Immunological and serological diversity of Neisseria gonorrhoeae: gonococcal serotypes and their relationship with immunotypes

Abstract

Strains of gonococcus were shown to be immunologically heterologous. Serum bactericidal activity generally correlated with induced immunity to gonococcal challenge as detected by the guinea pig subcutaneous chamber model. Sera devoid of bactericidal activity reflected the lack of cross-protection in subcutaneous chambers. Factors affecting the bactericidal assay described in this report include (i) source of complement, (ii) concentration of test antigen and complement activity, and (iii) presence of calcium and magnesium ions and bovine serum albumin in diluent. Poor correlation was observed between agglutinating activity of the immune sera and protection.Knowledge of immunological properties of gonococcal strains is essential both for the understanding of gonococcal immunity and for the development of vaccines. Immunological diversity of nine strains of Neisseria gonorrhoeae was demonstrated by cross-protection in guinea pig subcutaneous chambers (4). Since the procedure described for immunotyping is elaborate and is not suitable for large-scale screening or typing of clinical isolates, attempts were made to correlate the results of immunotyping with those obtained by experimental serotyping procedures. Our results are presented in this report.MATERIALS AND METHODS Bacterial cultures. The nine strains of Neisseria gonorrhoeae used in this study were described previously (4).Gonococcal immune sera. Antisera for each of the nine gonococcal strains were prepared in Hartley strain guinea pigs weighing approximately 300 g each. The guinea pigs were injected weekly for 11 weeks by the subcutaneous route with type 1 (Ti) living cultures containing 109 colony-forming units (CFU). Blood was obtained by cardiac puncture with a 25-gauge needle from animals under light anesthesia.Bactericidal antibody assay. The appropriate gonococcal strains were grown on GC base (GCB) medium supplemented with IsoVitaleX (Baltimore Biological Laboratories, Baltimore, Md.) overnight at 37°C in a candle jar. Bacterial growth was suspended in glass tubes (13 by 100 mm) containing Trypticase soy broth (TSB) (30 g/liter of distilled water). The suspension was adjusted to an optical density of 0.5 units with a Leitz spectrophotometer at 535 nm and was then diluted 1:100 in TSB for use in the bactericidal assay. The diluted suspension contained approximately 10" CFU per ml.The diluent used throughout the assay was phosphate-buffered saline (PBS), pH 7.2, containing 0.006% MgCl2 6H20, 0.004% CaCl2 2H20, and 1% bovine serum albumin. Guinea pig complement (Texas Biologicals, Fort Worth, Tex., lot 507) or fresh chimpanzee serum was used. Complement preparations were pretested to determine that the bacteria used were not nonspecifically killed in the complement. Complement was stored at -70°C until used.All sera to be tested, including reactive and nonreactive controls, were heated for 30 min at 56°C. Serial twofold dilutions were made by using microtiter loops to transfer 0.025 ml of each serum and the controls to microtiter U-bottom plates (Ames, E...

Characterization of the immune response in subcutaneous chambers of guinea pigs immunized with a ribosomal preparation from Neisseria gonorrhoeae

“…These data suggest that antibodies directed against gonococcal lipopolysaccharide were responsible for the bactericidal activity seen in the chamber fluids of immune animals in the present study and that this antibodycomplement-mediated bactericidal activity plays a major role in the protection of these chambers against N. gonorrhoeae infection. This conclusion is in agreement with suggestions made by other investigators using this animal model (5,6,27).…”

“…Previous work with the guinea pig subcutaneous (s.c.) chamber model has demonstrated the ability of gonococcal ribosomal preparations to elicit a protective immune response to a subsequent challenge with Neisseria gonorrhoeae (8,9). In agreement with others (1,5,6,15,27), the protective response seen in the s.c. chamber model correlated with the presence of hemagglutinating and bactericidal antibodies in the serum of immunized animals. However, the nature of the protection in the chamber fluid has not been fully investigated.…”

“…The outer membrane complex from gonococci has also been used as a protective immunogen in gonococcal infections in guinea pigs (5,6). The protection observed in these chambers can be correlated with the presence of serum bactericidal antibody (6,15,16,27); however, the antibacterial mechanisms within the chamber responsible for this protection have not been fully elucidated. Previous results in our laboratories have shown that immunization of guinea pigs with ribosomal preparations conferred protection against chamber infection with N. gonorrhoeae (8,9).…”

Role of outer envelope contamination in protection elicited by ribosomal preparations against Neisseria gonorrhoeae infection

“…The bactericidal assay employed in these studies was a modification of the method of Wong et al (25): microtiter plate wells were blanked with 25 PI of diluent (0.006% MgCl2.6H20, 0.004% CaCl2.2H20, and 1% bovine serum albumin [pH 7.2]). Heat-inactivated (56°C, 30 min) test serum (25 pl) was added to the first well and serially diluted.…”

“…Immediately after dilution, 25 ,ul of a 1/10 dilution of guinea pig sera previously tested as being negative for antigonococcal activity was added to each well as a p1l of a 107-colony-forming units per ml inoculum of gonococci and incubated at ambient temperature for 90 min. After the incubation, 10 p1 of the reaction mixture was plated on GC agar plates (Difco) containing VCN inhibitor (BBL Microbiology Systems), allowed to air dry for 20 min with the cover on, and incubated overnight at 370C in a 5% C02 atmosphere.…”