JP2021504478A - Combination therapy and prevention for reproductive tract infections - Google Patents

Abstract

Provided are methods for treating and reducing the recurrence of reproductive duct infections such as gonococcal infection. The method comprises topical administration of IL-12 incorporated into a polymeric microsphere. Also provided is a method for reducing the incidence of gonococcal-induced germline infections by administration of gonococcal-derived outer membrane vesicle preparations and IL-12 incorporated into polymeric microspheres. [Selection diagram] None

Description

Cross-reference of related applications

This application claims the priority of U.S. Patent Application No. 16 / 138,526 filed on September 21, 2018, which is a partial continuation of U.S. Patent Application No. 15 / 824,700 filed on November 28, 2017. However, their respective disclosures are incorporated herein by reference.

The present invention was completed with government support under grant AI074791 granted by the National Institutes of Health. The government has certain rights to the invention

The present invention relates to compositions comprising outer membrane vesicles derived from IL-12 and Neisseria gonorrhoeae, as well as methods of using such compositions for the treatment of reproductive tract infections.

Gonorrhea-induced genital (genital) infections cause gonorrhea, the second most common infection reported in the United States, affecting more than 300,000 people annually, but the actual incidence is that number. It is believed to be at least twice as much as. The global incidence of gonorrhea is estimated to exceed 100 million cases annually. Women are burdened with infection because untreated gonorrhea ascends to the upper genital tract, causing pelvic inflammatory disease and fallopian tube scarring, leading to the risk of infertility and potentially life-threatening ectopic pregnancy. .. However, the majority of infected women (various, up to 50% or more) are asymptomatic, which increases the risk of spreading the infection to sexual contacts. In contrast, men usually become aware of the infection within a few days and are urged to seek treatment. Newborns can infect the eye as a result of passing through the infected birth canal, leading to blindness if left untreated. Untreated gonorrhea is also known to increase the risk of acquiring and transmitting HIV by up to 5-fold. Although treatment depends on antibiotics, N. gonorrhoeae rapidly becomes resistant to each class of antibiotics used against N. gonorrhoeae, including the most recent fluoroquinolones (ciprofloxacin), and is currently recommended. The antibiotics used are cephalosporins. However, resistance to these has begun to emerge, and it has become gonococcal multidrug resistance. Despite various efforts, vaccines against N. gonorrhoeae are currently unavailable. Vaccine efforts are complicated by the widespread antigenic variability of Neisseria gonorrhoeae, with most major surface antigens, including lipooligosaccharides (LOS), porins, pilin, and turbid proteins (Opa), undergoing phase variation expression (LOS,). Receives Opa, pili), allelic variation (porin, Opa), or recombinant expression (pilin). As such, options for the treatment and control of disease are becoming limited. A mysterious but well-known feature of gonorrhea is that recovery from infection does not lead to protective immunity against reinfection and often repeats infection.

The present disclosure provides a method of reducing the risk of developing gonococcal infection by administering to an individual an gonococcal antigen and IL-12 incorporated into a polymeric microsphere. The gonococcal antigen may be in the form of adventitial vesicles or microvesicles. Other forms of antigen (eg, purified or semi-purified) can also be used. N. gonorrhoeae antigen preparations (eg, OMV) and IL-12 microspheres in a single composition or different compositions, by the same or different routes, at the same time or at different times, for the same period and in the same delivery regimen, or It can be delivered for different periods and with different delivery regimens. For example, N. gonorrhoeae OMV and IL-12 microspheres can be delivered intravaginally or intranasally.

In one aspect, the present disclosure provides a method for the treatment of cervical and vaginal infections by topical application of IL-12 incorporated into polymeric microspheres. Although not intended to be bound by any particular theory, topical application of IL-12 incorporated into polymeric microspheres to the mucosal surface enhances the body's own immune response to pre-existing infections, The result is believed to result in reduction or elimination of the infection and / or generation of immunity against recurrent infections. In one embodiment, the amount is sufficient to promote a Th1-induced response to the infectious microorganism. The amount of IL-12 may be sufficient to provide a therapeutic effect, a preventive effect, or both against the causative microorganism. Infections that can be treated by this method include, but are not limited to, infections caused by Neisseria gonorrhoeae, Chlamydia trachomatis, or both. An example of a polymer that can be used for microencapsulation of IL-12 is polylactic acid.

In one aspect, the disclosure provides a composition comprising OMV prepared from Neisseria gonorrhoeae and microspheres (ms) containing IL-12, suitable for vaginal delivery.

In one aspect, the disclosure provides a kit for intravaginal delivery of OMV and IL-12-containing microspheres prepared from Neisseria gonorrhoeae. OMV and IL-12ms may be present as separate compositions or the same composition. The kit may include multiple doses of OMV and IL-12 compositions, as well as instructions for administration, which may include instructions for frequency, length of dosing regimen, mode of dosing, and the like.

For a more complete understanding of the nature and purpose of the invention, the following detailed description will be referred to in conjunction with the accompanying drawings.

[Fig. 1]
FIG. 1 is a graph showing the effect of intravaginal treatment with 1 μg of IL-12 encapsulated in polylactic acid (PLA) microspheres on the course of vaginal infection by Neisseria gonorrhoeae in mice.

[Fig. 2]
FIG. 2 is a graph showing the effect of intravaginal administration of IL-12 microspheres during primary gonococcal infection on the course of secondary gonococcal infection in mice (FIG. 1).

[Fig. 3]
FIG. 3 is a graph showing the effect of intravaginal treatment with 1 μg of soluble IL-12-vs.-microencapsulated IL-12 on the course of vaginal infection by Neisseria gonorrhoeae in mice.

[Fig. 4]
FIG. 4 is a graph showing the effect of intravaginal IL-12 microsphere (ms) treatment on primary gonococcal infection in BALB / c mice.
(A) IL-12ms dose optimization experiment. Microspheres containing the prescribed dose of IL-12 were administered to n = 8 mice each on days 0, 2, 4, 6 and 8. Gonorrhea (Ngo) loading was monitored daily by vaginal swab culture. Significant differences in infection load were observed between mice treated with 2.0 μg (p <0.01), 1.0 μg (p <0.01), or 0.5 μg (p <0.05) of microencapsulated IL-12 and controls. (ANOVA).
(B) Time course of infection in mice treated with IL-12ms, soluble IL-12, IL-17ms, or control ms, or untreated mice; cytokine dose = 1.0 μg to -1, 1, 3, 5, 7 Administered on day; n = 8 mice in each group. There was a significant difference in infection load between mice treated with IL-12ms (p <0.01) or IL-17ms (p-0.01) and controls (ANOVA).
Data from the experiment shown in (C) B were plotted as the percentage of mice that remained infected under the indicated cytokine treatment. In mice treated with IL-12ms (p <0.0001) or IL-17ms (p <0.001), infection was cleared significantly faster than controls (Kaplan-Meier).
(D) Cytokine expression in sham-infected mice or isolated ILN cells from infected mice treated with IL-12ms, IL-17ms, or control ms; n = 7 mice in each group. The expression of IFN-γ, IL-4, and IL-17 in CD4 ⁺ T cells isolated 5 days after infection was analyzed by flow cytometry.
(E) IFN-γ, IL-4, and IL-17 mRNA levels in vaginal tissue collected on day 3 from pseudo-infected mice or infected mice treated with IL-12ms, IL-17ms, or control ms. RT-PCR analysis; n = 7 mice in each group. The expression level of the cytokine gene detected by the RT-PCR method was normalized to the expression of β-actin and set to 1.0 in the sham-infected group.
(F) Phenotypic profile of vaginal cells isolated on day 5 from sham-infected mice or infected mice treated with IL-17 ms or control ms; n = 7 mice in each group.
Anti-gonococcal IgA and IgG antibody responses of (G) vaginal and (H) sera in sham-infected mice or mice infected with IL-12ms, IL-17ms, or control ms treatment; n = 7 mice in each group. Vaginal lavage fluid and serum were collected on the 15th day of inoculation, and gonococcal-specific and total IgA and IgG were measured by ELISA. The results from one of the representatives of the three independent experiments are shown. For D to H, # p <0.05; * p <0.01 (one-sided t-test).

[Fig. 5]
FIG. 5 is a graph showing the effect of intravaginal IL-12 microsphere (ms) treatment during primary infection on secondary gonococcal infection.
(A) Secondary infection in mice that received IL-12ms, soluble IL-12, IL-17ms, or control ms during the primary infection, or previously pseudo-infected mice with or without IL-12ms. Changes over time; n = 8 mice in each group. There was a significant difference in infection load between mice previously treated with IL-12ms (p-0.01) and controls (ANOVA).
(B) The experimental data shown in A was plotted as the percentage of mice that remained infected after reinfection with the labeling treatment during primary infection. Infection disappeared significantly faster in mice previously treated with IL-12 ms (p <0.0001) than controls (Kaplan-Meier).
(C) Day 5 from re-infected mice treated with IL-12ms, IL-17ms, or control ms during primary infection, or from mice pseudo-infected in both primary and secondary phases (“pseudo-reinfection”). Flow cytometric analysis of cytokine expression in ILN CD4 ⁺ T cells isolated in 1; n = 7 mice per group.
(D) Intravaginal IFN-γ, IL-4, and vaginal IFN-γ, IL-4, collected on day 3 from pseudore-infected mice or reinfected mice treated with IL-12ms, IL-17ms, or empty ms during primary infection. RT-PCR analysis of IL-17 mRNA levels; n = 7 mice in each group. The expression level of the cytokine gene detected by RT-PCR was normalized to the expression of β-actin and was 1.0 in the pseudo-reinfection group.
Anti-gonococcal IgA and IgG of (E) vaginal and (F) serum against secondary infection in pseudo-re-infected mice or re-infected mice treated with IL-12ms, IL-17ms, or empty ms during primary infection Antibody response; n = 7 mice in each group. Vaginal lavage fluid and serum were collected 15 days after inoculation, and gonococcal-specific and total IgA and IgG were measured by ELISA. The results from one of the representatives of the three independent experiments are shown. For C to F, # p <0.05; * p <0.01 (one-sided t-test).

[Fig. 6]
FIG. 6 shows that intravaginal (I.vag) immunization with Neisseria gonorrhoeae OMV + IL-12 / ms induced resistance to genital infections with Neisseria gonorrhoeae, resulting in an immune response.
a: Mice were immunized with OMV (protein 40 μg) + control (empty) ms or IL-12 / ms (1 μg IL-12) from FA1090 strain three times at 7-day intervals; control mice were empty ms or IL. Immunized with either -12 / ms alone. Two weeks after the last immunization, all mice were loaded with gonococcal strain FA1090 (5 × 10 ⁶ CFU) by i.vag. Infection and infection was monitored by vaginal swabbing and plating. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8 mice), * P <0.01 (ANOVA); Right panel: Percentage of animals remaining infected at each time point (%), P <0.01 ( Kaplan-Meier analysis, log-rank test, OMV + IL-12 / ms-vs.-OMV + empty ms)
b: Vaginal lavage fluid (left) and serum (right) antibodies against FA1090 strain in the samples collected after completion (15th day), N = 5 samples shown by mean ± SEM; # P <0.05, * P <0.01, Student's t-test.
c: Intracellular cytokine staining in CD4 ⁺ cells recovered from ILN at the end (15th day), N = 3 samples shown by mean ± SEM, ratio of CD4 ⁺ staining to each cytokine, * P <0.01 Student t Test d: Mice were immunized twice with gonococcal (Ngo) OMV (protein 40 μg) + empty ms or IL-12 / ms (1 μg IL-12) at 14-day intervals; control mice were immunized with empty ms alone or NTHI OMV ( Pseudoimmunization was performed with protein 40 μg) + IL-12 / ms (1 μg IL-12). Two weeks later, all mice were loaded with N. gonorrhoeae FA1090 (5 × 10 ⁶ CFU). Left panel: Gonorrhea recovery (CFU) (mean ± SEM, N = 8 mice), * P <0.01 (ANOVA, Gonorrhea OMV + IL-12 / ms-vs.-Gonorrhea OMV + empty ms); Right panel: Infected at each time point Percentage of animals up to, P <0.0001 (Kaplan-Meier analysis, log rank test, gonococcal OMV + IL-12 / ms-vs.-gonococcal OMV + empty ms).

[Fig. 7]
FIG. 7 shows the antibody reaction generated by immunization with N. gonorrhoeae OMV + IL-12 / ms before loading with N. gonorrhoeae.
a: Two weeks after final immunization with 1, 2 or 3 doses of gonococcal OMV (protein 40 μg) + IL-12 / ms (IL-12 1 μg), vaginal lavage fluid (left panel) and serum (right panel) antibody by ELISA Was measured. Control samples were obtained from mice immunized with empty ms (3 doses); yet another mouse was immunized with gonococcal OMV + empty ms three times. Data are shown as mean ± SEM, N = 5 samples, # P <0.05, * P <0.01 (compared to control sample) (ANOVA).
Antibody durations of vaginal lavage fluid (b) and serum (c) in mice immunized with 2 doses of FA1090 OMV + empty ms or IL-12 / ms; data shown by mean ± SEM, N = 5 samples; C = Control sample from non-immunized mice.

[Fig. 8]
a: When gonococcal OMV (protein 40 μg) + IL-12 / ms (IL-12 1 μg) was immunized 1, 2 or 3 times, 2 weeks after the last immunization, gonococcal OMV + IL-12 / ms T cell cytokine response in ILN cells induced by immunization. Control ILNs were obtained from mice pseudoimmunized with empty ms (3 doses) and yet another mouse was immunized with gonococcal OMV + empty ms three times. Data are shown for each cytokine as mean ± SEM, N = 3 samples,% of CD4 ⁺ or CD8 ⁺ cell staining. * P <0.01 (Student's t-test) compares immunization at IL-12 / ms-vs-air ms.
b: Duration of IFNγ response in CD4 ⁺ ILN cells 1-6 months after two immunizations with N. gonorrhoeae OMV + IL-12 / ms or OMV + empty ms. Data are shown as mean ± SEM, N = 3 samples,% of CD4 ⁺ cells stained for IFNγ. C = Control ILN from non-immune mice.

[Fig. 9]
FIG. 9 shows that resistance to the gonococcal (FA1090) challenge persisted for at least 6 months after immunization with 2 doses of gonococcal (FA1090) OMV + IL-12 / ms. Left panel: Gonorrhea recovery (CFU) (mean ± SEM, N = 8 mice), * P <0.01 (ANOVA, Gonorrhea OMV + IL-12 / ms-vs.-Gonorrhea OMV + empty ms); Right panel: Infected at each time point Percentage of animals up to, P <0.001 (Kaplan-Meier analysis, log rank test, gonococcal OMV + IL-12 / ms-vs.-gonococcal OMV + empty ms).

[Fig. 10]
FIG. 10 shows resistance to the heterologous gonococcal challenge.
a: One month after immunization with FA1090 OMV + IL-12 / ms or empty ms, mice were loaded with gonococcal strain FA1090 (family challenge) or MS11 strain (heterologous challenge). Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8 mice), * P <0.001 (ANOVA, shown for comparison); Right panel:% of animals remaining infected at each time point, For FA1090 challenge, P <0.02, IL-12 / ms-vs.-empty ms; for MS11 challenge, P <0.001, IL-12 / ms-vs-empty ms (Kaplan-Meier analysis, log rank test).
b: Mice immunized with MS11 OMV showed resistance to loading of Neisseria gonorrhoeae FA1090. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8 mice), P <0.01 (ANOVA); Right panel:% of animals remaining infected at each time point, P <0.01 (Kaplan-Meier analysis) , Log rank test).
c: Mice immunized with FA1090 OMV showed resistance to loading of Neisseria gonorrhoeae FA19. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8 mice), * P <0.01 (ANOVA, shown for comparison); Right panel:% of animals remaining infected at each time point, FA1090 For load, P <0.01, IL-12 / ms-vs.-empty ms; for FA19 load, P <0.0001, IL-12 / ms-vs.-empty ms (Kaplan-Meier analysis, log rank test), N = 8 mouse.
d: Mice immunized with FA19 OMV showed resistance to loading with Neisseria gonorrhoeae FA1090. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8 mice), P <0.01 (ANOVA); Right panel:% of animals remaining infected at each time point, P <0.01 (Kaplan-Meier analysis) , Log rank test).
e: Mice immunized with FA1090 OMV showed resistance to loading of clinical isolate GC68. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8 mice), P <0.01 (ANOVA); Right panel:% of animals remaining infected at each time point), P <0.01 (Kaplan-Meier) Analysis, log rank test).

[Fig. 11]
FIG. 11 shows the immunoproteomics of Neisseria gonorrhoeae OMV.
a: SDS-PAGE of OMV preparations derived from N. gonorrhoeae strains FA1090, MS11, and FA19 was stained with Coomassie blue.
b: Western blot analysis of mouse sera tested for gonococcal OMV preparations isolated by SDS-PAGE. Lane 1, FA1090 OMV + empty ms-immunized mouse-derived control serum, FA1090 OMV was tested; Lanes 2-4, FA1090 OMV + IL-12 / ms-immunized mouse-derived serum # 1, FA1090-derived OMV ( Tested against lane 2), MS11 (lane 3), or FA19 (lane 4); lane 5, FA1090 OMV + IL-12 / ms immunized mouse-derived serum # 2, FA1090-derived OMV. Antibody H5 (anti-porin PIB3) tested against lane 6, FA1090 OMV.
c-e: Proteome maps of Neisseria gonorrhoeae OMV from FA1090 (c), MS11 (d), and FA19 (e) are revealed by 2D electrophoresis and Flamingo fluorescence staining (left panel) and correspond to them. Immunoblots (right panel) were obtained by probe with mouse serum # 2. Immunoreactive spots subjected to MS / MS analysis are labeled as spots 1 and 2 (arrows). The molecular weight marker (kDa) is shown on the left.

[Fig. 12]
FIG. 12 shows resistance to challenges induced by immunization with N. gonorrhoeae OMV + IL-12 / ms (depending on IFNγ and B cells).
a: Infection course in FA1090 OMV-immunized IFNγ-ko mice-vs. wild-type mice (FA1090); left panel, gonococcal recovery (CFU) (mean ± SEM, N = 8 mice), P <0.01 (ANOVA) ); Right panel,% of animals remaining infected at each time point, P <0.0001 for wild-type mice, IL-12 / ms-vs-air ms (Kaplan-Meier analysis, log-rank test).
b: Infection course in FA1090 OMV-immunized μMT mice-vs.-wild mice (FA1090); left panel, gonococcal recovery (CFU) (mean ± SEM, N = 8 mice), P <0.01 (ANOVA); Right panel,% of animals remaining infected at each time point, P <0.0001 for wild-type mice, IL-12 / ms-vs.-empty ms (Kaplan-Meier analysis, log rank test).
Vaginal lavage fluid (c) and serum (d) antibody responses in IFNγ-ko-vs.-wild type (mean ± SEM, N = 5 samples) were analyzed at the end (day 13). IgA and IgG responses in vaginal lavage fluid and serum were significant in wild-type mice (P <0.05, Student's t-test, OMV + empty ms-vs.-OMV + IL-12 / ms), but not in IFNγ-ko mice. There wasn't.
e: T cell cytokine response (mean ± SEM, N = 3 samples) in μMT mouse-vs. wild-type mice was analyzed at the end (day 13). The IFNγ response to immunization with OMV + empty ms-vs.-OMV + IL-12 / ms was significant for both wild-type and μMT mice (P <0.01) (ANOVA).
f: Infection course in FA1090 OMV-immunized CD4-ko mice-vs.-wild-type mice (FA1090); left panel, gonococcal recovery (CFU) (mean ± SEM, N = 8 mice), # P <0.05, P <0.01 (ANOVA), shown for comparison; right panel,% of animals remaining infected at each time point, wild-type mouse IL-12 / ms-vs.-empty ms P <0.001, CD4-ko mouse IL-12 / ms-vs.-empty ms P <0.01 (Kaplan-Meier analysis, log rank test).
g: Infection course in FA1090 OMV-immunized CD8-ko mice-vs.-wild-type mice (FA1090); left panel, gonococcal recovery (CFU) (mean ± SEM, N = 8 mice), # P <0.05, P <0.01 (ANOVA), shown for comparison; right panel,% of animals remaining infected at each time point, wild-type mouse IL-12 / ms-vs.-empty ms P <0.001, CD8-ko mouse IL-12 / ms-vs.-empty ms P <0.02 (Kaplan-Meier analysis, log rank test).

[Fig. 13]
FIG. 13 is a reproduction of FIG. I.vag immunization with N. gonorrhoeae OMV + IL-12 / ms induced resistance to genital infections with N. gonorrhoeae, resulting in an immune response.
A: Mice were immunized with OMV (protein 40 μg) derived from FA1090 strain + control (empty) ms or IL-12 / ms (IL-12 1 μg) three times at 7-day intervals; control mice were empty ms or IL. Immunized with either -12 / ms alone. Two weeks after the last immunization, all mice were challenged with i.vag inoculation of Neisseria gonorrhoeae FA1090 (5 × 10 ⁶ CFU) and infection was monitored by vaginal swabbing and plating. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8 mice), P <0.01 (ANOVA, OMV + IL-12 / ms-vs.-OMV + empty ms); Right panel: remain infected at each time point Animal%, P <0.001 (Kaplan-Meier analysis, log rank test, OMV + IL-12-vs.-OMV + empty ms).
B: Vaginal lavage fluid (left) and serum (right) antibodies against FA1090 strain in the samples collected after completion (15th day) are shown as mean ± SEM, N = 5 samples.
C: Intracellular cytokine staining in CD4 ⁺ cells recovered from ILN at the end (day 15) is shown for each cytokine as mean ± SEM, N = 3 samples,% of CD4 ⁺ staining.
D: Mice were immunized twice at 14-day intervals with gonococcal (Ngo) OMV (protein 40 μg) + empty ms or IL-12 / ms (IL-12 1 μg); control mice were empty ms alone or NTHI OMV ( It was pseudoimmunized with protein 40 μg) + IL-12 / ms (IL-12 1 μg). Two weeks later, all mice were loaded with N. gonorrhoeae FA1090. Left panel: Gonorrhea recovery (CFU) (mean ± SEM, N = 8 mice), * P <0.001 (ANOVA, Gonorrhea OMV + IL-12 / ms-vs.-Gonorrhea OMV + empty ms); Right panel: Infected at each time point Percentage of animals up to, P <0.001 (Kaplan-Meier analysis, log rank test, gonococcal OMV + IL-12 / ms-vs.-gonococcal OMV + empty ms).

[Fig. 14]
FIG. 14 shows an example of a flow cytometry plot for the data shown in FIG. 6C. Intracellular cytokine staining of ILN cells after infection clearance (day 15) in immunized mice as shown. X-axis (FL4H): Cytokine fluorescence; Y-axis (PE2): CD4 fluorescence (AL); CD8 fluorescence (MP).

[Fig. 15]
FIG. 15 shows the response induced by immunization with OMV prepared from NTHI; samples were taken 2 weeks after immunization. A: Vaginal lavage antibody (mean ± SEM, N = 5 samples); B: Serum antibody (mean ± SEM, N = 5 samples); C: T cell cytokine in ILN cells (mean ± SEM, N = 3 samples) .. * P <0.01 vs-control sample (Student's t-test).

[Fig. 16]
FIG. 16 shows an IgG subclass (mean ± SEM, N = 5 samples) of anti-gonococcal antibody reaction in vaginal lavage fluid (A) and serum (B) induced by immunization with gonococcal OMV + IL-12 / ms. # P <0.05, * P <0.01 vs-control sample (Student's t-test).

[Fig. 17]
A: Specificity of T cell response in ILN to gonococcal antigen. ILN CD4 ⁺ T cells from mice immunized with FA1090 OMV + empty ms or IL-12 / ms were preloaded with CFSE and in the presence of antigen-presenting cells for 3 days in the presence (stim) or absence of FA1090 cells. Underneath, cultured in vitro. Proliferation was analyzed by flow cytometry after surface staining for CD4, and cytokine secretion was measured by ELISA. Data are shown as mean ± SEM, N = 7 samples. * P <0.01 vs-control sample (Student's t-test).
B: RT-PCR analysis of RNA extracted from vaginal tissue 3 days after final immunization (1, 2, or 3 doses) with OMV + empty ms or IL-12 / ms. Data are shown with mean ± SEM, N = 7 samples. * P <0.01 vs. -OMV + empty ms mouse sample (Student's t-test).
C: Persistence of IFNγ response in CD4 ⁺ ILN cells collected 1-6 months after immunization with OMV + IL-12 / ms. Data are shown as mean ± SEM, N = 7 samples.

[Fig. 18]
FIG. 18 shows the reaction in mice loaded with Neisseria gonorrhoeae FA1090 6 months after immunization with FA1090 OMV + IL-12 / ms. A: Vaginal lavage antibody (mean ± SEM, N = 5 samples), B: Serum antibody (mean ± SEM, N = 5 samples), C: Cytokine production by CD4 ⁺ ILN cells (mean ± SEM, N = 3 samples) .. Control sample of # P <0.05, * P <0.01 vs. pseudo-infected mice (Student's t-test).

[Fig. 19]
FIG. 19 shows the reaction in mice immunized with gonococcal OMV + empty ms or IL-12 / ms after heterologous gonococcal loading. A, B, C: FA1090 OMV-immunized and MS11-loaded mice; A: vaginal lavage antibody against MS11, B: serum antibody (mean ± SEM, N = 5 samples); C: cytokine response in CD4 ⁺ ILN cells (Average ± SEM, N = 3 samples). D, E, F: FA1090 OMV-immunized and FA19-loaded mice; D: vaginal lavage antibody against FA19, E: serum antibody (mean ± SEM, N = 5); F: cytokine response in CD4 ⁺ ILN cells ( Average ± SEM, N = 3 samples). Control sample of # P <0.05, * P <0.01 vs. pseudo-infected mice (Student's t-test).

[Fig. 20]
FIG. 20 is a replica of FIGS. 7A, B, F, G: I.vag immunization with gonococcal OMV + IL-12 / ms in immunodeficient mice.
A: Infection course in FA1090 OMV-immunized IFNγ-ko mice-vs.-wild-type mice (FA1090); left panel, gonococcal recovery (CFU) (mean ± SEM, N = 8 mice), P <0.01 (ANOVA) ); Right panel,% of animals remaining infected at each time point, P <0.001 for wild-type mice, IL-12 / ms-vs-air ms (Kaplan-Meier analysis, log-rank test).
B: Infection course in μMT mice immunized with FA1090 OMV (FA1090); left panel, gonococcal recovery (CFU) (mean ± SEM, N = 8 mice).
C: Infection course in CD4-ko mice immunized with FA1090 OMV (FA1090); left panel, gonococcal recovery (CFU) (mean ± SEM, N = 8 mice); right panel, animals remaining infected at each time point %, P <0.01 (Kaplan-Meier analysis, log rank test).
D: Infection course in FA1090 OMV-immunized CD8-ko mice-vs.-wild-type mice (FA1090); left panel, gonococcal recovery (CFU) (mean ± SEM, N = 8 mice); right panel, at each time point % Of animals remaining infected, P <0.01 (Kaplan-Meier analysis, log rank test).

[Fig. 21]
FIG. 21 shows the effect of in-immunization by gonococcal OMV (FA19 strain) + IL-12 / ms on gonococcal-induced infection in BALB / c mice. A: Gonorrhea recovery (CFU) (mean ± sem, N = 8 mice), P <0.01 (ANOVA, Gonorrhea OMV + IL-12 / ms-vs.-Gonorrhea OMV + empty ms); B: Remains infected at each time point Animal's%.

[Fig. 22]
FIG. 22 shows the effect of immunization of gonococcal OMV (FA1090 strain) + IL-12 / ms at different doses on heterologous gonococcal-induced infection by FA19 strain in BALB / c mice. High dose: 60 μg, medium dose: 30 μg, low dose: 15 μg OMV protein + 1 μg IL-12 / ms per dose. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8 mice); Right panel: Percentage of animals remaining infected at each time point.

[Fig. 23]
A: Comparison of the effect of intranasal or vaginal immunization by gonococcal OMV (30 μg protein; FA1090 strain) + 1 μg IL-12 / ms on heterologous gonococcal-induced infection by FA19 strain in BALB / c mice. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8-9 mice); Right panel:% of animals remaining infected at each time point. At the end (2 weeks after loading), B: serum antibody, C: vaginal lavage antibody, D: saliva antibody; * P <0.01 compared to controls against gonococci in samples taken after infection clearance (Student's t-test) ).

[Fig. 24]
FIG. 24 shows a comparison of the effects of intranasal or vaginal immunization by gonococcal OMV (30 μg protein; FA1090 strain) + 1 μg IL-12 / ms on allogeneic gonococcal-induced infection by FA1090 strain in BALB / c mice. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8-9 mice); Right panel:% of animals remaining infected at each time point.

[Fig. 25]
FIG. 25 shows the effect of intranasal immunization of Neisseria gonorrhoeae OMV (30 μg protein, FA1090 strain) + 1 μg IL-12 / ms against heterologous gonococcal-induced infection by MS11 strain in BALB / c mice. Mice received one OMV + IL-12 / ms or twice at 2-week intervals; control mice received two empty ms. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 9 mice); Right panel: Percentage of animals remaining infected at each time point.

[Fig. 26]
FIG. 26 shows the effect of intranasal immunization of gonococcal OMV (30 μg protein, MS11 strain) + 1 μg IL-12 / ms on heterologous gonococcal-induced infection by FA1090 strain in BALB / c mice. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 8-9 mice); Right panel:% of animals remaining infected at each time point.

[Fig. 27]
FIG. 27 shows the effect of intranasal immunization of gonococcal OMV (30 μg protein, MS11 strain) + 1 μg IL-12 / ms on heterologous gonococcal-induced infection by FA19 strain in BALB / c mice. Left panel: Neisseria gonorrhoeae recovery (CFU) (mean ± SEM, N = 9 mice); Right panel: Percentage of animals remaining infected at each time point.

The present invention is based on our work, which has helped elucidate how gonococci prevent the immune system from initiating an effective immune response against itself. A new approach is offered to overcome the ability of N. gonorrhoeae to suppress the immune response to itself.

In one aspect, this method reduces the likelihood that an individual will develop N. gonorrhoeae infection by administration of an antigen preparation from N. gonorrhoeae and IL-12 present in microspheres (ms). Including. An example of an antigen preparation derived from Neisseria gonorrhoeae is OMV. OMV and IL-12ms may be delivered intravaginally. In one embodiment, OMV and IL-12ms may be delivered intranasally. OMV and IL-12ms may be delivered by separate routes of administration. For example, one may be delivered intravaginally and the other intranasally. In one embodiment, OMV and IL-12ms are delivered by the same route of administration, eg, intranasally.

In one embodiment, OMV and IL-12ms are delivered in the same composition. The amount of OMV and IL-12ms per dose (single dose) can be the amount that elicits an immune response. OMV can range from 0.01 to 1 mg of protein per dose. In one embodiment, the OMV can range from 0.01 to 2 mg of protein per dose. In multiple embodiments, the OMV can be 10, 50, 100, 250, 500, 750, 1,000, 1250, 1,500, 1,750 or 2,000 μg of protein per dose. In one embodiment, the OMV can range from 15 to 300 μg protein per dose. In one embodiment, the OMV dose can be 50-1,000 μg protein. For a 70 kg human, this corresponds to an OMV dose of about 0.7-28 micrograms of protein per kg body weight. In one embodiment, the OMV can be 0.5-30 μg protein per kg body weight. IL-12 (in microspheres) can be 1-500 μg / dose. This corresponds to about 14 ng to 7 μg per kg of body weight (assuming 70 kg body weight). In one embodiment, IL-12 can be between 10 ng and 10 μg / kg body weight. In one embodiment, IL-12 can be 10, 50, 100, 200, 300, 400 or 500 μg / dose. In one embodiment, IL-12 can be 10-300 ng or 5-300 ng per kg body weight. In one embodiment, IL-12 can be 0.5-20 micrograms in microspheres per dose. The amount of microspheres can be determined based on the IL-12 load. The composition may be provided using carriers, buffers, etc., or may be lyophilized. The two components may be provided separately, combined immediately prior to administration, or administered separately. In one embodiment, the composition is completely free of free soluble IL-12. The amount of IL-12 leaking from the microsphere before administration is considered to be small. The presence of free soluble IL-12 is not considered to contribute to the effects and methods of the present invention. Rather, IL-12 encapsulated in the microsphere is required in the composition to obtain a protective effect.

The outer membrane vesicles can be prepared from the outer membrane of Neisseria gonorrhoeae. For example, OMV can be prepared from the outer membrane of a culture of N. gonorrhoeae. OMV can be obtained from Neisseria gonorrhoeae grown in broth or solid medium cultures. Preparations include separating bacterial cells from the culture medium (eg, by filtration or slow centrifugation), lysing the cells (eg, by adding a surfactant, osmotic shock, ultrasound treatment, cavitation, homozygous). Separation of the outer membrane fraction from cytoplasmic molecules (eg, by filtration; or by differential precipitation or aggregation of outer membrane and / or outer membrane vesicles, or by affinity separation; or fast centrifugation. (By separation) can be included.

The composition can be administered multiple times, preferably at intervals. For example, at least two doses can be administered with an interval of at least one week. The interval may be 1 to 3 weeks. In one embodiment, the two doses are used at intervals of about two weeks.

In one embodiment, the IL-12 preparation of the present invention is a sustained release preparation. In one embodiment, IL-12 is delivered in a state of being incorporated into polymeric microparticles (also referred to herein as microspheres) (also referred to herein as encapsulated or microencapsulated). In one embodiment, the microparticles are biodegradable and biocompatible. Techniques for preparing such microspheres are known in the art. See, for example, U.S. Pat. Nos. 6,143,211; 6,235,244; 6,616,869; and 7,029,700 (these disclosures are incorporated herein by reference with respect to methods and compositions for the preparation of microspheres). Will be). In one embodiment, phase inversion techniques are used to prepare microencapsulated IL-12. Generally, the biodegradable polymer is dissolved in a solvent (such as dichloromethane or other organic solvent) and then micronized IL-12 (ie, a lyophilized mixture of IL-12 and an excipient such as polyvinylpyrrolidone). Is added to the polymer dissolved in the solvent to form a mixture. A non-solvent (eg, alcohol or hexane) is then introduced, causing the spontaneous formation of microencapsulated IL-12. Examples of biodegradable polymers include lactic acid and glycolic acid polymers, polyanhydrides, poly (ortho) esters, polyurethanes, poly (butyric acid), poly (lactide), poly (caprolactone), poly (hydroxybutyrate). , Poly (lactide-co-glycolide) and poly (lactide-co-caprolactone), as well as natural polymers. In one embodiment, the microsphere is composed of a polymer of lactic acid (polylactic acid (PLA)).

In one embodiment, the IL-12-containing microspheres slowly degrade over time by hydrolysis to release encapsulated IL-12. Microspheres may be suspended prior to use and delivered in acceptable buffered saline. In one embodiment, sustained release of IL-12 over a period of time, such as about 4 days, is locally present without raising the concentration of IL-12 in the local tissue or circulation to potentially harmful levels. Allows continuous stimulation of immune cells. Microspheres are made from biodegradable materials. In one embodiment, the hydrolyzate of microspheres is lactic acid, a harmless product of normal metabolism. PLA is a component of absorbable sutures and has been used for decades for this purpose and is therefore considered safe. Microencapsulated IL-12 has been shown to be stable at ambient temperature storage and have a long shelf life.

Microspheres range from 10 nm to 10 microns. Microspheres may be suspended in a pharmaceutically acceptable medium, such as physiological buffer. In one embodiment, the filling amount of IL-12 is 0.1-10 μg per 1 mg of particles. In one embodiment, the filling amount is 1-5 μg IL-12 per 1 mg of particles. In one embodiment, the filling amount is greater than or equal to about 2.5 μg of IL-12 per 1 mg of particles.

The IL-12 preparation can be used in an amount that provides a therapeutic and / or prophylactic effect. The effective dose in mice was observed to be IL-12 1 μg. Determining the effective dose for humans is within the scope of clinicians and other individuals involved in the treatment of such infections. In general, the dose administered depends on a variety of factors, including the severity of infection, the weight of the individual, health and age. Such factors can be easily determined by the clinician. In one embodiment, the dose can be between 1 μg and 200 μg of IL-12 per day. In some embodiments, the doses are 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 175 and 200 μg (all integer values between 1 and 200 μg and all in between). IL-12 (including range). The required dose may be less when used in combination with an antibacterial agent.

The administration may be repeated as needed. For example, administration may be repeated once a day, multiple times a day, or at longer intervals (eg, every 2-4 days, weekly or monthly). In one embodiment, administration is repeated at intervals of one day to one month (28, 29, 30 or 31 days) or more and all intervals between them. The treatment regimen may be repeated as needed. In some embodiments, the dose is administered every 2, 3, 4, 5, 6, 7, 10, or 14 days (or longer).

In one embodiment, the invention provides a method of treating a reproductive tract infection in a female subject by intravaginal administration of IL-12 incorporated into a polymeric microsphere. Infections that can be treated by this method include bacteria, fungi, parasites, viruses and the like. In one embodiment, this amount is sufficient to promote a Th1-induced response to the infectious microorganism. In one embodiment, this amount is sufficient to provide a therapeutic, prophylactic, or both effect on the causative microorganism. As used herein, the term "treated" or "treated" means reducing or eliminating an infection. If the root cause of the infection is reduced, the infection is considered to be reduced.

In one embodiment, the methods of the invention are useful for treating reproductive tract infections such as cervical and vaginal infections caused by bacteria such as gonorrhea. The method comprises providing a topical (intravaginal) application of the cytokine interleukin-12 (IL-12) incorporated into a biodegradable biocompatible microsphere. In one embodiment, the dose is sufficient to promote a Th1-induced immune response to gonococcal infection. In one embodiment, the invention provides a method for the treatment and / or prevention of cervical gonococcal infection (ie, gonorrhea) by topical administration of IL-12 microspheres. Although not intended to be bound by any particular theory, this method is believed to work, at least in part, by reversing the ability of N. gonorrhoeae to interfere with the host's immune response.

In one embodiment, the IL-12 formulation is delivered topically to the mucosal surface of an individual's reproductive tract. In one embodiment, the individual has not yet received IL-12 or has not been administered IL-12 prior to the initiation of the method. In one embodiment, the individual has not been administered IL-12 by any other mode of administration. In one embodiment, the formulation is free of other therapeutic agents, free of other prophylactic agents, or free of other agents useful for both treatment and prevention. In one embodiment, the formulation is free of infection-causing microorganisms (eg, in inactivated form) or antigens from it, and the individual has been administered an inactivated microorganism or antigens from it. Not and / or not administered. In another embodiment, the formulation may be delivered to an individual who has already been treated (other than IL-12) for a reproductive tract infection (eg, gonococcal infection).

In one embodiment, the invention further comprises the step of administering an antibacterial agent to an individual. For example, in one embodiment, the methods of the invention identify an individual suffering from or diagnosed with a gonad infection, locally in the gonad (eg, intravaginally). , A step of delivering a composition comprising IL-12 in a biodegradable polymeric microsphere, and optionally one, either therapeutically effective, prophylactically effective, or both therapeutically and prophylactically effective. The step of administering the above antibacterial agent (antibiotic, antifungal agent, antiviral agent, etc.) to an individual is included. The antibacterial agent can be administered before, during, or after administration of the IL-12 preparation. An example of such treatment is the administration of antibacterial agents such as antibiotics. Examples of suitable antibiotics used for reproductive tract infections include fluorquinolones, cephalosporins, azithromycin, ceftriaxone, doxycycline, and cefixime.

In one embodiment, administration of the microencapsulated IL-12 described herein reduces gonococcal infection. In one embodiment, the infection is eliminated. The presence or absence of infection or the level of infection can be tested by routine microbiological methods (such as culture and testing). In one embodiment, the infection can be tested by obtaining a vaginal swab and testing for the presence of bacteria (eg, by the ability to form colonies) or by nucleic acid amplification methods.

In another embodiment, administration of the microencapsulated IL-12 described herein reduces gonococcal infection and increases the risk of recurrent gonococcal infection after discontinuation of treatment with microencapsulated IL-12. Reduce. Although not intended to be bound by any particular theory, the prophylactic effect of IL-12 is believed to be achieved by stimulation of the immune system. In one embodiment, administration of IL-12 does not significantly increase the level of IL-12 in the systemic circulation. In one embodiment, serum levels of IL-12 do not increase above 50 picograms / ml.

For vaginal application, the formulations of the present invention can be applied and delivered to products that act as carriers. For example, the formulation may be incorporated into or out of inserts, applicators, tablets, suppositories, vaginal rings, vaginal sponges, tampons, etc., and may then be delivered via them. The formulations may also be delivered in the form of liquids, creams, gels, lotions, ointments, pastes, sprays and the like.

The pharmaceutical formulation may optionally contain a pharmaceutically acceptable carrier, buffer, diluent, solubilizer or emulsifier, and various salts. Such additives are well known in the art. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042).

The advantage of topical application of microencapsulated IL-12 as described herein is that it can provide lasting effects while avoiding potential systemic toxicity issues.

In one embodiment, the invention is used to treat a genital Chlamydia trachomatis infection (Chlamydia). Chlamydia is another sexually transmitted disease (STD) that occurs more frequently than gonorrhea and is the most frequently reported infection in the United States, with 3 million people annually (more than 92 million people worldwide). It is believed to extend to. It is also a major cause of pelvic inflammatory disease and sequelae (risk of infertility and ectopic pregnancy) in women. Therefore, in one embodiment, topical (intravaginal) application of IL-12 incorporated into polymeric microspheres is used to promote a local Th1 immune response for treatment and prevention against Chlamydia trachomatis. .. In one embodiment, the methods of the invention are used to treat genitourinary infections caused by Neisseria gonorrhoeae and Chlamydia trachomatis. This may be advantageous in the STD clinic, as gonorrhea and chlamydia present with similar signs and symptoms, and the differential diagnosis may depend on the identification of the causative organism. In addition, mixed infections of both are common. In other embodiments, other reproductive tract infections can also be treated with (intravaginal) application of microencapsulated IL-12 to enhance local immunity to them.

In other embodiments, topical application of microencapsulated IL-12 is used in the treatment of other local mucosal infections in which a normal immune response is inadequate to eliminate them. Examples include bronchitis and chronic obstructive pulmonary disease (airway), otitis media (middle ear infection, which is the most frequent reason for pediatric visits in the United States), Helicobacter pylori infection (which causes gastric ulcer and results in gastric cancer). (Get), and perhaps periodontal disease, which afflicts most adults after age 35, and is a major cause of tooth loss in adults.

In one embodiment, IL-12 can be administered with a vaccine containing a gonococcal antigen. For example, IL-12 can be administered with a locally administered non-viable gonococcal vaccine. As described in Example 3, the inventors gave mice i.vag by administering the Neisseria gonorrhoeae outer membrane vesicle (OMV) preparation with or without IL-12 / ms. I was immunized. OMV contains the majority of natural conformational surface antigens that have not been denatured by heat or chemical inactivation. This result demonstrates the generation of a Th1-induced antibody-dependent defense immune response that lasts for at least several months and is effective against antigenically diverse gonococcal strains. Immunization can also be performed via the intranasal route, as described in Example 4.

OMV and IL-12 microspheres may be administered to female or male subjects. When administered to a male subject, the composition may be delivered intranasally, and when administered to a female subject, the composition may be delivered intravaginally and / or intranasally.

The following examples are provided to further illustrate the present disclosure. These examples are not intended to be limiting.

[Example 1]
The present invention has been demonstrated in a mouse model of vaginal gonococcal infection. Details of the mouse model can be found in "Jerse, Infect. Immun. 67: 5699-5708; 1999".

Intravaginal administration of IL-12 microspheres (1 μg) to mice on days 0, 2, and 4 of temporary gonococcal infection (day 0) resulted in infection compared to control mice administered empty microspheres. Clearance was promoted (see Figure 1).

FIG. 1 shows the effect of intravaginal treatment (day 0, day 2, and day 4) with 1 μg of IL-12 encapsulated in PLA microspheres on the course of gonococcal vaginal infection in mice. Data shown as mean ± SEM cfu of gonococci recovered from daily vaginal swabs; 8 mice in each group. Control mice received empty microspheres. Mice were treated with antibiotics on day 14 and then rested for secondary infection (see Figure 2).

Mice treated with IL-12 microspheres during primary gonococcal infection were recovered, treated with antibiotics (ceftriaxone) on day 14, rested, and then re-infected with gonococcus one month later. The secondary infection was cleared faster than the control mice treated with empty microspheres during the primary infection (see Figure 2). Usually, the secondary infection is considered to disappear with the same dynamics as the primary infection, and the expression of antibody reaction is hardly or completely observed.

FIG. 2 shows the effect of intravaginal administration of IL-12 microspheres during primary gonococcal infection (see FIG. 1) on the course of secondary gonococcal infection in mice. Control mice were treated with empty microspheres. Data shown as mean ± SEM cfu of gonococci recovered from daily vaginal swabs; N = 8 mice in each group.

Furthermore, the effect of intravaginal treatment with microencapsulated IL-12 (IL-12 microspheres) on the course of mouse reproductive tract infection by Neisseria gonorrhoeae was compared with soluble IL-12. For this purpose, 1 μg of IL-12 was given in a free soluble form (ie, every other day until the infection disappeared) 0, 2, 4, 6, 8, and 10 days after infection with N. gonorrhoeae. (Dissolved in sterile phosphate buffered saline) was injected intravaginally into a group of 8 mice and compared directly with IL-12 microsphere-treated mice and vehicle-only controls. Mice treated with IL-12 microspheres cleared the infection within 7 days, much faster than the control group, whereas mice treated with soluble IL-12 cleared the infection at the same rate as the control group (Fig. 3). reference). Data shown as mean ± SEM cfu of gonococci recovered from daily vaginal swabs; N = 8 mice in each group.

The results indicate that topical intravaginal treatment with soluble IL-12 did not affect the course of infection, but IL-12 microspheres promoted clearance as described above.

[Example 2]
This example describes another series of experiments exemplifying the effectiveness of vaginal application of IL-12 microspheres for gonococcal vaginal infections.

Materials and Methods Mice: BALB / c mice were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained under standard conditions at the Laboratory Animal Facility at the University of Buffalo. All animal use protocols have been approved by the Animal Care and Use Committee of the University of Buffalo.

Bacteria: Neisseria gonorrhoeae FA1090, hemoglobin, concentrated medium (BD Diagnostic Systems, Franklin Lakes, NJ) were cultured on GC agar supplemented with the Isovitalex ^{(registered trademark)} is. Growth was checked for colony morphology consistent with Opa protein and pili expression, gonococci were collected from plates and cell densities were determined. Opa expression was as follows: Opa A, B / D / G, E / K.

Microspheres : Cytokines were encapsulated in polylactic acid (PLA) microspheres using the Phase Inversion Nanoencapsulation (PIN) technique as follows. Briefly, recombinant IL-12 (mouse or human) is mixed in water with an excipient containing sucrose (0.1%, w / w) and polyvinylpyrrolidone and then lyophilized. The lyophilized material is dissolved in tert-butyl alcohol (TBA) and mixed with polylactic acid (PLA) resomer dissolved in TBA (micronized IL-12 and PLA solution ratio 1: 3, vol. : Vol). The solution is then poured into 100-fold volume heptane to induce particle formation. The particles are then filtered and lyophilized. Three formulations were prepared: (a) control microspheres free of cytokines or antibodies; (b) mouse IL-12 (0.25 μg / particle 1 mg); and (c) mouse IL-17 (0.25 μg / particle 1 mg). ..

Mouse vaginal infection model : Female mice aged 7 to 9 weeks were infected with live gonococcus FA1090 intravaginally on day 0 as described above. Vaginal mucus was quantitatively cultured daily on GC agar supplemented with selective antibiotics, and the amount of bacterial colonies formed was measured. The detection limit was 100 CFU (recovery per mouse). Vaginal treatment with a microsphere preparation is performed from day 0 to day 8 by injecting a 40 μl suspension of microsphere containing IL-12 or IL-17 in PBS or a control microsphere. It was done every other day until the eyes.

Cell isolation and flow cytometry : Mice were sacrificed and iliac lymph nodes (ILNs) and genital tracts were aseptically resected. ILN was cleaved in Hanks buffered salt solution to release cells. Vaginal single cell suspensions were prepared by enzymatic digestion. The isolated cells were washed twice with staining buffer, then incubated with the antibodies shown for 30 minutes on ice, washed twice and analyzed on a FACSCalibur cytometer. For measurement of intracellular cytokine expression, cells, a protein transport inhibitor phorbol myristate acetate - ionomycin -GOLGISTOP ^{(TM) (eBioscience, San Diego, CA} ) for 5 hours restimulated with, then fixed / a transmission processing solution Cytofix / Cytoperm was fixed ^(R) (eBioscience). Antibodies to mouse CD4, CD8, CD19, CD11b, CD11c, NKG2D, Gr-1, IFN-γ, IL-4, and IL-17A bound to fluorescein isothiocyanate, phycoerythrin, or allophycocyanin were purchased from eBioscience.

Cytokine ELISA : IL-12p70, IFN-γ, IL-4, IL-5, and IL-17A concentrations in serum or vaginal lavage samples were measured in triplicate using an ELISA kit purchased from eBioscience.

Real-time RT-PCR: Total cellular RNA of all vaginal collected from mice, RNEASY ^(TM), RNA Purification mini kit isolated (Qiagen, Valencia, CA) in a single, iScript ^(TM) cDNA Synthesis Kit (Bio-Rad , Hercules, CA) was transcribed into cDNA. For real-time monitoring of PCR, using SYBR ^{(registered trademark)} Green Dye (Bio-Rad) which is a fluorescent dye, with ICYCLER IQ ^(TM) Real-Time PCR Detection System (Bio-Rad), real-time RT -PCR was performed. Relative quantification of target genes was analyzed based on the threshold cycle (Ct) determined by Bio-Rad IQ ^TM 5 optical system software.

Serum and mucosal antibody assays : Saliva, vaginal lavage fluid, and serum samples were taken from individual mice 15 days after inoculation. Gonorrhea-specific IgA, IgG, and IgM in saliva, serum, and vaginal lavage fluid, and total IgA, IgG, and IgM concentrations in secretions were measured by ELISA.

Statistical analysis : Represent the data as mean ± standard error of mean (SEM). Data on the effects of IL-12-, IL-17-, anti-TGF-β-, anti-IL-10 load ANOVA-vs-air ANOVA treatment on vaginal gonococcal infections by Bonferroni corrected test of pairwise comparison It was analyzed using repeated measures analysis of variance (ANOVA). Kaplan-Meier analysis by log-rank test was also used to compare infection clearance. Data from in vitro experiments were analyzed by unpaired two-sided t-test to compare the mean values between the two selected groups. P <0.05 was considered statistically significant.

[result]
Intravaginal administration of IL-12 microsphere protects mice from gonococcal infection
To investigate the therapeutic effect of IL-12-loaded microspheres, female BALB / c mice were infected with gonococci and vaginal swab cultures were used to monitor bacterial levels daily. Preliminary dose range experiments show that an intravaginal infusion of microspheres containing 1.0 μg IL-12 is sufficient to accelerate clearance of infection compared to treatment with empty microspheres. It was shown that there was no further enhancement of clearance with 2.0 μg microencapsulated IL-12, and lower doses were progressively less effective (Fig. 4A).

Untreated or empty microsphere-treated mice cleared the infection in about 15 days (FIGS. 4B, C). Intravaginal injection of an optimal 1.0 μg dose of microencapsulated IL-12 significantly reduced the amount of gonococci that could be recovered from day 4, and these mice were 7,8 more than empty microsphere-treated or untreated mice. The infection was cleared by the earliest day (Fig. 4B, C). The infection did not recur after treatment was discontinued on day 7. In contrast, intravaginal administration of free-soluble IL-12 had no effect on enhancing gonococcal clearance (FIGS. 4B, C).

Intravaginal administration of IL-17-loaded microspheres at the optimal dose (1.0 μg) also accelerated clearance for gonococcal infection, but to a lesser extent than IL-12 microspheres given on the same schedule (Fig. 4B, FIG. C).

Treatment with IL-12 microsphere enhances Th1 and antibody response to vaginal gonococcal infection
To elucidate the underlying mechanism of the therapeutic effect of IL-12, we characterized the local immune response to genital gonococcal infection in mice treated with IL-12-loaded microspheres or empty microspheres. Seven mice in each group 3, 5, 7, and 14 days after inoculation with N. gonorrhoeae or vehicle alone for the purpose of evaluation by flow cytometry to detect intracellular IFN-γ, IL-4, IL-17. A single cell suspension was prepared from ILN and vagina. From day 3 post-inoculation, IL-17 ⁺ / CD4 ⁺ T cells were observed in locally excreted ILN, production peaked on day 5 and persisted for the duration of infection. On day 5, about 22% of CD4 ⁺ T cells present in the ILN of control-treated infected mice were IL-17 ⁺ , whereas IFN-γ ⁺ was only about 3.5%, IL-4 ⁺ /. Almost no CD4 ⁺ T cells were detected (Fig. 4D). IL-12 microsphere treatment significantly enhanced the Th1 immune response against N. gonorrhoeae, as indicated by a significant increase in the number of IFN-γ ⁺ / CD4 ⁺ T cells (Fig. 4D). In contrast, IL-12 microspheres responded to Th2 or Th17 because the numbers of IL-4 ⁺ / CD4 ⁺ and IL-17 ⁺ / CD4 ⁺ T cells in ILN were similar between treatment groups. Not changed (Fig. 4D). As a result of RT-PCR analysis, the expression of IFN-γ was increased in the vagina of infected mice after treatment with IL-12 microspheres, but the expression of IL-4 or IL-17 mRNA was not increased (Fig. 4E). .. IL-17 microspheres improved gonococcal infection, although this treatment was not associated with enhanced Th1 or Th2 response (Fig. 4D, E), but the influx of Gr-1 ⁺ neutrophils into the genital tract. There was an increase (Fig. 4F).

In addition, the IL-12p70, IFN-γ, IL-4, and IL-17 concentrations in the vaginal lavage fluid and serum collected 7 days after inoculation were measured by the ELISA method. IL-12 (176.5 ± 48.6 pg / ml) was detected in the vaginal lavage fluid of infected mice treated with IL-12 microspheres. Since low levels of IL-12 (41.7 ± 10.7 pg / ml) were found in the sera of these mice, the effect of IL-12 microsphere treatment on gonococcal infection was mainly the transfer of cytokines into the circulating blood. It was suggested that it was not caused by. Consistent with flow cytometry, IFN-γ was present in vaginal lavage fluid (32.6 ± 9.8 pg / ml) and serum (43.3 ± 11.5 pg / ml) of infected mice treated with IL-12 microspheres, but IL. -4 and IL-17 were not detected. None of these cytokines were detected in control-treated infected mice.

IL-12 can stimulate the humoral immune response either IFN-γ-dependently or directly. Therefore, it was investigated whether treatment with IL-12 microspheres during gonococcal infection leads to the production of anti-gonococcal antibodies in vaginal lavage fluid, saliva, and serum collected 15 days after inoculation. IgM antibodies were low and there was little difference between the experimental groups (data not shown). No salivary gonococcal-specific antibodies were detected in any of the mouse groups (data not shown). Gonorrhea infection in control-treated mice did not significantly increase gonococcal-specific IgA or IgG antibodies in either vaginal lavage fluid or serum. However, IL-12 microsphere treatment increased vaginal and serum specific IgG antibody production (Fig. 4G, H), as well as vaginal specific IgA antibody production (Fig. 4G).

Treatment with IL-12 microspheres induces defensive pre-immunity against secondary gonococcal infection In addition, it was evaluated whether IL-12 microsphere treatment provides protective immunity against immune memory generation and reinfection. IL-12-loaded microspheres or empty microspheres were administered to a group of mice infected with gonococci, and after following the course of infection, mice were administered ceftriaxone (300 μg ip) on the 15th day to completely eliminate gonococci. Confirmed that it was eliminated. An additional group of pseudo-infected mice treated with IL-12 microspheres was used to assess the potential for sustained effects of IL-12 in the absence of infection. After 5-6 weeks, all mice were inoculated with the same strain of N. gonorrhoeae, but no further treatment was performed. As previously observed, the primary infection of control-treated mice did not protect the mice against subsequent secondary attacks: the duration and bacterial abundance of secondary gonococcal infection in previously empty microsphere-treated mice. , Same as the primary infection of age-matched naive mice (FIGS. 5A, B). In contrast, intravaginal administration of IL-12-loaded microspheres during primary infection protected mice from secondary infection. Reinfected mice that received IL-12 microspheres during the primary infection resisted the challenge more effectively than controls (FIGS. 5A, B). However, treatment of pseudo-infected mice in the past with IL-12 microspheres did not induce protection against subsequent infections (FIGS. 5A, B). The results also ruled out the possibility that the remaining microspheres still affect the secondary infection with N. gonorrhoeae.

ILN cell and vaginal flow cytometry and RT-PCR analysis collected on days 5 and 3 of the secondary infection showed that the protective effect of past IL-12 microsphere treatment against secondary gonococcal infection was Th1 ( It was also shown to be associated with a significant enhancement of the IFN-γ) response (FIGS. 5C, D). In addition, in IL-12 microsphere-treated mice, a strong specific secondary antibody reaction was observed after reloading with gonococci. Gonorrhea-specific IgA and IgG antibodies in the vaginal lavage fluid of re-infected mice previously treated with IL-12 microspheres, as well as IgG antibodies in serum, were significantly higher than in the control group (FIGS. 5E, F).

In contrast to the effects of IL-12 microsphere treatment, IL-17 microsphere treatment during primary gonococcal infection did not provide protective immunity against secondary gonococcal infection and did not elicit a history of T cells or antibody response ( 5A-F).

[Example 3]
This example describes that this experimental vaccine induces a Th1-induced immune response and resistance to gonococcal infection in a mouse model.

result

Vaginal immunization of mice with gonococcal OMV + IL-12 / ms accelerates clearance of gonococcal-induced infection
A group of 8 female BALB / c mice was immunized with gonococcal OMV (FA1090 strain; protein 40 μg) + IL-12 / ms (IL-12 1 μg) or OMV + control (empty) ms with i.vag; Two additional control groups were pseudoimmunized with IL-12 / ms or empty ms alone. Immunization was repeated after 1 and 2 weeks, and after 2 weeks, all mice were challenged by intravenous infusion of gonococcal FA1090 (5 × 10 ⁶ CFU) i.vag. Control (pseudo-immunized) mice, or mice immunized with OMV + empty ms, began to clear the infection 7 days after the challenge and all by 15 days; the central day of clearance was 10-13 days. .. There was no significant difference in clearance rate between these three control groups (Fig. 6a). However, mice immunized with OMV + IL-12 / ms began to clear the infection from day 6 and all cleared by day 9; clearance compared to day 12 of mice immunized with OMV + empty ms. Median = 7.5 days (P <0.01, Kaplan-Meier; Table 1 and Figure 6a). This experiment was repeated twice more and similar results were obtained (see Table 1 and FIG. 13). Further examples of replication of this finding can be found in subsequent experiments reported below. For example, FIGS. 6d, 9, 10a and c) (and for other N. gonorrhoeae strains, FIGS. 10b, d and e), and FIGS. 12a, b, f and g (in C57BL / 6 mice).

Serum and vaginal lavage samples taken after clearance (at the end, 15 days after inoculation) were assayed by ELISA for antibodies against intact gonococci (FA1090). This is because the expression levels of IgG and IgA antibodies in vagina and serum were highest in mice immunized with OMV + IL-12 / ms, but the expression levels of these antibodies were much higher in mice immunized with OMV + empty ms. It was shown to be low (Fig. 6b). Non-immunized, pseudo-infected mice showed no detectable antibody beyond the assay background at the initiating dilution, were immunized with empty ms alone, and infected mice also did not express detectable antibody ( FIG. 6b). Simultaneously collected iliac lymph node (ILN) mononuclear cells were stained for surface CD4 expression and intracellular cytokines and analyzed by flow cytometry. This is because only mice immunized with OMV + IL-12 / ms produced CD4 ⁺ / IFNγ ⁺ (and CD8 ⁺ / IFNγ ⁺ ) T cells, but expressed a significant number of CD4 ⁺ / IL-4 ⁺ T cells. It was revealed that there were no mice (see FIG. 6c; see also FIG. 14). However, regardless of immunization, all mice infected with N. gonorrhoeae expressed CD4 ⁺ / IL-17 ⁺ T cells (FIGS. 6c and 14).

Further experiments were performed to determine the minimum number of immunizations required to induce immune resistance to infection. A single dose of OMV + IL-12 / ms given with i.vag did not consistently develop resistance to the challenge, but at 2-week intervals of OMV (protein 40 μg) + IL-12 / ms (IL-12 1 μg). Two doses in were sufficient to induce similar resistance to infection. Median clearance = 8 days (Fig. 6d; Table 1). Furthermore, control immunization with OMV + IL-12 / ms prepared from untyped Haemophilus influenzae (NTHI) did not induce resistance to N. gonorrhoeae, with a median clearance of 13 days (Fig. 6d; Table 1). This induced antibodies against NTHI but not against Neisseria gonorrhoeae (FIGS. 15a and b), producing IFNγ-producing CD4 ⁺ and CD8 ⁺ cells in ILN (FIG. 15c).

Intravaginal immunization with gonococcal OMV + IL-12 / ms is performed after immunization with gonococcus OMV + IL-12 / ms one, two, or three times, which induces a sustained gonococcal-specific antibody reaction and Th1 cell reaction, and before the challenge. Serum, vaginal lavage fluid, and ILN were collected from immune and control mice 2 weeks after final immunization to reveal local and systemic immune responses. Serum anti-gonococcal IgM antibodies were low and there was little difference between the experimental groups. IgA and IgG antibodies were not detected beyond the background in vaginal lavage fluid or serum samples of mice administered with empty ms only. Intravaginal immunization with three doses of gonococcal OMV + empty ms increased anti-gonococcal IgA and IgG antibodies in the vagina and serum, but to a lesser extent than immunization with OMV + IL-12 / ms (Fig. 7a). In contrast, immunization with a single dose of OMV + IL-12 / ms induced low levels of anti-gonococcal antibody in both serum and vaginal lavage fluid; while the second dose increased antibody production. No further increase was seen after 3 doses (Fig. 7a). Antibodies were not detected above control levels against E. coli or NTHI, suggesting that they are specific to N. gonorrhoeae. Assays for IgG subclass antibodies in both vaginal lavage fluid and serum showed IgG2a predominance, low IgG1 and IgG2b levels, and low IgG3 levels (FIG. 16). Production of anti-gonococcal IgA antibody and IgG antibody in vaginal lavage fluid and serum peaked 3 months after immunization with a double inoculation of OMV + IL-12 / ms and was still detectable 6 months after immunization (Fig.). 7b and c).

Flow cytometric analysis of ILN cells revealed an increase in the number of IFNγ ⁺ / CD4 ⁺ and IFNγ ⁺ / CD8 ⁺ T cells in mice immunized with OMV + IL-12 / ms compared to those of control mice ( FIG. 8a). As observed in the antibody response, one immunization was sufficient to induce IFNγ production, which was further increased by two immunizations; no further increase by three immunizations. In contrast, immunization with OMV + IL-12 / ms did not significantly increase the number of IL-4 ⁺ / CD4 ⁺ and IL-17 ⁺ / CD4 ⁺ T cells compared to controls (Fig. 8a). ). CFSE was preloaded into CD4 ⁺ cells isolated from ILN to determine if the induced IFNγ ⁺ / CD4 ⁺ (and IFNγ ⁺ / CD8 ⁺ ) T cells were specific for the gonococcal antigen. The cells were cultured in vitro for 3 days in the presence of antigen-presenting cells with or without gonococcal OMV or as a control, and their growth was evaluated by flow cytometry. ILN-derived CD4 ⁺ cells from immune mice proliferated significantly and produced significantly more IFNγ in response to in vitro stimuli than cells from control mice (Fig. 17A). No production of IL-4 was observed, but IL-17 was produced by CD4 ⁺ T cells cultured with Neisseria gonorrhoeae OMV (Fig. 17A). IFNγ production by ILN CD4 ⁺ T cells remained elevated for 4 months after immunization, although levels declined slowly (Fig. 8b).

In addition, 3 days after the last immunization, the vagina was resected from the euthanized mice and RNA was extracted from all tissues. In RT-PCR analysis, gonococcal OMV + IL-12 / ms immunization significantly enhanced IFNγ mRNA expression, but not IL-4 or IL-17 mRNA expression compared to controls. It was shown that (Fig. 17B). IFNγ mRNA expression in vaginal tissue and IFNγ production by ILN CD4 ⁺ cells remained elevated up to 2 months after i.vag immunization with Neisseria gonorrhoeae OMV + IL-12 / ms (Fig. 17C). These findings support the cytokine expression results obtained in cells from excreted ILN.

Duration of Vaccine-Induced Infection Resistance To assess the duration of immunization, a group of 8 mice was immunized with Neisseria gonorrhoeae OMV + IL-12 / ms and immunized with 2, 4, or 6 A month later, I challenged with the same gonococcal strain (FA1090). Compared to age-matched controls that were immunized with pseudoimmunization or OMV + empty ms, mice immunized with OMV + IL-12 / ms were challenged 2 or 4 months after immunization. It was resistant to gonococcal infection; the median clearance of the control group was 11 to 11.5 days, whereas that of immunized mice was 7 days (Table 1 = Supplementary Table 1). Similar results were obtained in repeated experiments when mice were challenged 6 months after immunization; median clearance of controls = 9.5-10 days versus 6.5 in mice immunized with OMV + IL-12 / ms. It was a day (Fig. 9; Table 1). After completion, anti-gonococcal antibodies were detected in serum and vaginal lavage fluid (FIGS. 18A and B). IFNγ (not IL-4-) secreted CD4 ⁺ T cells were present in ILN (Fig. 18C). Notably, the antibody and IFNγ responses detected after the challenge were higher than before the challenge (compared to FIGS. 7b, c, and 8b), implying memory recall. As previously observed, IL-17 secretory T cells were always found after challenge with N. gonorrhoeae, regardless of immunization (Fig. 18C). Mice were not evaluated for a longer period of time as they became more resistant to gonococcal infection as they age.

Immunization induces resistance to heterologous strains of N. gonorrhoeae An important consideration for any vaccine should be that it should be effective against strains with different pathogens as well as strains that are antigenically homologous to the immune strain. Is. N. gonorrhoeae is well known for its antigenic variability in which most of its surface antigens are involved via multiple molecular mechanisms. Therefore, we determined whether i.vag immunization in one strain of N. gonorrhoeae OMV was as effective against challenges by other strains as it was by the same strain. First, mice (8 in each group) were immunized with i.vag using OMV + IL-12 / ms or empty ms prepared from FA1090 strain, and 1 month later, gonococcal strain FA1090 or MS11 (5 × 10 ⁶ CFU). ) Was challenged. Immunization with FA1090 OMV induced resistance to challenge with FA1090 or MS11 to the same extent (Fig. 10a; Table 2). After challenge and clearance, the antibody was elevated to similar levels against MS11 and the Th1 response exhibited by IFNγ ⁺ / CD4 ⁺ T cells in ILN was similarly enhanced (FIGS. 19A, B, and C). .. In a reciprocal manner, immunization with MS11 OMV (+ IL-12 / ms) induced resistance to the challenge in the FA1090 strain (Fig. 10b; Table 2).

Both N. gonorrhoeae strains FA1090 and MS11 carry the same major type of porin (PorB.1B), although they have different subtypes. Therefore, further experiments were performed with the FA19 strain (PorB.1A) to determine if the major porin type is essential for immune resistance. Immunization with FA1090 OMV (+ IL-12 / ms) induced resistance to the challenge with FA19 (Fig. 10c; Table 2). The antibody response assayed at the end showed cross-reactivity to FA19 and IFNγ ⁺ / CD4 ⁺ T cells in ILN were elevated (FIGS. 19D, E, and G). Reciprocally, immunization with FA19 OMV induced resistance to the challenge with the FA1090 strain (Fig. 10d; Table 2). Other immunization and challenge combinations (ie, MS11 to FA19 and vice versa) similarly showed similar cross-resistance (Table 2).

N. gonorrhoeae FA1090, MS11, and FA19 strains are widely used in many laboratories and have been extensively subcultured since their originals were isolated. As a result, mutations may have been acquired and some of their characteristics may have changed. Therefore, we also challenged immune mice with a novel clinical strain that has been minimally passaged in vitro since isolation. Mice immunized with FA1090 OMV + IL-12 / ms were also resistant to the challenges of clinical isolates GC68 (PorB.1B; FIG. 10e; Table 2) and GC69 (PorB.1A; Table 2).

Antigens Targeted by Immune-Inducing Antibodies The protein profiles of FA1090, MS11, and FA19 OMV were similar when tested by one-dimensional (1D) SDS-PAGE, but with obvious quantitative and qualitative mutations. (Fig. 11a). Western blot analysis of sera from one FA1090 OMV + IL-12 / m-immunized mouse (# 1) against FA1090, MS11 or FA19 OMV isolated by SDS-PAGE migrates at approximately 35-80 kDa. The band-reactive IgG antibody was clarified and the reactivity to the band present in OMV of all three strains was shown (Fig. 11b, lanes 2-4). Since the H5 antibody reacted with bands of similar mobility, one of these bands of about 35 kDa may correspond to porin (Fig. 11b, lane 6). Another serum (# 2) showed strong reactivity to three bands migrating at about 45-65 kDa (Fig. 11b, lane 5). An immunoproteomics approach consisting of two-dimensional (2D) SDS-PAGE separation and parallel 2D SDS-PAGE of OMV followed by immunoblotting (2D-immunoblot) and mass spectrometry to identify antigens of approximately 45-65 kDa. Using. The three 2D protein maps of OMV revealed by Flamingo staining showed significant differences in the OMV proteome between a large number of protein species and the FA1090, MS11, and FA19 strains (FIGS. 11c, d, and e). .. In contrast, the blotted protein maps show 45 kDa and 43 kDa, respectively, and two spots (spots 1 and 2) with masses corresponding to pI 5.2 and 5.5 (FA1090 OMV; Figure 11c), or an approximate mass of 45 kDa and One spot with pI 5.2 (Spot 1) (MS11 and FA19 OMV; FIGS. 11d and e) is shown. Mass spectrometry of trypsin peptides obtained from Spot 1 and Spot 2 (FIGS. 11c, d, and e) are translation elongation factor-Tu (EF-Tu) as a top hit and a putative periplasmic polyamine binding protein, respectively. , PotF3 (Table 3) was clarified. EF-Tu emerged as the most reliable antigen because it was immunoreactive in all three 2D-immunoblots, with the highest reliability (scores 485.0-947.1) and coverage (scores 485.0-947.1) and coverage (scores 485.0-947.1) for all OMV formulations. It was identified in 64.2-90.6) (Table 3).

Immune resistance to Neisseria gonorrhoeae depends on IFNγ and antibodies
IFNγ (IFNγ) is used to determine whether the protective effect of IL-12 / ms-enhanced OMV immunization depends on the IFNγ or antibody response, or the immunity determined by CD4 ⁺ or CD8 ⁺ T cells. -ko) Immunization experiments were performed using mutant C57BL / 6 mice deficient in B cells (μMT), CD4 ⁺ T cells (CD4-ko), or CD8 ⁺ T cells (CD8-ko). A group of 8 C57BL / 6 wild-type (control) and immunodeficient mice were immunized with FA1090 OMV + IL-12 / ms or empty ms and challenged with gonococcus FA1090 (5 × 10 ⁶ CFU) one month later. .. The course of vaginal gonococcal infection was unchanged in unimmunized immunodeficient mice compared to wild-type controls. All wild-type and immunodeficient mice began to reduce the amount of recoverable gonococci from days 7-11 and cleared the infection by days 12-14 (median 9-13). This was similar to the BALB / c mice used in the experiments described in the previous section (FIGS. 12a, b, f, g; Table 4).

In contrast to wild-type mice, IFNγ-ko or μMT mice immunized with OMV + IL-12 / ms did not accelerate clearance for gonococcal infection compared to those immunized with OMV + empty ms (Figure). 12a and b; Table 4; FIGS. 20A and B). Thus, deficiency of either IFNγ or B cells suppressed the adjuvant effect of IL-12 / ms on the development of immune resistance to gonococcal infection. OMV + IL-12 / ms-induced production of gonococcal-specific vaginal and serum IgA and IgG antibodies in wild-type mice was suppressed in IFNγ-ko mice (FIGS. 12c and d), and ILN in immunized IFNγ-ko mice. The production of IFNγ by cells was not observed as expected (data not shown). Similarly, there was no detectable antibody response to immunization in μMT mice (data not shown). In contrast, the number of IFNγ ⁺ / CD4 ⁺ T cells in ILN of μMT mice immunized with N. gonorrhoeae OMV + IL-12 / ms was unaffected and had no IL-4 response, but altered IL-17 response. There was no (Fig. 10e = Fig. 5E). These findings indicate that the resistance induced by immunization with N. gonorrhoeae OMV + IL-12 / ms depends on both IFNγ and B cells, the latter probably producing N. gonorrhoeae-specific antibodies.

The immune protective effect of Neisseria gonorrhoeae OMV + IL-12 / ms was incompletely reduced in CD4-ko and also in CD8-ko mice compared to wild-type controls (FIGS. 12f and g; Table 4; FIGS. 20C and D). Partially decreased. These findings suggest that the need for CD4 ⁺ T cells to develop immune resistance can be partially supplemented by other cells, such as CD8 ⁺ or NK cells, which can also produce IFNγ. However, CD8 ⁺ cells did not appear to be less important for protective immunity.

Discussion We have demonstrated for the first time that an immune-resistant vaccine-induced state against gonococcal infection is reliably produced by the intact mammalian immune system. This state of immunity was thought to be dependent on the production of antibodies by B cells and mainly by the production of IFNγ by CD4 ⁺ T cells. I.vag vaccination of mice with gonococcal OMV + IL-12 / ms (as an adjuvant) induced serum and vaginal IgG and IgA antibodies against gonococcal antigens, and CD4 ⁺ and CD8 ⁺ T cells secreting IFNγ in excreted ILN. .. Both the Th1 cell reaction and the antibody reaction persisted for several months after immunization, and were able to induce resistance to gonococcal attack for at least 6 months, evoking immune memory. I.vag immunization with N. gonorrhoeae OMV alone, with or without adjuvant (empty) ms, did not involve detectable IFNγ production, showed no significant resistance to evoked infections, and elicited only a weak antibody response. There wasn't. Control immunization with OMV + IL-12 / ms prepared from NTHI induced an IFNγ response, but did not produce antibodies showing immune resistance or cross-reactivity with gonococci. Therefore, IFNγ appears to be required for resistance to N. gonorrhoeae, but not sufficient without specific antibodies.

It is reported herein that IL-12 / ms administered with i.vag as an adjuvant, along with the gonococcal OMV vaccine, enhances Th1-induced defense immunity revealed by a significantly shortened course of genital gonococcal infection. .. It should be emphasized that free-soluble IL-12 was ineffective and that IL-12 encapsulated in ms was required for the adjuvant effect with OMV.

Given the widespread well-known antigenic variation exhibited by N. gonorrhoeae, resistance unexpectedly extended not only to homologous strains for which the OMV vaccine was prepared, but also to heterologous strains. From this result, immunization with OMV derived from FA1090 strain equally enhanced resistance to MS11 strain and FA19 strain (and vice versa), and in addition to these "laboratory strains", clinical gonococci It is shown that the isolates are also resistant. Among the major gonococcal surface antigens, FA1090, MS11, and FA19 have been found to differ in their porin (PorB) molecules. FA1090 and MS11 have different subtypes of PorB.1B, and FA19 has PorB.1A (Elkins et al., Mol. Microbiol. 14, 1059-1075 (1994)). Although not well characterized, the Opa proteins encoded in their genomes are different (Hobbs et al., Front. Microbiol. 2, 123 (2011), Cole et al., PLoS One 4, e8108 ( 2009)), and their LOS are different (Erwin et al., J. Exp. Med. 184, 1233-1241 (1996)). The Opa protein and LOS glycan chains are also phase-variable, resulting in the expression of different antigenic epitopes (Apicella, MA et al. Phenotypic variation in epitope expression of the Neisseria gonorrhoeae lipooligosaccharide. Infect. Immun. 55, 1755-1761. (1987)).

Consistent with cross-defense immunity, ELISA analysis of immune-induced antibodies reveals detectable, quantitatively similar levels of antibodies against different strains for both IgG and IgA in serum and vaginal lavage fluid. did. Antibodies were not detected against E. coli or NTHI, suggesting that they are specific to N. gonorrhoeae, and they were not produced by immunization with OMV prepared from NTHI. However, Western blot analysis of serum IgG antibodies revealed evidence of antigens shared between different strains of N. gonorrhoeae. Bands migrating at 45-65 kDa migrated at higher molecular weights than major gonococcal outer membrane proteins such as porin and Opa in the range 30-40 kDa.

Our study identified two novel gonococcal vaccine candidates: EF-Tu in FA1090, MS11, and FA19 OMV, and PotF3 in FA1090. Both proteins have been identified in cell envelope and quantitative proteomics profiling of OMVs from four common gonococcal isolates (Zielke et al., Molec. Cell. Proteomics 13, 1299-1317 (2014)). EF-Tu is of particular interest as it has been identified in both spots and all analyzed OMVs. EF-Tu is a cytoplasmic GTP-binding protein and is generally recognized as an essential factor in protein synthesis.

The present disclosure demonstrates that an individual can be immunized against N. gonorrhoeae by administration of i.vag, an inactivated vaccine (OMV), with the Th1-induced adjuvant IL-12 / ms. These findings show the feasibility of a vaccine against Neisseria gonorrhoeae despite past setbacks, and these findings also shed light on the types of immune responses that need to be induced to generate protective immunity. ing.

Method
Mice Wild-type BALB / c and C57BL / 6 mice, for C57BL / 6 ^{background, B6.129S7-Ifng tm1Ts / J (} IFNγ- ^{deficiency), B6.129S2-Ighm tm1Cgn / J} (B cell - deficient; as μMT All mice were purchased from Jackson Laboratories (Bar Harbor, ME), including (known), B6.129S2-Cd4 ^tm1Mak / J (CD4-deficient), and B6.129S2-Cd8a ^tm1Mak / J (CD8-deficient) mice. did. Unless otherwise stated, BALB / c mice were used in the experiments. Mice were managed at the BSL2 facility, a laboratory animal facility at the University of Buffalo, fully accredited by AAALAC. All animal use protocols have been approved by the Animal Care and Use Committee of the University of Buffalo.

Bacterial gonorrhoeae strain FA1090 is maintained at Dr. Janne Cannon (University of North Carolina at Chapel Hill), MS11 strain is maintained at Dr. Daniel Stein (University of Maryland), and FA19 strain and clinical isolates are maintained at the University of North Carolina at Chapel Hill. Obtained from a collection of clinical strains. For use in a mouse infection model, N. gonorrhoeae strains 9087 and 336 were transformed with the streptomycin-resistant rpsL gene from the FA1090 strain to generate GC68 and GC69 strains, respectively. E.coli K12 was provided by Dr. Terry Connell (University at Buffalo). Unclassifiable Haemophilus influenzae (NTHI) strain 6P24H1 was provided by Dr. Timothy Murphy (University at Buffalo). Gonorrhea was cultivated on a GC agar supplemented with hemoglobin and ISOVITALEX® ⁽ BD Diagnostic Systems, Franklin Lakes, NJ), a concentrated medium, and the resulting growth was colonized to match Opa protein and pili expression. I checked the morphology. NTHI was cultured on GC agar supplemented with hemoglobin only. E. coli was cultured in BHI agar. Bacteria were harvested from plates and cell densities were determined (Liu et al., Mucosal Immunol. 5, 320-331 (2012)).

IL-12 microsphere mouse IL-12 (Wyeth, Philadelphia, PA) phase-reversed nanocapsules as previously described, except that bovine serum albumin was replaced with sucrose (0.1%, w / w). Encapsulated in polylactic acid microspheres using chemical techniques (Egilmez et al., Methods Mol. Med. 75, 687-696 (2003)). Empty microspheres were prepared in the same way without IL-12.

Neisseria gonorrhoeae outer membrane vesicle (OMV)
After culturing on supplemental GC agar for 18-22 hours, gonococci were harvested from a plate in ice-cold lithium acetate buffer (pH 5.8) and passed 10-12 times through a 25-gauge needle to shear the outer membrane from the bacteria. .. The suspension was spun in a microcentrifuge tube at 13,000 RPM for 1 minute. The supernatant was collected and centrifuged at 107,000 xg for 2 hours. The pellet was washed with 50 mM Tris-HCl (pH 8.0) and resuspended in PBS. Proteins were assayed with the Micro BCA protein kit (Thermo Scientific, Rockford, IL) or the RC DC protein assay kit (Bio-Rad, Hercules, CA).

Immunization schedule and mouse vaginal infection model
A group of 8 female mice 7-9 weeks old with various strains of gonococcal OMV (protein 40 μg) + IL-12 / ms (1 μg IL-12) or empty ms (total volume 40 μl PBS) i.vag was immunized and the control group was pseudoimmunized with IL-12 / ms or empty ms alone. Mice were immunized 1-3 times at 7-14 day intervals as shown. After an additional 2 weeks to 6 months, immunized mice were subjected to as previously described (Jerse, Infect. Immun. 67, 5699-5708 (1999; Liu et al., J. Infect. Dis. 208, 1821-). 1829 (2013)), 5 × 10 ⁶ CFU infected with live gonococci (0.5 mg Pfizer, Philadelphia, PA) was used as estradiol administered subcutaneously on days -2, 0, and 2. accompanied by modification of). vaginal swabs taken daily, hemoglobin, Isovitalex ^{(registered trademark)} (enriched media) and selective antibiotics (vancomycin, streptomycin, nisin, colistin, and trimethoprim) quantified on GC agar supplemented with The amount of bacterial colony formed was determined. The detection limit was 100 colony forming units (CFU) recovered per mouse. Gonorrhea recovery was "blinded" for experimental treatment. Counted by individuals, all experiments were repeated 2 or 3 times for verification.

Serum and Mucosal Antibody Assays Vaginal lavage fluid and serum samples were taken from mice at the time indicated (Liu et al., MBio 2: (2011)). Gonorrhea in vaginal lavage fluid and serum-specific IgA, IgG, IgM, and IgG subclass antibodies IgG1, IgG2a, IgG2b, and IgG3 were used in whole serum using undiluted vaginal lavage fluid and 10-fold diluted starting dilution. Measured by ELISA on a plate coated with gonococcus ^17,26 . Total IgA, IgG, and IgM concentrations in secretions were assayed by ELISA on plates coated with anti-IgA, -IgG, or -IgM antibodies (Southern Biotech, Birmingham, AL). A standard curve was established using H5 mouse monoclonal antibody (specific for gonococcal serotype PIB3) or affinity purified mice IgA, IgG and IgM (Southern Biotech). Bound antibodies were detected with alkaline phosphatase-bound goat anti-mouse IgA, IgG, IgM, IgG1, IgG2a, IgG2b, or IgG3 antibody (Southern Biotech) and p-nitrophenyl phosphate substrate (Southern Biotech). Plates were read with a VersaMax microplate reader using SoftMax software (Molecular Devices, Sunnyvale, CA) or an EL _X 800 universal microplate reader using KC Junior software (Bio-Tek Instruments, Winooski, VT). Antibody data were expressed as relative (-fold increase) to antibody levels detected in control samples (derived from pseudoimmune mice) assayed simultaneously.

Flow cytometry The isolated cells were washed twice with staining buffer, then incubated with the antibodies shown for 30 minutes on ice, washed and analyzed on a FACSCalibur cytometer. For intracellular staining, it was fixed first with Cytofix / Cytoperm cells ^{(registered trademark)} (eBioscience). Antibodies to mouse CD4, CD8, IFNγ, IL-4, and IL-17A bound to FITC, PE, or allophycocyanin were purchased from eBioscience.

Isolation and culture of lymphocytes Mononuclear cells were isolated from ILN aseptically collected using Histopaque 1083 (Sigma-Aldrich, St Louis, MO) density gradient centrifugation and from 2 or 3 mice. Pooled to provide sufficient numbers of cells for culture. CD4 ⁺ T cells were purified by negative selection using the Dynamic CD4 cell isolation kit (Invitrogen, Carlsbad, CA). Cells were cultured in 24-well culture plates at a density of 2 × 10 ⁶ cells / ml in the presence of the same number of mitomycin C inactivated spleen cells, either unstimulated or with 2 × 10 ⁷ gonococcal cells. Worked as an APC.

Growth assay cells were labeled with carboxymethylfluorescein succinimide ester (CFSE; Sigma-Aldrich). CFSE-labeled cells were then washed twice in PBS, recounted, and stimulated as described above. Cultured cells were harvested and then stained with allophycocyanin-binding anti-mouse CD4 antibody. Data were obtained by gating the CD4 ⁺ cell population with a FACSCalibur cytometer. Cell proliferation was measured using attenuation of CFSE fluorescence.

Cytokine ELISA
IFNγ, IL-4, and IL-17A levels were measured in triplicate using an ELISA kit purchased from eBioscience.

Real-time RT-PCR
Total cellular RNA of all vaginal collected from mice, isolated RNEASY ^(TM) RNA Purification mini kit (Qiagen, Valencia, CA), ISCRIPT TM cDNA Synthesis Kit (Bio-Rad, Hercules, CA ) using cDNA Transferred to. For real-time monitoring of PCR, at SYBR ICYCLER IQ ^(TM) detection system using a ^(R) Green dye (Bio-Rad) (Bio- Rad), it was performed real-time RT-PCR. The primers used were:
IFNγ, 5'-TACTGCCACGGCACAGTCATTGAA-3'(SEQ ID NO: 1), 5'-GCAGCGACTCCTTTTCCGCTTCCT-3' (SEQ ID NO: 2);
IL-4, 5'-GAAGCCCTACAGACGAGCTCA-3'(SEQ ID NO: 3), 5'-ACAGGAGAAGGGACGCCAT-3' (SEQ ID NO: 4);
IL-17A, 5'-TCAGGGTCGAGAAGATGCTG-3'(SEQ ID NO: 5), 5'-TTTTCATTGTGGAGGGCAGA-3' (SEQ ID NO: 6);
β-actin 5'-CCTAAGGCCAACCGTGAAAAG-3'(SEQ ID NO: 7), 5'-GAGGCATACAGGGACAGCACA-3'(SEQ ID NO: 8)
Relative quantification of target genes was analyzed based on the threshold cycle (Ct) determined by Bio-Rad IQ ^TM 5 optical system software.

Western blot gonococcal OMV preparations were boiled in sodium dodecyl sulfate (SDS) loading buffer containing 2-mercaptoethanol for 5 minutes. Protein quantification was performed using the RC DC protein assay kit. 10 micrograms of protein from each sample were separated on a 10% polyacrylamide SDS electrophoresis gel. Protein bands were transferred onto nitrocellulose membranes using an electrophoretic transcription system (Bio-Rad, Hercules, CA, USA). Nitrocellulose membranes were blocked overnight at 4 ° C. with PBS containing 3% skim milk and then vaginal wash diluted 1:20 with serum samples diluted 1: 200 or with PBS containing 3% skim milk. Incubated with sample for 2 hours. Specific antibodies bound to the Neisseria gonorrhoeae OMV preparation were detected using horseradish peroxidase-bound goat anti-mouse IgG (Santa Cruz Biotechnology, Paso Robles, CA) at a dilution of 1: 4000. Images were collected with the ChemiDoc MP imaging system (Bio-Rad) using the Pierce detection kit for chemiluminescence detection.

Immunoproteomics
Protein concentration in OMV was measured using the DC Protein Assay Kit (Bio-Rad). Samples of OMV [300 μg and 50 μg proteins, respectively for two-dimensional (2D) SDS-PAGE-MS / MS analysis and immunoblotting] were precipitated overnight in 90% acetone and 2 in 100% ice-cold acetone. Washed twice and air dried. Protein pellet in 200 μL rehydration buffer (7M urea, 2M thiourea, 2% CHAPS, 2% ASB-14, 1% DTT, 2mM TBP, 2% 3-10 IPG buffer, trace amount of Orange G) ReadyStrip IPG strips (Bio-Rad) at pH 4-7 were used to rehydrate overnight at 25 ° C. Isoelectric focusing using PROTEAN ^(TM) i12 ^TM IEF system (Bio-Rad), and carried out a total 26,000Vh following settings: 50 .mu.A current limiting, 8000 V fast lamp for 26,000Vh, 750V hold. Two-dimensional (2D) SDS-PAGE was performed using a Criterion TGX Any kD gel (Bio-Rad). Proteins were stained overnight with flamingo fluorescent staining (Bio-Rad) and spots were visualized using the ChemiDoc imaging system (Bio-Rad). For immunoblotting, the separated proteins were transferred onto PVDF membranes using the TurboBlott transfer system (Bio-Rad). Membranes are blocked in PBS Tween containing 5% milk for 2 hours and probed by overnight incubation with immunized mouse serum, followed by incubation with anti-mouse HRP-binding antibody (Bio-Rad). Was done. Spots were visualized using the Clarity Western ECL substrate and the ChemiDoc MP imaging system (Bio-Rad). Proteins on the membrane were stained with Novex Reversible Membrane Protein Stain (InVitrogen) and the location of selected "anchor" spots was overlaid with a flamingo-stained 2D gel. The matching spot was excised and the protein was trypsinized. Samples containing the extracted peptides were desalted with ZipTip C ₁₈ (Millipore, Billerica, MA), eluted with 70% acetonitrile / 0.1% TFA and dried in SpeedVac. Thermo Scientific Easy-nLC II (Thermo Scientific, Waltham, MA) Nano HPLC in which desalted peptides were placed in 2% acetonitrile in 0.1% formic acid (20 μL) and coupled to a hybrid Orbitrap Elite ETD (Thermo Scientific) mass spectrometer. Analyzed by LC / ESI MS / MS using the system (2 μL). In-line desalting is performed using a reverse phase trap column (100 μm × 20 mm) packed with Magic C ₁₈ AQ (5 μm 200 Å resin; Michrom Bioresources, Auburn, CA), followed by direct placement on an electrospray ion source. Peptide separation was performed on a reverse phase column (75 μm × 250 mm) packed with Magic C ₁₈ AQ (5 μm 100 Å resin; Michrom Bioresources). A 30 minute gradient of 7% to 35% acetonitrile in 0.1% formic acid was used for chromatographic separation at a flow rate of 400 nL / min. The heating capillary temperature was set to 300 ° C. and a spray voltage of 2750 V was applied to the electrospray tip. The Orbitrap Elite instrument is operated in data-dependent mode, with MS survey scans in Orbitrap (AGC target value 1 million, resolution 240,000, and injection time 250 ms) and MS / MS spectrum acquisition in linear ion traps (AGC target value). It was automatically switched between 10,000 and injection time 100 ms). The 20 strongest ions from the Fourier Transform (FT) full scan were selected for fragmentation in a linear trap with collision-induced dissociation at 35% normalized collision energy. Selected ions were dynamically eliminated for 15 seconds with an exclusion mass of ± 0.5 with a wrist size of 500 and a mass width of ± 0.5. Data analysis was performed using Proteome Discover 1.4 (Thermo Scientific). All identified peptides were searched against the gonococcal database (FA1090, FA19, and MS11) combined with the database of common pollutants (thegpm.org/crap/), cRAP.fasta; Make a list of proteins commonly found in proteomics experiments that are present by accidental or unavoidable contamination. Trypsin was set as the enzyme with maximum missed cleavages set to 2. The precursor ion tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.8 Da. Variable modifications included oxidation of methionine (+15.995 Da) and carbamide methyl (+57.021 Da) on cysteine. Data was searched using Sequest HT. All search results were performed through the Percolator for scoring.

Statistical analysis data is expressed as mean ± standard error of mean (SEM). Data on the effect of immunization on gonococcal recovery after inoculation were analyzed using a two-way ANOVA for repeated measurements by Fisher's protected minimal significant post-test. In addition, Kaplan-Meier analysis by log-rank test was used to compare infection clearance (defined as the first of three consecutive days of zero recovery) between treatment groups. For immune response data, mean values between the two groups were compared using an independent two-sided t-test, or ANOVA by Bonferroni post-test was used for multiple comparisons. P <0.05 was considered statistically significant. Statistical analysis was performed using Microsoft Excel or Prism 5 (GraphPad Software, San Diego, CA).

[Example 4]
This example describes intranasal administration of OMV and IL-12ms. Female mice (8 in each group) were intranasally immunized with OMV (40 μg protein, FA19 strain) + IL-12 / ms (1 μg IL-12) or empty ms on days 0 and 14. Two weeks later, all mice were vaginal challenged with 5 × 10 ⁶ CFU gonococcal strain FA1090. Vaginal swabs collected daily were diluted and quantitatively cultured on GC agar plates containing selective antibiotics. The results show that mice immunized with OMV + IL-12ms clear infection significantly faster than controls (p <0.01, FIG. 21). In addition, bacterial colonization was significantly lower in mice immunized with OMV + IL-12ms.

[Example 5]
Studies were conducted to better explain the protective effect of compositions containing OMV and IL-12 microspheres delivered via the nasal route of administration, and the comparison between the intranasal and intravaginal routes of administration.

Mice were prepared with different doses (15 μg, 30 μg, or 60 μg) of OMV prepared from the OMV protein of N. gonorrhoeae strain FA1090, with microencapsulated IL-12 (IL-12 / ms; 1 μg IL-12), or. Intranasal (in) immunization with IL-12 / ms alone. Immunization was repeated 2 weeks later. Two weeks after the final dose, mice were vaginal challenged with live gonococcal strain FA19 and the course of infection was observed by daily vaginal swabing and plating as described above. As shown in FIG. 22, mice immunized with a low dose of 15 μg OMV did not show accelerated clearance of infection compared to controls immunized with IL-12 / ms alone (median clearance time:: 10 days in both groups). Mice immunized with OMV at the usual dose (medium dose) of 30 μg or increased to 60 μg cleared the infection significantly faster than the control group (median clearance 6 days and 7.5 days, respectively) (P <0.001, Kaplan-Meier). , Log rank test).

Intranasal (i.n.) immunization and intravaginal (i.vag.) immunization were compared as follows. Mice are immunized with 30 μg of Neisseria gonorrhoeae FA1090 OMV + IL-12 / ms (1 μg IL12) or empty microspheres (ms) either in or i.vag. and repeated immunization after 2 weeks. It was. Two weeks later, mice were challenged with live gonococcal strain FA19 (heterologous challenge) and the course of infection was observed by daily vaginal swabbing and plating as described above. As shown in FIGS. 23A to 23D, mice immunized with OMV + IL-12 / ms i.n. cleared the infection in the same manner as mice immunized with i.vag. After clearance of infection (2 weeks after challenge), serum, vaginal lavage fluid, and saliva samples were taken to assay IgG and IgA anti-gonococcal antibodies by ELISA. Mice i.n. immunized with IL-12 / ms removed infection to the same extent as i.vag. Immunized mice with the same vaccine (Fig. 23A). Mice i.n. immunized with OMV + IL-12 / ms produced IgG and IgA antibodies in serum (Fig. 23B) and vaginal lavage fluid (Fig. 23C) at higher levels than i.vag. Immunized mice. Mice immunized with OMV + IL-12 / ms i.n. also expressed high levels of salivary IgA antibody (Fig. 23D).

When challenged with FA1090, the same strain used to prepare mice for OMV (allogeneic challenge), mice immunized with OMV + IL-12 / ms (shown in Figure 3) were i.vag. Immunized. The infection was removed to the same extent as in mice (Fig. 24).

Mice were immunized with gonococcal FA1090 OMV (30 μg protein) + IL-12 / ms (1 μg IL-12) once or twice i.n., and control mice were administered empty ms. Two weeks after the second immunization, mice were challenged with gonococcus MS11 i.vag. (Heterogeneous challenge). As shown in FIG. 25, mice immunized twice with OMV + IL-12 / ms are more infected than once immunized mice (median clearance time: 9 days) and control mice (median clearance time: 7 days). Removed promptly (median clearance time: 4 days); P = 0.0155 (Kaplan-Meier analysis, log rank test).

Mice were immunized with OMV (30 μg protein) + IL-12 / ms (1 μg IL-12) or empty ms prepared from Neisseria gonorrhoeae MS11 and challenged with the heterologous Neisseria gonorrhoeae strain FA1090. As shown in FIG. 26, mice immunized with MS11 OMV + IL-12 / ms were compared with mice immunized with MS11 OMV + empty ms (median clearance time: 11 days) or non-immunized control mice (clearance). Median time: 12 days) removed infection faster (median clearance time: 7 days); P = 0.0231 (Kaplan-Meier analysis, log rank test).

Mice were i.n. immunized with OMV (30 μg protein) + IL-12 / ms (1 μg IL-12) or empty ms prepared from Neisseria gonorrhoeae MS11 and challenged with the heterologous Neisseria gonorrhoeae strain FA19. As shown in FIG. 27, mice immunized with MS11 OMV + IL-12 / ms were compared with mice immunized with MS11 OMV + empty ms (median clearance time: 9 days) or non-immunized control mice (clearance). The infection was cleared faster (median clearance time: 5 days) compared to median time (10 days). P = 0.0007 (Kaplan-Meier analysis, log rank test).

It should be noted that the gonococcal strains used in these experiments express different porin types. Neisseria gonorrhoeae PorB is a major outer membrane protein of Neisseria gonorrhoeae, represented by two major types, PorB-1A and PorB-1B, each of which has many subtypes. The FA1090 strain has a porin type PorB-1B, and the MS11 strain also has a porin type PorB-1B, but it is a different subtype from the FA1090, and the FA19 strain has a porin type PorB-1A. In addition, these strains express different Opa proteins and lipooligosaccharide structures, thus indicating a wide variety of gonococcal antigenic variants.

Although the present invention has been described with reference to one or more specific embodiments, it is understood that other embodiments of the invention are possible without departing from the spirit and scope of the invention.

Claims (16)

A method for reducing the risk of gonococcal infection in an individual, in which IL-12 incorporated into a high molecular weight microsphere and outer membrane vesicles (OMV) derived from gonococcus are reproduced. A method comprising the step of intranasally administering to said individual in an amount effective to reduce the risk of contracting a tube infection. The method of claim 1, wherein the IL-12 microspheres and OMV are delivered in the same composition. The method of claim 1, wherein IL-12 microspheres and OMV are administered multiple times over a period of up to 3 weeks. The method of claim 3, wherein IL-12 microspheres and OMV are administered 2 to 4 times with an interval of at least about 1 week between administrations. The method of claim 4, wherein IL-12 microspheres and OMV are administered twice, with an interval of about 2 weeks between administrations. The method of claim 1, wherein the OMV is in the range of 0.01-2000 μg protein per dose. The method of claim 6, wherein the OMV is in the range of 0.01-1000 μg protein per dose. The method of claim 1, wherein the OMV is in the range of 0.5-30 μg per kg body weight. The method of claim 1, wherein IL-12 is in the range of 1-500 μg per dose. The method of claim 1, wherein IL-12 is in the range of 10 ng to 10 μg per kg body weight. The method of claim 1, wherein the OMV is in the range of 0.01-2000 μg protein per dose and IL-12 is in the range of 1-500 μg per dose. A composition for intranasal administration, containing Neisseria gonorrhoeae outer membrane vesicles (OMV) and IL-12, which is incorporated into polymeric microspheres in pharmaceutical carriers. Stuff. The composition of claim 12, wherein the OMV is in the range of 0.01-2000 μg protein per dose and IL-12 is in the range of 1-500 μg per dose. The composition according to claim 12, wherein the composition is substantially free of soluble IL-12. A kit for intranasal vaccination of individuals against gonococcal infection.
It contains multiple doses of a composition suitable for intranasal administration containing Neisseria gonorrhoeae outer membrane vesicles (OMV) and IL-12.
IL-12 has been incorporated into polymeric microspheres in pharmaceutical carriers.
A kit comprising an instruction manual for administration of the composition, wherein the amount of OMV per dose is 0.01-2000 μg protein, the amount of IL-12 per dose is 1-500 μg. A kit for intranasal vaccination of individuals against gonococcal infection.
i) Compositions suitable for intranasal administration containing Neisseria gonorrhoeae outer membrane vesicles (OMV): where the amount of OMV per dose is 0.01-2000 μg;
ii) Composition suitable for intranasal administration containing IL-12: where IL-12 is incorporated into a polymeric microsphere and the amount of IL-12 per dose is 1-500 μg; as well as
iii) A kit that optionally contains instructions for combining i) and ii) and for administering the combined composition.