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EP3716995A1 - Polythérapie et prophylaxie contre des infections du tractus génital - Google Patents

Polythérapie et prophylaxie contre des infections du tractus génital

Info

Publication number
EP3716995A1
EP3716995A1 EP18884342.9A EP18884342A EP3716995A1 EP 3716995 A1 EP3716995 A1 EP 3716995A1 EP 18884342 A EP18884342 A EP 18884342A EP 3716995 A1 EP3716995 A1 EP 3716995A1
Authority
EP
European Patent Office
Prior art keywords
mice
omv
gonorrhoeae
infection
plus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18884342.9A
Other languages
German (de)
English (en)
Other versions
EP3716995A4 (fr
Inventor
Michael W. Russell
Yingru LIU
Nejat K. Egilmez
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research Foundation of the State University of New York
Original Assignee
Research Foundation of the State University of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US15/824,700 external-priority patent/US20180071379A1/en
Priority claimed from US16/138,526 external-priority patent/US20190008941A1/en
Application filed by Research Foundation of the State University of New York filed Critical Research Foundation of the State University of New York
Publication of EP3716995A1 publication Critical patent/EP3716995A1/fr
Publication of EP3716995A4 publication Critical patent/EP3716995A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A61K2039/55538—IL-12

Definitions

  • the invention relates to compositions comprising IL-12 and outer membrane vesicles from Neisseria gonorrhoeae , and methods for using such compositions for treatment of genital tract infections.
  • Genital tract infection by Neisseria gonorrhoeae gives rise to gonorrhea, which is the second most frequent reportable infectious disease in the US affecting >300,000 individuals per annum, although the real incidence is believed to be at least double that number.
  • the worldwide incidence of gonorrhea is estimated to be >100 million cases per year. Women bear the brunt of the infection, because untreated gonorrhea can ascend into the upper reproductive tract and give rise to pelvic inflammatory disease and tubal scarring, leading to infertility and risk for ectopic pregnancy which can be life-threatening.
  • gonorrhoeae has quickly become resistant to each class of antibiotics used against it, including most recently the fluorquinolones (ciprofloxacin), and the currently recommended antibiotics are cephalosporins. However, resistance to these has begun to emerge, making N. gonorrhoeae multiple-drug-resistant. Despite various efforts, no vaccine against N. gonorrhoeae is currently available.
  • Vaccine efforts are complicated by the extensive antigenic variability of N gonorrhoeae , in which most major surface antigens, including lipooligosaccharide (LOS), porin, pilin, and the opacity proteins (Opa) are subject to phase-variable expression (LOS, Opa, pilus), allelic variation (porin, Opa), or recombinatorial expression (pilin).
  • LOS lipooligosaccharide
  • Opa opacity proteins
  • This disclosure provides a method of reducing the risk of developing N gonorrhoeae infections by administering to an individual N gonorrhoeae antigens and IL-12 incorporated in polymeric microspheres.
  • the N gonorrhoeae antigens may be in the form of outer membrane vesicles or microvesicles. Other forms of antigens (such as purified or semi- purified) may also be used.
  • the N gonorrhoeae antigen preparation (such as OMVs) and the IL-12 microspheres may be delivered in a single composition or different compositions, by the same route or different routes, at the same time or different times, over a same time period and delivery regimen or different time period and delivery regimens.
  • the N gonorrhoeae OMVs and IL-12 microspheres can be delivered intravaginally, or may be delivered intranasally.
  • this disclosure provides a method for treatment of cervico- vaginal infections by local application of IL-12 incorporated in polymeric microspheres. While not intending to be bound by any particular theory, it is considered that application of IL-12 incorporated in polymeric microspheres locally to mucosal surfaces enhances the body’s own immune response against an existing infection resulting in reduction or elimination of that infection and/or generation of immunity against repeat infection. In one embodiment, the amount is sufficient to promote Thl- driven response against the microorganisms causing the infection. The amount of IL-12 may be sufficient to provide a therapeutic effect, a prophylactic effect, or both against the causative microorganisms.
  • Infections that can be treated by the present method include, but are not limited to, those that are caused by N gonorrhoeae, C. trachomatis or both.
  • An example of a polymer that can be used for microencapsulation of IL-12 is polylactic acid.
  • this disclosure provides a composition comprising OMVs prepared from N. gonorrhoeae and IL-12 containing microspheres (ms) suitable for intravaginal delivery.
  • this disclosure provides a kit for intravaginal delivery of OMVs prepared from N gonorrhoeae and IL-12 containing microspheres.
  • the OMVs and IL-12 ms may be present as separate compositions or the same composition.
  • the kit may comprise multiple doses of the OMV composition and the IL-12 composition and instructions for administration, which may include instruction on frequency, length of administration regimen, mode of administration and the like.
  • Fig. 1 is a graph showing the effects of intravaginal treatment with 1 pg of IL-
  • PLA polylactic acid
  • Fig. 2 is a graph showing the effects of intravaginal treatment with IL-12 microspheres during primary infection with N gonorrhoeae (Fig. 1) on the course of secondary vaginal infection with N gonorrhoeae in mice.
  • Fig. 3 is a graph showing the effects of intravaginal treatment with 1 pg of soluble vs. microencapsulated IL-12 on the course of vaginal infection with N gonorrhoeae in mice.
  • FIG. 4 is a graph showing the effect of intravaginal IL-12 microsphere (ms) treatment on primary gonococcal infection in BALB/c mice.
  • FIG. 5 is a graph showing the effect of intravaginal IL-12 microsphere (ms) treatment during primary infection on secondary gonococcal infection.
  • B Data from the experiment shown in A plotted as percentage of mice remaining infected after reinfection under the indicated treatments during primary infection.
  • Fig. 6 shows intravaginal (I.vag) immunization with gonococcal OMV plus
  • IL-l2/ms induced resistance to genital infection with N. gonorrhoeae , and generated an immune response a: Mice were immunized 3 times at 7-day intervals with OMV (40pg protein) from strain FA1090 plus control (blank) ms or IL-l2/ms (lpg IL-12); control mice were sham-immunized with either blank ms, or with IL-l2/ms alone. Two weeks after the last immunization, all mice were challenged by i.vag. inoculation with N gonorrhoeae strain FA1090 (5 x 10 6 CFU), and infection was monitored by vaginal swabbing and plating.
  • OMV 40pg protein
  • Intracellular cytokine staining in CD4 + cells recovered from ILN at termination (day 15), shown as mean ⁇ SEM, N 3 samples, % of CD4 + staining for each cytokine; * P ⁇ 0.01 Student’s t.
  • Mice were immunized twice at a l4-day interval with gonococcal (Ngo) OMV (40pg protein) plus blank ms or IL-l2/ms (lpg IL-12); control mice were sham-immunized with blank ms alone or with NTHI OMV (40pg protein) plus IL-l2/ms (lpg IL-12). Two weeks later, all mice were challenged with N.
  • gonorrhoeae FA1090 (5 x 10 6 CFU).
  • Fig. 7 shows antibody responses generated by immunization with gonococcal
  • OMV plus IL-l2/ms prior to gonococcal challenge a: Vaginal wash (left panel) and serum (right panel) antibodies assayed by ELISA 2 weeks after the last immunization with 1, 2, or 3 doses of gonococcal OMV (40pg protein) plus IL-l2/ms (lpg IL-12).
  • Fig. 8 shows: a: T cell cytokine responses in ILN cells induced by
  • Fig. 9 shows resistance to gonococcal (FA1090) challenge persisted for at least 6 months after immunization with two doses of gonococcal (FA1090) OMV plus IL- l2/ms.
  • CFU recovery of N. gonorrhoeae
  • ANOVA 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 were resistant to challenge with N gonorrhoeae FA19.
  • Fig. 11 shows immunoproteomics of gonococal OMV.
  • Lane 1 control serum from a mouse immunized with FA1090 OMV plus blank ms, tested against FA1090 OMV; lanes 2-4, serum #1 from a mouse immunized with FA1090 OMV plus IL-l2/ms, tested against OMV from FA1090 (lane 2), MS11 (lane 3), or FA19 (lane 4); lane 5, serum #2 from a mouse immunized with FA1090 OMV plus IL-l2/ms, tested against OMV from FA1090; lane 6, antibody H5 (anti- porin PIB3) tested against FA1090 OMV.
  • IgA and IgG responses in vaginal wash and serum were significant ( ⁇ 0.05, Student’s /, OMV plus blank ms vs.
  • Fig. 13 is a replicate of Fig. 1 : I.vag. immunization with gonococcal OMV plus IL-l2/ms induced resistance to genital infection with N. gonorrhoeae and generated an immune response.
  • A Mice were immunized 3 times at 7-day intervals with OMV (40pg protein) from strain FA1090 plus control (blank) ms or IL-l2/ms (lpg IL-12); control mice were sham-immunized with either blank ms, or with IL-l2/ms alone. Two weeks after the last immunization, all mice were challenged by i.vag.
  • N gonorrhoeae strain FA1090 (5 x 10 6 CFET), and infection was monitored by vaginal swabbing and plating.
  • Left panel: recovery (CFU) of N. gonorrhoeae (mean ⁇ SEM, N 8 mice), P ⁇ 0.01 (ANOVA,
  • gonococcal OMV plus blank ms right panel: % of animals remaining infected at each time point, P ⁇ 0.001 (Kaplan-Meier analysis, log-rank test, gonococcal OMV plus IL-l2/ms vs. gonococcal OMV plus blank ms).
  • Fig. 14 shows examples of flow cytometry plots for data shown in Fig. 6C.
  • A-L CD8 fluorescence
  • M-P CD8 fluorescence
  • Fig. 15 shows responses induced by immunization with OMV prepared from
  • NTHI samples collected 2 weeks after immunization as shown.
  • Fig. 16 shows IgG subclass of anti-gonococcal antibody responses (mean
  • Fig. 17 shows: A: Specificity of T cell responses in ILN for gonococcal antigen.
  • Fig. 18 shows responses in mice challenged with N. gonorrhoeae FA1090 6 months after immunization with FA1090 OMV plus IL-l2/ms.
  • A: Vaginal wash antibodies (mean ⁇ SEM, N 5 samples)
  • B: serum antibodies (mean ⁇ SEM, N 5 samples)
  • C: cytokine production by CD4 + ILN cells (mean ⁇ SEM, N 3 samples).
  • Fig. 19 shows responses in mice immunized with gonococcal OMV plus balnk ms or IL-l2/ms after heterologous gonococcal challenge.
  • A, B, C Mice immunized with FA1090 OMV and challenged with MS11;
  • B: serum antibodies to MS11 (mean ⁇ SEM, N 5 samples);
  • Fig. 20 is a replicate of Fig. 7A,B,F,G: I.vag. immunization with gonococcal
  • Fig. 21 shows the effect of i.n. immunization with gonococcal OMV (strain
  • Fig. 22 shows the effect of immunization with different doses of gonococcal
  • OMV strain FA1090
  • Low dose 60pg
  • mid dose 30pg
  • low dose l5pg of OMV protein per dose, plus lpg IL-l2/ms.
  • Fig. 23 shows A: comparison of the effects of intranasal or intravaginal immunization with gonococcal OMV (30pg protein; strain FA1090) plus lpg IL-l2/ms on heterologous gonococcal challenge infection with strain FA19 in BALB/c mice.
  • B serum
  • C vaginal wash
  • D salivary antibodies against N. gonorrhoeae in samples collected after clearance of the infection at termination (2 weeks after challenge); * P ⁇ 0.0l compared with controls (Student / test).
  • Fig. 24 shows a comparison of the effects of intranasal or intravaginal immunization with gonococcal OMV (30pg protein; strain FA1090) plus lpg IL-l2/ms on homologous gonococcal challenge infection with strain FA1090 in B ALB/c mice.
  • Fig. 25 shows the effect of intranasal immunization with gonococcal OMV
  • mice (30pg protein, strain FA1090) plus lpg IL-l2/ms on heterologous gonococcal challenge infection with strain MS11 in B ALB/c mice. Mice were given either one dose of OMV plus IL-l2/ms, or two doses with a 2 week interval; control mice were given 2 doses of blank ms.
  • Fig. 26 shows the effect of intranasal immunization with gonococcal OMV (30 pg protein, strain MS11) plus lpg IL-l2/ms on heterologous gonococcal challenge infection with strain FA1090 in BALB/c mice.
  • Fig. 27 shows the effect of intranasal immunization with gonococcal OMV
  • the present invention is based on our studies which have helped to unfold the ways in which N gonorrhoeae prevents the immune system from mounting effective immune responses against it. We provide here a novel approach to overcome the ability of N.
  • the method comprise reducing the likelihood of an individual contracting a N. gonorrhoeae infection by administration of an antigenic preparation from N gonorrhoeae and IL-12 present in microspheres (ms).
  • An example of an antigenic preparation from N gonorrhoeae is OMVs.
  • the OMVs and the IL-12 ms may be delivered intravaginally.
  • the OMVs and the IL-12 ms may be delivered intranasally.
  • the OMVs and the IL-12 ms may be delivered by separate routes of
  • administration For example, one may be delivered intravaginally and the other intranasally.
  • the OMVs and the IL-12 ms are delivered by the same route of administration, e.g, intranasally.
  • the OMVs and the IL-12 ms are delivered in the same composition. Amounts of OMVs and IL-12 ms per dose can be such that an immune response is elicited.
  • the OMVs can be in the range of 0.01 to 1 mg of protein per dose. In one embodiment, the OMVs can be in the range of 0.01 to 2 mg of protein per dose. In
  • OMVs can be 10, 50, 100, 250, 500, 750, 1,000, 1250, 1,500, 1,750 or 2,000 pg protein per dose. In an embodiment, OMVs can be in the range of 15-300 pg protein per dose. In one embodiment, the OMV dose can be 50 to 1,000 pg protein. For a 70 kg human, that corresponds to a OMV dose of about 0.7 to 28 microgram protein per kg body weight. In one embodiment, the OMVs can be from 0.5 to 30 pg protein/kg body weight.
  • the IL-12 (in microspheres) can be 1 to 500 pg/dose. That corresponds to about 14 ng to 7 pg/kg body weight (assuming a 70 kg body weight).
  • IL-12 can be 10 ng to 10 pg/kg body weight. In one embodiment, the IL-12 can be 10, 50, 100, 200, 300, 400 or 500 pg/dose. In an embodiment, the IL-12 can be 10 to 300 ng or 5 to 300 ng per kg body weight. In an embodiment, IL-12 can be 0.5 to 20 micrograms in microspheres per dose. Determining the amount of microspheres can be done based on the loading of IL-12.
  • the compositions may be provided in carriers, buffers and the like or may be lyophilized. The two components may be provided separately, and can be combined just before administration or may be administered separately. In one embodiment, the composition does not have any added free soluble IL-12.
  • any leaked IL-12 from the microspheres prior to administration is considered to be insignificant. Even if free soluble IL-12 is present, it is not considered to contribute to the present effects and method. Rather, encapsulated IL-12 in microspheres was required in the composition to see the protective effects.
  • Outer membrane vesicles can be prepared from the outer membranes of N. gonorrhoeae.
  • OMVs can be prepared from the outer membranes of a cultured strain of Neisseria gonorrhoeae spp.
  • OMVs can be obtained from a N gonorrhoeae grown in broth or solid medium culture.
  • the preparation may comprise separating the bacterial cells from the culture medium (e.g. by filtration or by a low-speed centrifugation and the like), lysing the cells (e.g. by addition of detergent, osmotic shock, sonication, cavitation, homogenization and the like) and separating an outer membrane fraction from cytoplasmic molecules (e.g. by filtration; or by differential precipitation or aggregation of outer membranes and/or outer membrane vesicles, or by affinity separation methods; or by a high speed centrifugation).
  • compositions can be administered preferably as multiple doses with an interval in between.
  • at least two doses can be administered with an interval of at least one week in between.
  • the interval may be from one to three weeks.
  • two doses are used with an interval of about 2 weeks in between.
  • the IL-12 formulations of the present invention are sustained release formulations.
  • IL-12 is delivered as incorporated (also referred to herein as encapsulated or microencapsulated) in polymeric microparticles (also referred to herein as microspheres).
  • the microparticles are biodegradable and biocompatible. Preparation techniques for such microspheres are known in the art. See for example, U.S. Patent numbers 6,143,211; 6,235,244; 6,616,869; and 7,029,700, the disclosures of which pertaining to methods and compositions for preparation of microspheres are incorporated herein by reference.
  • a phase inversion technique is used to prepare microencapsulated IL-12.
  • a biodegradable polymer is dissolved in a solvent (such as dichloromethane or other organic solvent) and then a mixture is formed by adding micronized IL-12 (i.e. lyophilized mixtures of IL-12 and excipient such as polyvinyl pyrrolidone) to the polymer dissolved in the solvent.
  • a non-solvent such as alcohol or hexane
  • spontaneous formation of microencapsulated IL-12 is then introduced causing spontaneous formation of microencapsulated IL-12.
  • biodegradable polymers include polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide) and poly(lactide-co- caprolactone), and natural polymers.
  • the microspheres are composed of a polymer of lactic acid (polylactic acid (PLA)).
  • the IL-12 containing microspheres degrade by hydrolysis slowly over time, releasing the encapsulated IL-12.
  • the microspheres are suspended before use and can also be delivered in an acceptable buffered physiological saline solution.
  • slow release of IL-12 over a period of time such as approximately 4 days allows for continuous stimulation of locally present immune cells without elevating the
  • the microspheres are made of biodegradable materials.
  • the hydrolytic product of the microspheres is lactic acid, a harmless product of normal metabolism.
  • PLA is a component of absorbable sutures and has been in use for that purpose for many decades, and is therefore considered safe.
  • Microencapsulated IL-12 has been shown to be stable in storage at ambient temperatures and to have a long shelf-life.
  • the microspheres are in the range of 10 nm to 10 microns.
  • the microspheres may be suspended in pharmaceutically acceptable medium such as a physiological buffer.
  • the loading of IL-12 is from 0.1 to 10 pg per mg of the particles. In one embodiment, the loading is from 1 to 5 pg IL-12 per mg of the particles. In one embodiment, the loading is from about 2.5 pg IL-12 per mg of the particles.
  • the IL-12 formulations can be used in amounts that will result in therapeutic and/or prophylactic effects.
  • An effective dose in mice was observed to be 1 pg of IL-12. Determining the effective dosage for humans is within the purview of clinicians and other individuals involved in the treatment of such infections. Generally, the amount administered depends upon various factors including the severity of the infection, the weight, health and age of the individual. Such factors can be readily determined by a clinician.
  • the dose may be from 1 pg to 200 pg of IL-12 per day. In some embodiments, the dose is 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 175 and 200 pg of IL-12 per dose and all integers between 1 and 200 pg and all ranges therebetween. The dosage required may be less if used in conjunction with an antimicrobial agent.
  • the dosage may be repeated as necessary.
  • the administration may be repeated daily, multiple times in a day, or at longer intervals, such as at intervals of 2- 4 days, weekly or monthly.
  • the administration is repeated at intervals from 1 day to 1 month (28, 29, 30 or 31 days) or beyond that and all intervals therebetween.
  • the treatment regimen may be repeated as necessary.
  • the dosage is administered every 2, 3, 4, 5, 6, 7, 10, or 14 days, or longer.
  • the present invention provides a method of treating genital tract infections in a female subject by intravaginal application of IL-12 incorporated in polymeric microspheres.
  • the infections that can be treated by this method include bacterial, fungal, parasitic, viral and the like.
  • the amount is sufficient to promote Thl- driven response against the microorganisms causing the infection.
  • the amount is sufficient to provide a therapeutic effect, a prophylactic effect, or both against the causative microorganisms.
  • the term“treated” or“treatment” as used herein means to reduce or eliminate an infection. An infection is considered to be reduced when the underlying cause of the infection is reduced.
  • the method of the present invention is useful for treating genital tract infections, such as cervico-vaginal infections, caused by bacteria, such as N. gonorrhoeae.
  • the method comprises the steps of providing local (intravaginal) application of the cytokine interleukin- 12 (IL-12) incorporated in biodegradable, biocompatible
  • the dose is sufficient to promote Thl-driven immune responses against infection with N. gonorrhoeae.
  • the invention provides a method for therapy or prophylaxis or both for cervico-vaginal gonococcal infection (i.e., gonorrhea) by means of local administration of IL-12 microspheres. While not intending to be bound by any particular theory, it is considered that this method works, at least in part, by reversing the ability of N gonorrhoeae to interfere with the host’s immune responses.
  • the IL-12 formulation is delivered locally to the mucosal surface of the genital tract of an individual.
  • the individual is not already receiving IL-12, or has not been administered IL-12 prior to the initiation of the present method.
  • the individual is not receiving IL-12 via any other
  • the formulation contains no other therapeutic agent, no other prophylactic agent, or no other agent that is both therapeutic and prophylactic.
  • the formulation does not contain the infection causing microorganism (such as in an inactivated form) or an antigen therefrom, and the individual has not been and/or is not being administered the inactivated microorganism or an antigen therefrom.
  • the formulation may be delivered to an individual who is already receiving treatment (other than IL-12) for genital tract infection (such as gonococcal infection).
  • the invention further comprises the step of administering an antimicrobial agent to the individual.
  • the method of this invention comprises the steps of identifying an individual who is suffering from or has been diagnosed with an infection of the genital tract, delivering to the genital tract locally (such as intravaginally) a composition comprising a therapeutically effective, a prophylactically effective, or both therapeutically and prophylactically effective amount of a composition comprising IL-12 in biodegradable polymeric microspheres, and optionally administering to the individual one or more antimicrobial agents (such as antibiotics, antifungal or antiviral agents).
  • the antimicrobial agents may be administered prior to, during or after the
  • the administration of the microencapsulated IL-12 as described here reduces the N. gonorrhoeae infection.
  • the infection is eliminated.
  • the presence or absence of infection or the level of infection may be tested by routine microbiological methods (such as culture and testing).
  • the infection may be tested by obtaining vaginal swab and testing for the presence of bacteria (such as by the ability to form colonies), or by nucleic acid amplification methods.
  • the administration of the microencapsulated IL-12 as described here reduces the N. gonorrhoeae infection and reduces the risk of repeat infection of N gonorrhoeae after the treatment with microencapsulated IL-12 has been stopped. While not intending to be bound by any particular theory, it is considered that the prophylactic effect of IL-12 is achieved by stimulation of the immune system.
  • the administration of IL-12 does not significantly increase the level of IL-12 in the systemic circulation.
  • the serum level of IL-12 does not increase to greater than 50 pi cogram s/ml.
  • the formulations of the present invention can be delivered as applied to an article of manufacture acting as a carrier.
  • the formulations may be incorporated into or onto and then delivered via an insert, an applicator, tablet, suppository, vaginal ring, vaginal sponge, tampon and the like.
  • the formulation may also be delivered in the form of a liquid, cream, gel, lotion, ointment, paste, spray and the like.
  • the pharmaceutical formulations may optionally include pharmaceutically acceptable carriers, buffers, diluents, solubilizing or emulsifying agents, and various salts.
  • pharmaceutically acceptable carriers such as, e.g., Remington's Pharmaceutical Sciences, l8th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042).
  • An advantage of local application of microencapsulated IL-12 as described herein is that it can provide a sustained effect while avoiding problems of potential systemic toxicity.
  • the present invention is used for treating genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital genital
  • Chlamydia trachomatis infection Chlamydia
  • Chlamydia is another sexually transmitted disease (STD) of even more frequent occurrence than gonorrhea, and is the most frequently reported infectious disease in the ETS, thought to affect up to 3 million individuals per annum (>92 million worldwide). It is also a major cause of pelvic inflammatory disease in women and its sequelae (infertility and risk for ectopic pregnancy). Therefore in one embodiment, local (intravaginal) application of IL-12 incorporated in polymeric microspheres is used to promote local Thl- immune responses for therapy and prophylaxis against C. trachomatis. In one embodiment, the method of the present invention is used to treat urinogenital infections due to N.
  • gonorrhoeae and C. trachomatis This may be advantageous in the STD clinic setting because gonorrhea and chlamydia present with similar signs and symptoms, and the differential diagnosis may depend on identifying the causative organism.
  • mixed infections with both are common.
  • other genital tract infections could also be treated with (intravaginal) application of microencapsulated IL-12 to enhance local immunity against them.
  • microencapsulated IL-12 is used in the treatment of other local mucosal infections where the normal immune response is insufficient to eliminate them.
  • examples include: bronchitis and chronic obstructive pulmonary disease (respiratory tract), otitis media (middle ear infection, which is the most frequent reason for pediatric office visits in the US), Helicobacter pylori infection (which causes gastric ulcer and can lead to gastric cancer), and possibly periodontal disease (which afflicts most adults from age 35 onwards and is the main cause of tooth loss in adults).
  • the IL-12 may be administered with a gonococcal antigen containing vaccine.
  • IL-12 can be administered with a locally administered non living gonococcal vaccine.
  • OMV gonococcal outer membrane vesicle
  • the results demonstrate the generation of a Thl -driven, antibody-dependent, protective immune response that persists for at least several months and is effective against antigenically diverse strains of N gonorrhoeae.
  • immunization can also be carried out via intranasal route.
  • the OMV and the IL-12 microspheres may be administered to female or male subjects.
  • the compositions When administered to a male subject, the compositions may be delivered intranasally, and when administered to a female subject, the compositions may be delivered intravaginally and/or intranasally.
  • mice that were treated with IL-12 microspheres during primary vaginal gonococcal infection were allowed to recover, treated with antibiotic (ceftriaxone) on day 14, rested and then reinfected one month later with N gonorrhoeae , the secondary infection was cleared faster than in control mice given blank microspheres during primary infection (See Fig. 2). Normally, secondary infection is considered to clear with the same kinetics as primary infection, and there is little or no antibody response developed.
  • 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 (See Fig. 3).
  • Data shown as mean ⁇ SEM cfu of N gonorrhoeae recovered from vaginal swabs taken daily; N 8 mice per group.
  • This example describes another set of experiments that illustrate the effectiveness of intravaginal application of IL-12 microspheres on N. gonorrhoeae vaginal infection.
  • mice BALB/c mice were purchased from Jackson Laboratories (Bar Harbor,
  • N. gonorrhoeae FA1090 were cultured on GC agar supplemented with hemoglobin and ISO VIT ALEX ® , an enrichment medium (BD Diagnostic Systems, Franklin Lakes, NJ). Growth was checked for colony morphology consistent with Opa protein and pilus expression, and gonococci were harvested from plates and the cell density was determined. Opa expression as was: Opa A, B/D/G, E/K.
  • Microspheres Cytokines were encapsulated into poly-lactic acid (PL A) microspheres using the Phase Inversion Nanoencapsulation (PIN) technology as follows. Briefly, recombinant IL-12 (mouse or human) is mixed with excipients including sucrose (0.1%, w/w) and polyvinylpyrrolidone in water and then is lyophilized. The lyophilized material is dissolved in tertyl butyl alcohol (TBA) and is mixed with polylactic acid (PLA) resomer dissolved in TBA (1 to 3 ratio, vokvol for micronized IL-12 and PLA solution). This solution is then poured into lOOx volume of heptane to induce formation of the particles.
  • PIN Phase Inversion Nanoencapsulation
  • control microspheres containing no cytokine or antibody (a) control microspheres containing no cytokine or antibody; (b) murine IL-12 (0.25 pg/mg particles); and (c) murine IL-17 (0.25 pg/mg particles).
  • Mouse Vaginal Infection Model Female mice between 7 and 9 weeks old were infected vaginally on day 0 with live N gonorrhoeae FA1090 as previously described. Vaginal mucus was quantitatively cultured daily on GC agar supplemented with selective antibiotics to determine the bacterial colonization loads. The limit of detection was 100 CFU recovered per mouse. Intravaginal treatments with microsphere preparations were given every second day from day 0 to day 8, by instillation of 40pl suspensions in PBS of microspheres containing IL-12 or IL-17, or control microspheres.
  • FACSCalibur cytometer For determination of intracellular cytokine expression, cells were restimulated with phorbol myristate acetate-ionomycin-GOLGISTOP ® , a protein transport inhibitor (eBioscience, San Diego, CA) for 5 h, and then fixed with
  • CYTOFIX/CYTOPERM ® a fixation/permeabilization solution, (eBioscience).
  • Antibodies to mouse CD4, CD8, CD19, CDl lb, CDl lc, NKG2D, Gr-l, IFN-g, IL-4, and IL-17A conjugated with fluorescein isothiocyanate, phycoerythrin, or allophycocyanin were purchased from eBioscience.
  • Cytokine ELISA IL-l2p70, IFN-g, IL-4, IL-5, and IL-17A levels in serum or vaginal wash samples were measured in triplicate using ELISA kits purchased from eBioscience.
  • Real-time RT-PCR Total cellular RNA of whole vaginas harvested from the mice was isolated with RNEASY ® , RNA purification Mini Kits (Qiagen, Valencia, CA), and was transcribed to cDNA using the ISCRIPTTM cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time RT-PCR was performed on an ICYCLER IQ ® real-time PCR detection system (Bio-Rad) using SYBR ® Green dye, a fluorescent dye, (Bio-Rad) for real-time monitoring of the PCR. Relative quantification of target genes was analyzed based on the threshold cycle (Ct) determined by Bio-Rad IQTM 5 optical system software.
  • Ct threshold cycle
  • IL-12 microsphere treatment markedly enhanced Thl immune responses to N gonorrhoeae , indicated by significantly increased numbers of IFN-y + /CD4 + T cells (Fig. 4D).
  • IL-12 microspheres did not change Th2 or Thl7 responses as the numbers of IL-4 + /CD4 + and IL-l7 + /CD4 + T cells in ILN were similar between the treated groups (Fig. 4D).
  • RT-PCR analyses showed that IFN-g, but not IL-4 or IL-17 mRNA expression was elevated in the vaginas of infected mice following IL-12 microsphere treatment (Fig. 4E). Although IL-17 microspheres ameliorated gonococcal infection, this treatment was not associated with enhanced Thl or Th2 responses (Fig. 4D, E), but there was increased influx of Gr-l + neutrophils into the genital tract (Fig. 4F).
  • IFN- g was present in the vaginal wash (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 was detected in control-treated infected mice.
  • IL-12 can stimulate humoral immune responses in an IFN-y-dependent manner or directly.
  • IL-12 microsphere treatment during N. gonorrhoeae infection led to the production of anti-gonococcal antibodies in vaginal wash, saliva, and serum collected 15 days after inoculation.
  • IgM antibodies were at low levels with little difference between experimental groups (data not shown). No salivary gonococcus- specific antibody was detected in any group of mice (data not shown).
  • N. gonorrhoeae infection of control-treated mice did not significantly elevate gonococcus-specific IgA or IgG antibodies in either vaginal washes or sera.
  • IL-12 microsphere treatment increased vaginal and serum specific IgG antibody (Fig. 4G, H), as well as vaginal specific IgA antibody production (Fig. 4G).
  • mice infected with N gonorrhoeae were treated with IL-l2-loaded or blank microspheres, and after the infection had run its course, the mice were treated with ceftriaxone (300 pg i.p.) on day 15 to ensure complete elimination of the gonococci.
  • ceftriaxone 300 pg i.p.
  • gonorrhoeae of the same strain without any further treatment.
  • primary infection of control-treated mice did not protect them against subsequent secondary challenge: the duration and bacterial burden of secondary gonococcal infection in previously blank microsphere-treated mice were the same as for the primary infection of age-matched naive mice (Fig. 5 A, B).
  • intravaginal treatment with IL-l2-loaded microspheres during primary infection protected the mice against secondary infection: reinfected mice that had been treated with IL-12 microspheres during the primary infection resisted the challenge more effectively than controls (Fig. 5A, B).
  • previous IL-12 microsphere treatment of sham-infected mice did not induce protection against subsequent infection (Fig. 5A, B). This result also excluded the possibility that any persisting microspheres still affected the secondary N. gonorrhoeae infection.
  • This example describes that the present experimental vaccine induces Thl- driven immune responses and resistance to Neisseria gonorrhoeae infection in a murine model.
  • mice with gonococcal OMV plus IL-12/ms accelerates clearance of challenge infection with N. gonorrhoeae.
  • Groups of 8 female BALB/c mice were immunized i.vag. with gonococcal OMV (strain FA1090; 40 pg protein) plus IL-l2/ms ( 1 pg IL-12), or with OMV plus control (blank) ms; two additional control groups were sham -immunized with IL-l2/ms or with blank ms alone. Immunizations were repeated 1 week and 2 weeks later, and all mice were challenged after a further 2 weeks by i.vag. instillation of N.
  • Table 1 Summary data from immunization experiments using homologous challenge (BALB/c mice).
  • mice immunized with OMV plus IL-l2/ms had developed the highest levels of vaginal and serum IgG and IgA antibodies, whereas those mice immunized with OMV plus blank ms developed much lower levels of these antibodies (Fig. 6b).
  • Mice that were unimmunized and sham-infected showed no antibodies detectable above assay background at the starting dilutions, and mice immunized with blank ms alone and infected also did not develop detectable antibodies (Fig. 6b).
  • Iliac lymph node (ILN) mononuclear cells collected at the same time were stained for surface CD4 expression and for intracellular cytokines, and analyzed by flow cytometry. This revealed that only mice immunized with OMV plus IL-l2/ms generated CD4 + /IFNy + (and CD8 + / ⁇ FNy + ) T cells, whereas no mice developed significant numbers of CD4 + /IL-4 + T cells (Fig. 6c; see also Fig. 14). However, all mice that were infected with N. gonorrhoeae regardless of immunization developed CD4 + /IL- l7 + T cells (Fig. 6c and Fig. 14).
  • Intravaginal immunization with gonococcal OMV plus IL-12/ms induces persistent gonococcus-specific antibody responses and Thl cellular responses.
  • serum, vaginal washes, and ILN were collected from immunized and control mice 2 weeks after the last immunization.
  • Serum anti -gonococcal IgM antibodies were at low levels with little difference between experimental groups. IgA and IgG antibodies were not detectable above background in vaginal wash or serum samples of mice given blank ms alone.
  • Intravaginal immunization with 3 doses of gonococcal OMV plus blank ms elevated vaginal and serum anti-gonococcal IgA and IgG antibodies but to a lesser degree than immunization with OMV plus IL-l2/ms (Fig.7a).
  • immunization with one dose of OMV plus IL-l2/ms induced low levels of anti-gonococcal antibodies in both serum and vaginal wash; a second dose elevated antibody production, but no further elevation was seen after 3 doses (Fig.7a).
  • OMV plus IL-l2/ms did not significantly increase the numbers of IL-4 + /CD4 + and IL-l7 + /CD4 + T cells relative to controls (Fig. 8a).
  • IFNy + /CD8 + T cells were specific for gonococcal antigens, CD4 + cells isolated from ILN were preloaded with CFSE, cultured in vitro for 3 days in the presence of antigen-presenting cells with gonococcal OMV or without stimulation as controls, and their proliferation was assessed by flow cytometry.
  • CD4 + cells from the ILN of immunized mice proliferated significantly more, and produced significantly more IFNy, in response to stimulation in vitro, than the cells from control mice (Fig. l7a). No production of IL-4 was seen, but IL-17 was generated by CD4 + T cells cultured with gonococcal OMV (Fig. l7a). IFNy production by ILN CD4 + T cells remained elevated, albeit at slowly declining levels, for 4 months after immunization (Fig. 8b).
  • vaginas were excised from euthanized mice 3 days after the last immunization and RNA was extracted from the whole tissue.
  • RT-PCR analysis showed that, in comparison to controls, immunization with gonococcal OMV plus IL-l2/ms significantly enhanced the expression of mRNA for IFNy but not for IL-4 or IL-17 (Fig. l7b).
  • IFNy mRNA expression in vaginal tissue, and production of IFNy by ILN CD4 + cells remained elevated for up to 2 months following i.vag. immunization with gonococcal OMV plus IL- l2/ms (Fig. l7c).
  • mice immunized with gonococcal OMV plus IL-l2/ms were challenged with the same strain (FA1090) of N. gonorrhoeae at 2, 4, or 6 months after immunization.
  • gonorrhoeae An important consideration for any vaccine is that it should be effective against different strains of the pathogen, as well as those antigenically homologous to the
  • mice 8 per group were immunized i.vag. with OMV prepared from strain FA1090 plus IL- l2/ms or blank ms, and were challenged one month later with N gonorrhoeae strains FA1090 or MS11 (5 x 10 6 CFU). Immunization with FA1090 OMV induced resistance to challenge with either FA1090 or MS11 to similar extents (Fig.
  • N. gonorrhoeae strains FA1090, MS11, and FA19 have been widely used in many laboratories and extensively subcultured since their original isolation. As a result, it is possible that they have acquired mutations and become altered in some of their
  • mice immunized with FA1090 OMV plus IL-l2/ms were also resistant to challenge with clinical isolates GC68 (a PorB.lB strain; Fig. lOe; Table 2) and GC69 (PorB. lA; Table 2).
  • the three 2D protein maps of OMV revealed by Flamingo staining showed numerous protein species and significant differences in the OMV proteome between FA1090, MS11, and FA19 strains (Figs. 1 lc, d, and e).
  • the blotted protein maps showed two spots (Spot 1 and Spot 2) of masses corresponding to 45kDa and 43kDa, and pi 5.2 and 5.5, respectively (FA1090 OMV; Fig.
  • EEZ44683 prolyl cis-trans fkpA, NGEG_01946, NG01225 109.5 44.1 28.94 5.86
  • EEZ48268 potF3, NGFG_01435, NGQ1494 794.15 74.0 41.29 5.96 Periplasmic substrate-binding
  • the course of vaginal gonococcal infection was not altered in unimmunized immunodeficient mice relative to wild-type controls. All wild-type and immunodeficient mice started to reduce the recoverable gonococcal load from day 7-11 and had cleared the infection by dayl2-l4 (median 9-13 days), similar to BALB/c mice used in experiments described in the previous sections (Figs. l2a, b, f, and g; Table 4).
  • mice with gonococcal OMV plus ⁇ L-l2/ms as an adjuvant induced serum and vaginal IgG and IgA antibodies against gonococcal antigens, and IFNy-secreting CD4 + and CD8 + T cells in the draining ILN. Both Thl cellular and antibody responses persisted for several months after immunization, and were capable of eliciting resistance to challenge with N. gonorrhoeae for at least 6 months, with the recall of immune memory. I.vag.
  • OMV vaccine enhances Thl -driven protective immunity revealed by a significantly shortened course of genital gonococcal infection. It should be emphasized that free soluble IL-12 is ineffective and and that IL-12 encapsulated in ms were required for the adjuvant effect with the OMVs.
  • Opa proteins encoded in their genomes differ (Hobbs et al., Front. Microbiol. 2, 123 (2011), Cole et al., PLoS One 4, e8l08 (2009), and their LOS are different (Erwin et al., ./. Exp. Med. 184, 1233-1241 (1996)).
  • Opa proteins and LOS glycan chains are also phase-variable, resulting in the expression of different antigenic epitopes (Apicella, M. A. et al. Phenotypic variation in epitope expression of the Neisseria gonorrhoeae lipooligosaccharide. Infect. Immun. 55, 1755-1761 (1987)).
  • the present disclosure provides demonstration that individuals can be immunized against A. gonorrhoeae by the i.vag. administration of a non-living vaccine (OMV) with a Thl-driving adjuvant, IL-l2/ms.
  • OMV non-living vaccine
  • IL-l2/ms Thl-driving adjuvant
  • mice All mice, including wild-type BALB/c and C57BL/6 mice, B6.129S7-
  • mice were used for the experiments unless otherwise specified. Mice were maintained in a BSL2 facility in the Laboratory Animal Facility at the ETniversity at Buffalo, which is fully accredited by AAALAC. All animal use protocols were approved by the Institutional Animal Care and Use Committee of the University at Buffalo.
  • strain MS11 was provided by Dr Daniel Stein (University of Maryland); strain FA19, and clinical isolates were obtained from the collection of clinical strains maintained at the University of North Carolina at Chapel Hill.
  • a gonorrhoeae strains 9087 and 0336 were transformed with the streptomycin-resistant rpsL gene from strain FA1090 to generate strains GC68 and GC69, respectively.
  • E. coli K12 was provided by Dr Terry Connell (University at Buffalo).
  • Non-typeable Haemophilus influenzae (NTHI) strain 6P24H1 was provided by Dr Timothy Murphy (University at Buffalo).
  • a gonorrhoeae was cultured on GC agar supplemented with hemoglobin and ISOVITALEX ® , an enrichment medium (BD Diagnostic Systems, Franklin Lakes, NJ) and the resultant growth was checked for colony morphology consistent with Opa protein and pilus expression.
  • NTHI was cultured on GC agar supplemented with hemoglobin only.
  • E. coli was cultured on BHI agar. Bacteria were harvested from plates and the cell density was determined (Liu et al., Mucosal Immunol. 5, 320-331 (2012)).
  • IL-12 microspheres [0115] IL-12 microspheres.
  • Murine IL-12 (Wyeth, Philadelphia, PA) was encapsulated into poly-lactic acid microspheres using the Phase Inversion Nanoencapsulation technology as previously described except that bovine serum albumin was replaced by sucrose (0.1 %, w/w) (Egilmez et al Methods Mol. Med. 75, 687-696 (2003)). Blank microspheres were prepared in the same way but without IL-12.
  • CFU colony-forming units
  • Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-mouse IgA, IgG, IgM, IgGl, IgG2a, IgG2b, or IgG3 antibody (Southern Biotech) and p- nitrophenylphosphate substrate (Southern Biotech). Plates were read in a VersaMax microplate reader with SoftMax software (Molecular Devices, Sunnyvale, CA) or an ELx800 LTniversal microplate reader with KC Junior software (Bio-Tek Instruments, Winooski, VT). Antibody data were expressed as relative (fold increase) to the antibody levels detected in control samples (from sham-immunized mice) assayed simultaneously.
  • FACSCalibur cytometer For intracellular staining, cells were first fixed with
  • CYTOFIX/CYTOPERM ® (eBioscience). Antibodies to mouse CD4, CD8, PTNGg, IL-4, and IL-17A conjugated with FITC, PE, or allophycocyanin were purchased from eBioscience.
  • CFSE carboxymethyl fluorescein succinimide ester
  • Cytokine ELISA ⁇ FNy, IL-4, and IL-17A levels were measured in triplicate using ELISA kits purchased from eBioscience.
  • Real-time RT-PCR Total cellular RNA of whole vaginas harvested from the mice was isolated with RNEASY ® RNA purification Mini Kits (Qiagen, Valencia, CA), and was transcribed to cDNA using the ISCRIPTTM cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time RT-PCR was performed on an ICYCLER IQ ® detection system (Bio-Rad) using SYBR ® Green Dye (Bio-Rad) for real-time monitoring of the PCR.
  • the primers used were as follows: IFNy, 5’-TACTGCCACGGCACAGTCATTGAA-3’(SEQ ID NO: 1), 5’- GCAGCGACTCCTTTTCCGCTTCCT-3’ (SEQ ID NO: 2); IL-4, 5’- GAAGCCCTACAGACGAGCTCA-3’(SEQ ID NO: 3), 5’- ACAGGAGAAGGGACGCC AT-3’ (SEQ ID NO: 4); IL-17A, 5’-
  • TCAGGGTCGAGAAGATGCTG-3’ (SEQ ID NO: 5), 5’ -TTTTCATTGTGGAGGGCAGA- 3’ (SEQ ID NO: 6); b-actin, 5’ -CCTAAGGCC AACCGTGAAAAG-3’ (SEQ ID NO: 7), 5’- GAGGCATACAGGGACAGCACA-3’ (SEQ ID NO: 8).
  • Ct threshold cycle
  • nitrocellulose membranes were blocked with PBS containing 3% skim milk overnight at 4°C before incubation for 2 h with serum samples diluted 1 :200, or vaginal wash samples diluted 1 :20 in PBS containing 3% skim milk.
  • Isoelectric focusing was carried out using the PROTEAN® il2TM IEF System (Bio-Rad) for a total of 26,000Vh with the following settings: 50mA current limit, 8000V rapid ramp for 26,000Vh, 750V hold.
  • the second dimension (2D) SDS-PAGE was performed using
  • TGX Any kD gels (Bio-Rad). The proteins were stained overnight in Flamingo fluorescent stain (Bio-Rad) and the spots were visualized using the ChemiDoc Imaging System (Bio-Rad). For immunoblotting, separated proteins were transferred onto PVDF membranes using the TurboBlott transfer system (Bio-Rad). The membranes were blocked for 2h in 5% milk in PBS Tween, and probed by overnight incubation with sera from immunized mice, followed by incubation with anti-mouse HRP-conjugated antibodies (Bio- Rad). Spots were visualized using Clarity Western ECL Substrate and ChemiDoc MP Imaging System (Bio-Rad).
  • the heated capillary temperature was set to 300°C and a spray voltage of 2750V was applied to the electrospray tip.
  • the Orbitrap Elite instrument was operated in the data- dependent mode, switching automatically between MS survey scans in the Orbitrap (AGC target value 1,000,000, resolution 240,000, and injection time 250 milliseconds) with MS/MS spectra acquisition in the linear ion trap (AGC target value of 10,000, and injection time lOOmsec).
  • the 20 most intense ions from the Fourier-transform (FT) full scan were selected for fragmentation in the linear trap by collision-induced dissociation with normalized collision energy of 35%. Selected ions were dynamically excluded for l5sec with a list size of 500 and exclusion mass by mass width ⁇ 0.5.
  • mice Female mice (8 per group) were immunized intranasally with OMV (40pg protein, strain FA19) plus IL-l2/ms (lpg IL-12) or blank ms on days 0 and 14. Two weeks later all mice were challenged intravaginally with 5 x 10 6 CFET of N gonorrhoeae strain FA1090. Vaginal swabs collected daily were diluted and cultured quantitatively on GC agar plates containing selective antibiotics. Results show that mice immunized with OMVs plus IL-12 ms cleared the infection significantly faster than the control groups (p ⁇ 0.01, Fig. 21). In addition, the bacterial colonization loads were significantly lower in the mice immunized with OMVs plus IL-12 ms.
  • mice were immunized intranasally (i.n.) with different doses of OMV prepared from N. gonorrhoeae strain FA1090: 15pg, 30pg, or 60 pg of OMV protein, together with microencapsulated IL-12 (IL-l2/ms; 1 pg of IL-12), or with IL-l2/ms alone. Immunizations were repeated 2 weeks later. Two weeks after the last dose, mice were challenged intravaginally with live N.
  • mice immunized with the low dose of l5pg OMV did not show accelerated clearance of the infection compared to the control group immunized with IL-l2/ms alone (median clearance times: 10 days for both groups).
  • Mice immunized with the usual (mid) dose 30pg or with an increased dose of 60pg OMV cleared the infection significantly faster (median clearance times 6 days and 7.5 days, respectively) than the control group (PO.OOl, Kaplan- Meier, log-rank test).
  • Intranasal (i.n.) immunization was compared with intravaginal (i.vag.) immunization as follows. Mice were immunized with 30pg of gonococcal FA1090 OMV (as protein) plus IL-l2/ms (lpg IL12) or blank microspheres (ms) either i.n. or i.vag. and immunizations were repeated after 2 weeks. Two weeks later, mice were challenged i.vag. with live N gonorrhoeae strain FA19 (heterologous challenge), and the course of infection was followed by daily vaginal swabbing and plating as described previously.
  • mice immunized with OMV plus IL-l2/ms i.n. cleared the infection comparably to mice immunized i.vag.
  • samples of serum, vaginal wash, and saliva were collected for assay of IgG and IgA anti -gonococcal antibodies by ELISA.
  • Mice immunized i.n. with IL-l2/ms cleared the infection comparably to those immunized i.vag. with the same vaccine (Fig 23 A).
  • mice were challenged with the same strain as used for the preparation of the OMV, FA1090 (homologous challenge), mice immunized with OMV plus IL-l2/ms i.n. (as described for Figure 3), cleared the infection comparably to those immunized i.vag. (Fig 24).
  • mice were immunized i.n. once or twice with gonococcal FA1090 OMV
  • mice (30pg protein) plus IL-l2/ms (lpg IL-12); control mice received blank ms. Two weeks after the second immunization, mice were challenged i.vag. with N gonorrhoeae MS11
  • mice were immunized i.n. with OMV (30pg protein) prepared from N.
  • mice were immunized i.n. with OMV (30pg protein) prepared from N gonorrhoeae MS11, plus either IL-l2/ms (lpg IL-12) or blank ms, and challenged with heterologous gonococcal strain FA19.
  • OMV (30pg protein) prepared from N gonorrhoeae MS11, plus either IL-l2/ms (lpg IL-12) or blank ms, and challenged with heterologous gonococcal strain FA19.
  • mice immunized with MS11 OMV plus IL-l2/ms cleared the infection faster (median clearance time: 5 days) compared to those immunized with MS11 OMV plus blank ms (median clearance time: 9 days), or compared to unimmunized control mice (median clearance time: 10 days).
  • P 0.0007 (Kaplan-Meier analysis, log-rank test)
  • strains of N gonorrhoeae used in these experiments express different porin types.
  • Gonococcal porin PorB is the major outer membrane protein in N gonorrhoeae , and is represented by two major types, PorB-l A and PorB-lB, each having many sub-types.
  • Strain FA1090 has porin type PorB-lB
  • strain MS11 also has porin type PorB-lB although a different sub-type from FA1090
  • strain FA19 has porin type PorB- 1 A.
  • these strains express different Opa proteins and lipooligosaccharide structures, thus they represent widely different antigenic variants of N gonorrhoeae.

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Abstract

L'invention concerne une méthode pour traiter et réduire la récurrence d'infections du tractus génital telles que des infections gonococciques. La méthode comprend l'application locale d'IL-12 incorporée dans des microsphères polymères. L'invention concerne également une méthode pour réduire l'incidence d'infections du tractus génital provoquées par N. gonorrhoeae par l'administration de préparations de vésicules de membrane externe de N. gonorrhoeae et d'IL-12 incorporée dans des microsphères polymères.
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