into non-professional phagocytic cells, such as epithelial cells, as a strategy of survival and multiplication. We have taken enteroinvasive micro-organisms such as Yersinia, Shigella and Salmonella as a paradigm of the significance of phagocytosis/antiphagocytosis in the development of an infection and on the elicitation of the host response.     

Immune response of the female rat genital tract after oral and local immunization with keyhole limpet hemocyanin conjugated to the cholera toxin B subunit.

The immune response of the female rat genital tract was evaluated with Lewis rats given primary and secondary immunizations with keyhole limpet hemocyanin (KLH) alone or coupled to the cholera toxin (CT) B subunit (CTB) by the oral or intravaginal-uterine route or a combination of routes. CT (2 to 5 micrograms) was administered as an adjuvant with the KLH-CTB conjugate. While a significant mucosal immunoglobulin A (IgA) response was induced by KLH, there were no significant differences among the immunized groups in the levels of IgA antibodies in salivary gland, gut, vaginal, and uterine secretions, with the exception that rats immunized only orally with the KLH-CTB conjugate lacked a detectable vaginal response. Levels of IgA antibodies to CT, however, were significantly increased in genital tract secretions of rats immunized locally versus orally with the KLH-CTB conjugate. Antibody activity of the IgG isotype against both KLH and CT was significantly elevated in genital tract secretions of rats immunized with KLH-CTB by the oral or intravaginal-uterine route and given genital tract boosters, in comparison with the results for the other groups. IgM antibody titers were generally negligible in the different secretions. An enzyme-linked spot-forming assay revealed IgA and IgG antibody-secreting cells in salivary gland and uterine tissues. A highly significant correlation between the numbers of antibody-secreting cells and antibody titers existed for uterine IgG but not IgA responses to KLH among the different groups of rats. In conclusion, a vigorous local immune response was induced after immunization of the female rat reproductive tract alone or in combination with peroral challenge with the KLH-CTB conjugate.     

Innate immune responses of epithelial cells following infection with bacterial pathogens

The ability to discriminate between pathogenic and non-pathogenic bacteria is extremely important for epithelial cells lining mucosal surfaces and is particularly so in colonic epithelial cells. Accumulating evidence suggests that bacterial recognition systems used by epithelial cells are very different from those in cells of the myeloid lineage and are likely to have developed to maintain mucosal surfaces in a state of homeostasis with the normal microbial flora. Bacterial invasion of epithelial cells or breach of the epithelial barrier provides a signal to epithelial cells to initiate inflammatory responses, which are key events for the clearance of the infecting microbe. Therefore, elucidation of the mechanisms by which epithelial cells recognize bacteria and bacterial products, and of the nature of the innate immune responses that are triggered by these factors are important for our understanding of both the immunology of mucosal surfaces and bacterial pathogenesis.     

Decreased antibody response following immunization with enantiomorphic synthetic polypeptides

The ability to decrease the antibody response to poly(Glu52Lys33Tyr15) in the DA and PVG strains of rats by prior immunization with its D-amino acid enantiomorph poly(DGlu48DLys38DTyr14) was tested as part of a continuing investigation into the role of optical isomerism in the ability of an antigen to induce an antibody response. The results showed that prior immunization with the D-amino acid polymer decreased the immune response to the isomeric L-polypeptide in both strains.     

Immune response to tumor antigens in a patient with colorectal cancer after immunization with anti-idiotype antibody.

An active vaccination protocol was performed on one patient with colon carcinoma as a pilot to a prospective randomized double-blind clinical trial with the vaccine SDZ SCV 106. This vaccine is an anti-idiotype goat antibody to the monoclonal antibody 17-1A, which is directed against the tumor antigen 17-1A. To study the effect of the therapy on the immune reactivity, several tests were performed to detect anti-tumor antibodies in the serum as well as in eluates of metastatic tissue. Furthermore metastases removed from the lung were examined by immunohistochemistry. The results suggest that the humoral and cellular immune reactivity against the tumor are enhanced.     

Incidence of bacterial respiratory pathogens and their susceptibility to common antibacterial agents.

Although most respiratory tract infections are caused by viruses, bacterial pathogens are responsible for higher morbidity and mortality. Because virtually nothing is known about the etiology of bacterial respiratory pathogens in Saudi Arabia, this study examined the incidence of these organisms in 5426 patients over a 1-year period. Of the bacterial pathogens isolated from 904 patients, the most common organism was Hemophilus influenzae (31%), followed by pneumococci (22%), Pseudomonas aeruginosa (16%), and others (31%). Because the first two organisms accounted for more than 50% of isolates, their susceptibility to commonly used antibiotics was also reviewed. The results are presented here.     

Generation of gamma interferon responses in murine Peyer's patches following oral immunization.

To date, oral immunizations have been shown to generate only Th2 responses in murine Peyer's patches (PP), raising the possibility that T cells present in PP may be capable of mounting only Th2 responses or that the microenvironment of PP does not favor the generation of Th1 cells. However, it is also possible that antigens that can generate Th1 responses have not yet been used for oral immunizations. This study shows that T cells in PP of mice immunized orally with live Salmonella typhimurium secrete large amounts of gamma interferon (IFN-gamma) when they are stimulated with bacterial sonicate in vitro. Moreover, oral challenge of mice with live bacteria 4 months after immunization elicits a secondary IFN-gamma response in PP and mesenteric lymph nodes. Parenteral immunization does not generate an IFN-gamma T-cell response in PP, and parenteral challenge of orally immunized mice does not elicit a secondary response in PP. However, oral challenge of intraperitoneally immunized mice elicits a secondary IFN-gamma response in PP and mesenteric lymph nodes, and intraperitoneal challenge of orally immunized mice elicits a secondary response in the spleen. The data suggest that memory T cells recirculate between mucosal and nonmucosal compartments and that they may be recruited to the site of antigenic challenge.