Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae

Abstract

All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage DNA sequences. To ascertain if phage encoded proteins could form the basis of a gonococcal vaccine, rabbits were orally infected with S. enterica Typhimurium strain χ3987 harboring phagemid NgoΦ6 fm. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by indirect fluorescent analysis and flow cytometry. The elicited sera was able to bind to several phage proteins. The sera also had bactericidal activity. These data demonstrate that N. gonorrhoeae filamentous phage can induce antibodies with anti-gonococcal activity and that phage proteins may be a candidate for vaccine development.

Figure 1. Localization of ORF9 in cellular compartments.

An aliquot of His-tagged ORF9 protein purified by metal affinity chromatography was separated on a 15% SDS-PAGE gel and stained with Coomassie brilliant blue R250. Lane M, PageRuler prestained protein ladder; lane 1, ORF9 protein. To demonstrate the mobility of ORF9, western blot analysis (lane 2) was performed using a mouse monoclonal anti-His IgG-alkaline phosphatase conjugate antibody to detect the His-tag found on ORF9. For analysis of the subcellular localization of ORF9 protein in E. coli Top10 cells with Ngoϕ6fm, cells were fractionated, enriched by chromatography on a metal affinity column and aliquots from the outer membrane (lane 3), cytoplasm-periplasm (lane 4) and inner membrane (lane 5) separated on an SDS-PAGE gel and stained with Coomassie brilliant blue R250. For this figure, lanes M and 1 were analyzed on one gel, lane 2 on a second gel, and lane 3–5 on a third gel. Gels 2 and 3 were run simultaneously, with one gel used for Coomassie staining and the second gel used for transfer to nitrocellulose.

Figure 2. Serum IgG antibodies levels specific for N. gonorrhoeae ORF9 protein.

Rabbits were immunized via oral administration of S. enterica sv. Typhimurium χ3987 carrying pBLΦ6. Sera obtained at day 38, 66 and 87 was analyzed by quantitative dot ELISA. His-tagged ORF9 protein (5 μg/ml) was affixed to nitrocellulose filters and incubated with different dilutions of rabbit sera. Binding of anti ORF9 IgG was detected with goat anti-rabbit IgG-alkaline phosphatase conjugate. Data are presented as mean ± S.D. of two separate experiments each performed in duplicate. The intensity of the color of the each spot was expressed as the change of the spot intensity compare to the negative control where spotting of ORF9 was omitted. For each point 4 spots were analyzed. The lines represent: o – o, pre-immunized sera; ●- ●, day 38 sera; Δ- Δ, day 66 sera; and ▲-▲, day 87 sera.

Figure 3. Reactivity of rabbit’s sera obtained after immunization with S. enterica sv. Typhimurium χ3987 (NgoΦ6fm) and N. gonorrhoeae proteins.

Phage particles were separated on SDS-PAGE gels and subjected to Western blot analysis. In lanes 1 and 3, extracts were derived from broth-grown gonococci. In lanes 2 and 4, extracts were from purified phage. Lanes 1 and 2 were incubated with day 66 sera and lanes 3 and 4 were incubated with pre-immunized rabbit sera.

Figure 4. Serum IgG and IgA antibodies levels.

Sera were analyzed by quantitative dot ELISA. N. gonorrhoeae cells (2 μl of 10⁸ cells/ml) diluted in PBS were spotted onto a nitrocellulose strip and allowed to dry. Panel (A) Titers of IgG polyclonal antibodies collected on days 0, 38, 66 and 87 after immunization. The intensity of the color of the each spot was expressed as the change of the spot intensity compare to the negative control where spotting of bacteria was omitted. For each point 4 spots were analyzed. Line: ●-●, day 87, o- o, day 66, Δ- Δ day 38, □- □, day 0). Panel (B) Titers of IgA polyclonal. The intensity of the color of the each spot was expressed as the change of the spot intensity compare to the negative control where spotting of bacteria was omitted. For each point 4 spots were analyzed. Line: ●-●, day 87, o- o, day 66, Δ- Δ day 38, □- □, day 0). Panel (C) The amount of antibodies was determined by comparing the optical density of specific anti-N. gonorrhoeae antibodies bound to the spots as presented in Panel A,B to a standard curve obtained with known quantities of purified mouse IgG reference antibodies. Standard curves were prepared by quantitative spot ELISA method. The amount of protein was quantified using GeneTools GBox (Syngen) program and expressed as the intensity of spot versus concentration of IgG or IgA protein. Since the measurement range of the ELISA dot was between dilution 2000 and 4000 the final determination of IgG concentration in all sera tested was based on of spot intensity values in this range. Determination of the IgG and IgA level against N. gonorrhoeae FA1090ΔФ6789 strain was based on similar experiments as in Panel A and Panel B (data not presented). Grey rectangle; anti-N. gonorrhoeae IgG, dark grey rectangle; anti-N. gonorrhoeae IgA, open rectangle; anti FA1090ΔФ6789 IgG, light rectangle; anti- FA1090ΔФ6789 IgA.

Figure 5. Immunofluorescent staining of N. gonorrhoeae FA1090.

Bacterial cells (2 × 10⁸) were probed with rabbit sera immunized with S. enterica sv. Typhimurium χ3987 (NgoΦ6fm) obtained with day 66 sera (Panel A1) or prebleed sera (panel B1). Binding was detected by reaction with an AlexaFluor Cy3 goat anti-rabbit IgG (Invitrogen, Carlsbad, California) secondary antibody (Molecular Probes). Nomarski images (Panels A2,B2) correspond to the same fields as (A1,B1).

Figure 6. Flow cytometry analysis of antibody binding to N. gonorrhoeae.

FA1090 cells were treated with buffer (panel A) pre-immunization sera (dilution 1:500) (panel B) or immunized sera (dilution 1:500) obtained after 66 day (panel C) followed by treatment with Cy3 goat anti-rabbit IgG (Life Technologies). The bacteria were analyzed by using FACS Calibur flow cytometer. Data were analyzed with CellQuest. Shown are representative histograms from three independent experiments. The bar in the figure represents the gate used to measure binding efficiency. The number in each figure corresponds to the percentage of the population that bound antibody.

Figure 7. Bactericidal properties of elicited antibody.

N. gonorrhoeae-specific bactericidal activity of sera from immunized rabbits was detected using an antibody complement-mediated bactericidal assay. Serum samples (heat inactivated at 56 °C for 30 min.) were mixed with gonococcal cells (3 × 10⁴ CFU/ml) and incubated at 37 °C and for 15 min. Normal undiluted human serum (final concentration of 4%) was added to the mixtures as a complement source and incubated for an additional 45 mins. The number colony forming units from these mixtures containing the immunized serum were counted and compared to those from negative controls (gonococci incubated with normal rabbit serum). N. gonorrhoeae MS11; ● - ●, N. gonorrhoeae FA62: Δ – Δ, N. gonorrhoeae FA1090: ▲ - ▲.