Artificial invaplex formulated with deacylated lipopolysaccharide

Abstract

Disclosed herein are artificial Invaplexes comprising deacylated lipopolysaccharides and methods of making and using thereof.

Claims

1. An artificial Invaplex comprising one or more invasin proteins complexed with a deacylated lipopolysaccharide from a gram-negative bacterial strain which is a strain of a Shigella spp.

2. The artificial Invaplex of claim 1, wherein the deacylated lipopolysaccharide lacks one or more fatty acid chains as compared to the corresponding wild-type lipopolysaccharide.

3. The artificial Invaplex of claim 1, wherein the deacylated lipopolysaccharide lacks one fatty acid chain as compared to the corresponding wild-type lipopolysaccharide.

4. The artificial Invaplex of claim 1, wherein the deacylated lipopolysaccharide lacks two fatty acid chains as compared to the corresponding wild-type lipopolysaccharide.

5. The artificial Invaplex of claim 1, wherein the deacylated lipopolysaccharide lacks more than two fatty acid chains as compared to the corresponding wild-type lipopolysaccharide.

6. The artificial Invaplex of claim 1, wherein the gram-negative bacterial strain is an msbB mutant strain, such as a msbB1 mutant strain, a msbB2 mutant strain, or a msbB1/msbB2 mutant strain.

7. The artificial Invaplex of claim 1, wherein the gram-negative bacterial strain is WR10, WR20, or WR30.

8. The artificial Invaplex of claim 1, wherein the one or more invasin proteins are IpaB and IpaC from Shigella spp.

9. The artificial Invaplex of claim 1, wherein the deacylated lipopolysaccharide was deacylated by enzymatic treatment or was obtained from a strain of a Shigella spp. that lacks one or more genes responsible for lipopolysaccharide acylation or has a loss-of-function mutation in one or more genes responsible for lipopolysaccharide acylation.

10. A composition comprising the artificial Invaplex of claim 1 and a pharmaceutically acceptable carrier.

11. The composition of claim 10, further comprising an immunogen.

12. The composition of claim 11, wherein the artificial Invaplex is an adjuvant for the immunogen.

13. The composition of claim 10, wherein the artificial Invaplex is present in an immunogenic amount.

14. A method for inducing an immune response in a subject, which comprises administering to the subject an immunogenic amount of the artificial Invaplex according to claim 1 or a composition thereof.

15. The method according to claim 14, wherein the immunogenic amount is administered by mucosal administration.

16. The method according to claim 14, wherein the immune response is a protective immune response.

17. The method according to claim 14, wherein the immune response is a balanced Th1/Th2 response as compared to that provided by a corresponding Invaplex comprising the corresponding wild-type lipopolysaccharide instead of the deacylated lipopolysaccharide.

18. The method or use according to claim 17, wherein the balanced Th1/Th2 response is a protective immune response.

Description

DESCRIPTION OF THE DRAWINGS

(1) This invention is further understood by reference to the drawings wherein:

(2) FIG. 1 is a flow diagram schematically showing the production of deacylated LPS. S. flexneri 2a msbB mutant strains were fermented, harvested, and washed to yield a cell paste that was phenol/water extracted to extract the LPS which was then subjected to dialysis to remove residual phenol. The dialyzed LPS was next dispensed and lyophilized to form the final LPS product used to make Invaplex.sub.AR-Detox.

(3) FIG. 2 is a flow diagram showing the final assembly process in which IpaB, IpaC, and deacylated LPS are combined to form a high molecular mass complex that is purified by ion-exchange chromatography.

(4) FIG. 3 is an SDS-PAGE gel of individual Invaplex.sub.AR components of each Invaplex exemplified herein stained with Coomassie blue.

(5) FIG. 4 is an SDS-PAGE gel of individual Invaplex.sub.AR components of each Invaplex exemplified herein stained with silver.

(6) FIG. 5 is a graph showing TNF- release from murine macrophages after incubation with 0.01 g of LPS purified from S. flexneri 2a strain 2457T (wild-type), the indicated msbB mutant strain, or 0.1 g of Invaplex preparations made therewith each given LPS.

(7) FIG. 6, FIG. 7, and FIG. 8 are graphs showing mean percent weight change of mice treated with 0.25 g, 2.5 g, and 25 g, respectively, of the given Invaplex.sub.AR preparation or control at 24, 48, and 72 hours after each intradermal immunization.

(8) FIG. 9, FIG. 10, and FIG. 11 are graphs of the mean scores of ruffled fur and hunched posture after intradermal immunization of the group administered with 0.25 g, 2.5 g, and 25 g, respectively, of the indicated Invaplex.sub.AR preparation at the indicated time points. As shown, the LPS of the Invaplex.sub.AR preparations for each set of bars in the graph from left to right are: WT-LPS, B1-LPS (from WR10), B2-LPS (from WR20), and B1/2-LPS (from WR30), and the last sets of bars are saline.

(9) FIG. 13 is a table summarizing administration site induration observed at administration site 1 and 2.

(10) FIG. 14, FIG. 15, and FIG. 16 are graphs showing the Shigella LPS-specific serum IgG endpoint titers on day 35 from mice intradermally immunized with 0.25 g, 2.5 g, and 25 g, respectively, of the indicated Invaplex.sub.AR preparation. As shown from left to right the LPS are: WT-LPS, B1-LPS (from WR10), B2-LPS (from WR20), and B1/2-LPS (from WR30), and the 5th bar is saline. Dotted lines represent GMT after immunization with Invaplex.sub.AR-WT.

(11) FIG. 17, FIG. 18, and FIG. 19 are graphs showing the Shigella Invaplex-specific serum IgG endpoint titers on day 35 from mice intradermally immunized with 0.25 g, 2.5 g, and 25 g, respectively, of the indicated Invaplex.sub.AR preparation. As shown, the LPS of the Invaplex.sub.AR preparations from left to right are: WT-LPS, B1-LPS (from WR10), B2-LPS (from WR20), and B1/2-LPS (from WR30), and the last bar is saline. Dotted lines represent GMT after immunization with Invaplex.sub.AR-WT.

(12) FIG. 20 is a table summarizing Shigella antigen-specific serum IgA and IgG endpoint titers on day 35 from mice intradermally immunized with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox.

(13) FIG. 21 is a graph showing the anti-IpaB serum IgG1 and IgG2a responses in mice immunized intradermally with 0.25 g of Invaplex.sub.AR assembled with LPS isolated from wild-type S. flexneri 2a, 2457T or msbB1, msbB2, or msbB1/2 mutant S. flexneri 2a strains. WT=Invaplex.sub.AR-WT, B1=Invaplex.sub.AR-Detox with LPS from WR10, B2=Invaplex.sub.AR-Detox with LPS from WR20, and B1B2=Invaplex.sub.AR-Detox with LPS from WR30.

(14) FIG. 22 is a table summarizing Shigella antigen-specific lung wash IgG endpoint titers on day 35 from mice intradermally immunized with Invaplex.sub.AR-Wt or Invaplex.sub.AR-Detox.

(15) FIG. 23 is a table with the results of protective efficacy of intranasal immunization with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox (msbB1/2 LPS) after intrarectal challenge with wild-type S. flexneri 2a 2457T.

(16) FIG. 24 is a table detailing Shigella LPS and Invaplex-specific serum IgA and IgG endpoint titers on day 42 from guinea pigs intranasally immunized with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox (msbB1/2 LPS).

(17) FIG. 25 is a table with Shigella IpaB and IpaC-specific serum IgA and IgG endpoint titers on day 42 from guinea pigs intranasally immunized with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox (msbB1/2 LPS).

(18) FIG. 26 is a table with Shigella antigen-specific ocular IgA endpoint titers on day 42 from guinea pigs intranasally immunized with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox(msbB1/2 LPS).

(19) FIG. 27 is a table that provides Shigella antigen-specific fecal IgA endpoint titers on day 35 from guinea pigs intranasally immunized with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox(msbB1/2 LPS).

(20) FIG. 28 is a table summarizing the protective efficacy after ocular challenge of guinea pigs immunized either intranasally, intramuscularly or intradermally with Invaplex.sub.AR-Detox. Guinea pigs (n=6-12/grp) were immunized on day 0, 14 and 28 with 100 l containing either 5 or 25 g of S. flexneri 2a Invaplex.sub.AR-Detox. On day 49, guinea pigs were challenged ocularly with 2.010.sup.8 cfu/eye of S. flexneri 2a strain 2457T.

(21) FIG. 29 is the mass spectrum of Lipid A isolated from 2457T showing a parental peak at 1798 m/z and a dephosphorylation peak at 1716 m/z (arrow).

(22) FIG. 30 is the mass spectrum of Lipid A isolated from WR30 showing a parental peak at 1590 amu and a dephosphorylation peak at 1502 amu (arrow).

DETAILED DESCRIPTION OF THE INVENTION

(23) The present invention provides Invaplex.sub.AR-Detox, which is an artificial Invaplex that contains a deacylated lipopolysaccharide (LPS), and methods of making and using thereof.

(24) As used herein, Invaplex.sub.AR-Detox refers to an artificial Invaplex made with deacylated LPS and Invaplex.sub.AR-WT refers to an artificial Invaplex made with wild-type LPS (WT-LPS). As used herein, Invaplex.sub.AR refers to an artificial Invaplex, i.e., a non-naturally occurring Invaplex.

(25) As used herein, deacylated LPS and detoxified LPS are used interchangeably to refer to an LPS obtained from a gram-negative bacterium that lacks at least one fatty acid chain as compared the corresponding WT-LPS. As used herein, wild-type LPS refers to LPS that has been obtained from wild-type gram-negative bacteria and has not been modified such that it lacks one or more of its native fatty acid chains. In some embodiments, the deacylated LPS lacks two fatty acid chains as compared to the corresponding WT-LPS.

(26) In some embodiments, the gram-negative bacterium is Shigella spp., e.g., S. boydii, S. dysenteriae, S. flexneri, and S. sonnei. In some embodiments, the gram-negative bacterium is a strain of Shigella. In some embodiments, the gram-negative bacterium is a strain of Shigella flexneri 2a. In some embodiments, the deacylated LPS is obtained from an msbB mutant. As used herein, an msbB mutant refers to a gram-negative bacterium that has been mutated to contain at least one defective msbB gene such that the LPS produced therefrom is missing at least one fatty acid chain as compared to that produced by the corresponding wild-type bacteria.

(27) In some embodiments, the msbB mutant is a msbB1 (WR10), msbB2 (WR20), or msbB1/msbB2 (WR30) mutant. The msbB1/msbB2 mutant is sometimes referred to as msbB1/2 mutant or a double mutant. In some embodiments, the msbB mutant is a mutant strain of Shigella. In some embodiments, the msbB mutant is a mutant strain of Shigella flexneri 2a. In some embodiments, the Shigella strain is WR10, WR20, or WR30.

(28) Invaplex.sub.AR-Detox according to the present invention can be used as an alternative to artificial invasin complex employed in compositions and methods in the art, e.g., WO 2008118118 and Shigella flexneri 2a Invaplex.sub.AR which is in clinical trials for intranasally vaccinating against Shigellosis Bacillary Dysentery (Clinical Trial No. NCT02445963).

(29) As disclosed herein, Invaplex.sub.AR-Detox is less reactogenic than Invaplex.sub.AR-WT after parenteral administration, yet the immunogenicity of Invaplex.sub.AR-Detox is similar to that of Invaplex.sub.AR-WT. Invaplex.sub.AR-Detox provides a more balanced Th1/Th2 immune response as compared to that of Invaplex.sub.AR-WT. As such, Invaplex.sub.AR-Detox may be more useful as a parenteral vaccine and more compatible with adjuvants as compared with Invaplex.sub.AR-WT. Additionally, because of its reduced reactogenicity, Invaplex.sub.AR-Detox may be administered to subjects in amounts higher than Invaplex.sub.AR-WT that are typically administered, and thereby provide a more potent immune response or reduce potential reactogenicity to a level that would allow the addition of an adjuvant, which may have reactogenicity, to increase the immune response.

(30) Therefore, in some embodiments, the present invention provides Invaplex.sub.AR-Detox as a vaccine. In some embodiments, the present invention provides an immunogenic composition which comprises at least one Invaplex.sub.AR-Detox as the immunogen. In some embodiments, the present invention provides a vaccine composition which comprises at least one Invaplex.sub.AR-Detox as an adjuvant. In some embodiments, the present invention is directed to vaccinating a subject against dysentery by administering to the subject an Invaplex.sub.AR-Detox. In some embodiments, the present invention is directed to a method of inducing a balanced Th1/Th2 immune response in a subject which comprises administering at least one Invaplex.sub.AR-Detox to the subject. In some embodiments, the at least one Invaplex.sub.AR-Detox is parenterally administered to the subject. In some embodiments, the at least one Invaplex.sub.AR-Detox is mucosally administered to the subject. In some embodiments, the present invention provides an improvement to Invaplex.sub.AR-WT, wherein the improvement is a deacylated LPS as a substitute for the WT-LPS in the Invaplex.sub.AR-WT.

(31) Compositions of the present invention, including pharmaceutical compositions and vaccines, include at least one Invaplex.sub.AR-Detox. The term pharmaceutical composition refers to a composition suitable for pharmaceutical use in a subject. A pharmaceutical composition generally comprises an effective amount of an active agent, e.g., at least one Invaplex.sub.AR-Detox according to the present invention, and a pharmaceutically acceptable carrier. The term effective amount refers to a dosage or amount sufficient to produce a desired result. The desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount, e.g., long-term survival, effective prevention of a disease state, and the like.

(32) At least one Invaplex.sub.AR-Detox according to the present invention may be administered, preferably in the form of pharmaceutical compositions, to a subject. Preferably the subject is mammalian, more preferably, the subject is human. Preferred pharmaceutical compositions are those comprising at least one Invaplex.sub.AR-Detox in a therapeutically effective amount or an immunogenic amount, and a pharmaceutically acceptable vehicle.

(33) Vaccines according to the present invention provide a protective immune response when administered to a subject. As used herein, a vaccine according to the present invention is a pharmaceutical composition that comprises an immunogenic amount of at least one Invaplex.sub.AR-Detox and provides a protective immune response when administered to a subject. The protective immune response may be complete or partial, e.g., a reduction in symptoms as compared with an unvaccinated subject. In some embodiments, a vaccine according to the present invention provides a subject with a protective immune response against infection by a Shigella spp., e.g., S. boydii, S. dysenteriae, S. flexneri, and S. sonnei. In some embodiments, a vaccine according to the present invention inhibits or reduces the degree of infection by a Shigella spp., e.g., S. boydii, S. dysenteriae, S. flexneri, and S. sonnei in a subject when administered to the subject.

(34) As used herein, an immunogenic amount is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the given immunogen, the degree of infection, the manner of administration, the general state of health of the subject, and the like. Typical immunogenic amounts for initial and boosting immunizations for therapeutic or prophylactic administration may range from about 0.1 g to 10 mg per kilogram of body weight of a subject of at least one Invaplex.sub.AR-Detox. In some embodiments, the immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject of 70 kg body weight ranges from about 5 g to about 1 mg of at least one Invaplex.sub.AR-Detox. In some embodiments, the immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject of 70 kg body weight ranges from about 10-500 g of at least one Invaplex.sub.AR-Detox. In some embodiments, the immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject of 70 kg body weight ranges from about 40-60 g of at least one Invaplex.sub.AR-Detox. In some embodiments, the immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject of 70 kg body weight ranges from about 200-300 g of at least one Invaplex.sub.AR-Detox. In some embodiments, the immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject of 70 kg body weight ranges from about 450-550 g of at least one Invaplex.sub.AR-Detox. Examples of suitable immunization protocols include an initial immunization injection (time 0), followed by booster injections at 2 and 4 weeks, which these initial immunization injections may be followed by further booster injections at 1 or 2 years if needed.

(35) As used herein, a therapeutically effective amount refers to an amount that may be used to treat, prevent, or inhibit a given disease or condition, such as infection by a Shigella spp., in a subject as compared to a control. Again, the skilled artisan will appreciate that certain factors may influence the amount required to effectively treat a subject, including the degree of infection by a Shigella spp., previous treatments, the general health and age of the subject, and the like. Nevertheless, therapeutically effective amounts may be readily determined by methods in the art. It should be noted that treatment of a subject with a therapeutically effective amount or an immunogenic amount may be administered as a single dose or as a series of several doses. The dosages used for treatment may increase or decrease over the course of a given treatment. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using dosage-determination tests and/or diagnostic assays in the art. Dosage-determination tests and/or diagnostic assays may be used to monitor and adjust dosages during the course of treatment.

(36) The compositions of the present invention may include an adjuvant. As used herein, an adjuvant refers to any substance which, when administered in conjunction with (e.g., before, during, or after) a pharmaceutically active agent, such as a Invaplex.sub.AR-Detox according to the present invention, aids the pharmaceutically active agent in its mechanism of action. Thus, an adjuvant in a vaccine according to the present invention is a substance that aids the at least one Invaplex.sub.AR-Detox in eliciting an immune response. Suitable adjuvants include incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1-2-dipa-lmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, MTP-PE), and RIBI, which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by methods in the art.

(37) Pharmaceutical compositions of the present invention may be formulated for the intended route of delivery, including intravenous, intramuscular, intra peritoneal, subcutaneous, intraocular, intrathecal, intraarticular, intrasynovial, cisternal, intrahepatic, intralesional injection, intracranial injection, infusion, and/or inhaled routes of administration using methods known in the art. Pharmaceutical compositions according to the present invention may include one or more of the following: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. The compositions and formulations of the present invention may be optimized for increased stability and efficacy using methods in the art. See, e.g., Carra et al. (2007) Vaccine 25:4149-4158, which is herein incorporated by reference.

(38) The compositions of the present invention may be administered to a subject by any suitable route including oral, transdermal, subcutaneous, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the preferred route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular Invaplex.sub.AR-Detox used.

(39) As used herein, a pharmaceutically acceptable vehicle or pharmaceutically acceptable carrier are used interchangeably and refer to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration and comply with the applicable standards and regulations, e.g., the pharmacopeial standards set forth in the United States Pharmacopeia and the National Formulary (USP-NF) book, for pharmaceutical administration. Thus, for example, unsterile water is excluded as a pharmaceutically acceptable carrier for, at least, intravenous administration. Pharmaceutically acceptable vehicles include those known in the art. See, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY. 20.sup.th ed. (2000) Lippincott Williams & Wilkins. Baltimore, Md., which is herein incorporated by reference.

(40) The pharmaceutical compositions of the present invention may be provided in dosage unit forms. As used herein, a dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the one or more Invaplex.sub.AR-Detox calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the given Invaplex.sub.AR-Detox and desired therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

(41) Toxicity and therapeutic efficacy of an Invaplex.sub.AR-Detox according to the instant invention and compositions thereof can be determined using cell cultures and/or experimental animals and pharmaceutical procedures in the art. For example, one may determine the lethal dose, LC.sub.50 (the dose expressed as concentrationexposure time that is lethal to 50% of the population) or the LD.sub.50 (the dose lethal to 50% of the population), and the ED.sub.50 (the dose therapeutically effective in 50% of the population) by methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD.sub.50/ED.sub.50. An Invaplex.sub.AR-Detox that exhibits large therapeutic indices is preferred. While an Invaplex.sub.AR-Detox that results in toxic side-effects may be used, care should be taken to design a delivery system that targets such compounds to the site of treatment to minimize potential damage to uninfected cells and, thereby, reduce side-effects.

(42) The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Preferred dosages provide a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration utilized. Therapeutically effective amounts and dosages of at least one Invaplex.sub.AR-Detox according to the present invention can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. Additionally, a dosage suitable for a given subject can be determined by an attending physician or qualified medical practitioner, based on various clinical factors.

(43) In some embodiments, the present invention is directed to kits which comprise at least one Invaplex.sub.AR-Detox, optionally in a composition, packaged together with one or more reagents or drug delivery devices for preventing, inhibiting, reducing, or treating infection by a Shigella spp. in a subject. Such kits include a carrier, package, or container that may be compartmentalized to receive one or more containers, such as vials, tubes, and the like. In some embodiments, the kits optionally include an identifying description or label or instructions relating to its use. In some embodiments, the kits comprise the at least one Invaplex.sub.AR-Detox, optionally in one or more unit dosage forms, packaged together as a pack and/or in drug delivery device, e.g., a pre-filled syringe. In some embodiments, the kits include information prescribed by a governmental agency that regulates the manufacture, use, or sale of Invaplex.sub.AR-Detox and compositions thereof according to the present invention.

(44) The following examples are intended to illustrate but not to limit the invention.

(45) LPS Production

(46) WT-LPS and deacylated LPS, obtained from S. flexneri 2a and msbB mutants (i.e., S. flexneri 2a strains having msbB deletions), were purified, lyophilized, and dried as schematically shown in FIG. 1. Then the lyophilized LPS was used to make Invaplex.sub.AR-Detox and Invaplex.sub.AR-WT.

(47) Gram-Negative Strains

(48) The S. flexneri 2a wild-type and msbB mutant strains are summarized in Table 1:

(49) TABLE-US-00001 TABLE 1 Name Genotype Other info 2457T Wild-type S. flexneri (Wild-type) 2a 2457T WR10 msbB1 single deletion msbB1 is (msbB1) mutant of 2457T chromosomally encoded WR20 msbB2 single deletion msbB2 is plasmid (msbB2) mutant of 2457T encoded WR30 msbB1 msbB2 double (msbB1/msbB2) deletion mutant of 2457T

(50) The 2457T, WR10, WR20, and WR30 strains are described in Ranallo et al. (2010) Infect. Immun. 78:400-412. The strains 2457T, WR10, WR20, and WR30 and their LPS products are sometimes notated with wild-type, msbB1, msbB2, and msbB1/msbB2, respectively. msbB1/msbB2 is sometimes shortened to msbB1/2. Cell banks for each strain were prepared and stored frozen. Strains lacking msbB (late acyltransferase) have reduced lipid A acylation which reduces the net toxicity of the LPS. The S. flexneri msbB mutant strains, make an underacylated (i.e., deacylated) LPS with either a tetra or penta-acylated rather than a hexa-acylated lipid A as found in WT-LPS.

(51) Production of Artificial Invaplex

(52) Each artificial Invaplex was assembled from purified, recombinant IpaB, IpaC, and purified deacylated LPS from the desired species of Shigella, mixed in a molar ratio of 8 IpaC to 1 IpaB, and with an LPS total protein mixing ratio of approximately 2.2. FIG. 2 schematically shows the final assembly process in which IpaB, IpaC, and deacylated LPS are combined to form a high molecular mass complex that is purified by ion-exchange chromatography. Fractions containing IpaB, IpaC, and deacylated LPS are pooled and stored at 80 C. for evaluation.

(53) Analysis of Invaplex.sub.AR-Detox and Invaplex.sub.AR-WT

(54) As it was unknown whether the deacylated LPS would prevent the formation of Invaplex, once each assembled Invaplex was purified by ion-exchange chromatography each was analyzed for quantity, quality, and identity of the individual components, as well as the molecular weight of the given Invaplex prior to use in animals or in vitro studies.

(55) Four individual lots of Invaplex.sub.AR were manufactured using research grade, purified deacylated LPS from each msbB mutant strain or WT-LPS strain, the same research-grade, purified IpaB and IpaC, as well as the same mobile phases and mixing diluents. The 4 lots are as follows: Lot PB (msbB2), Lot PC (msbB1/msbB2), Lot PD (msbB1), and Lot PE (WT-LPS). Characteristics of the four Invaplex.sub.AR lots are shown in Table 2:

(56) TABLE-US-00002 TABLE 2 SEC- Product Lot Molar IpaC:IpaB HPLC LPS (LPS Total Yield Ratio Ratio by Hydrodynamic Retention Endotoxin (g/mg Source) (Concentration) IpaC:IpaB Densitometry Radius by DLS (mins) (EU/ml) Invaplex) PB 31.45 mg 36:1 9.2:1 22.9 1.9 16.04 5.7 10.sup.7 3353 (msbB2) (1.7 mg/ml) PC 31.05 mg 25:1 5.7:1 18.2 1.2 16.37 8.1 10.sup.7 3522 (msbB1/2) (2.3 mg/ml) PD 30.55 mg 28:1 8.7:1 23.5 3.9 16.00 6.2 10.sup.7 4769 (msbB1) (1.3 mg/ml) PE 29.6 mg 25:1 6.8:1 26.7 7.3 15.84 5.6 10.sup.7 3500 (WT-LPS) (1.6 mg/ml)

(57) Each lot of Invaplex.sub.AR-Detox was found to be comparable to a reference standard, i.e., S. flexneri 2a Invaplex.sub.AR-WT Lot NYNZ, which was manufactured using similar IpaB and IpaC components and a WT-LPS-to-protein mixing ratio of 2.2. These results suggest that each Invaplex.sub.AR made with deacylated LPS is structurally similar to Invaplex.sub.AR-WT. Each Invaplex formulation was analyzed by SDS-PAGE stained with Coomassie blue (FIG. 3) or silver (FIG. 4) to assess the protein and LPS content and by SEC-HPLC to estimate the size of each Invaplex.sub.AR complex. No differences in silver-stained LPS ladders were identified and all were comparable to the reference standard S. flexneri 2a Invaplex.sub.AR Lot NYNZ by SDS-PAGE analysis. SEC-HPLC showed single 215 nm peaks; however, Lots PB, PC, and PD, constructed from deacylated LPS, took slightly longer to resolve and were thus considered slightly smaller as compared to Lot PE constructed using WT-LPS. Specifically, the retention times were as follows: Lot PB=16.04 minutes, Lot PC=16.37 minutes, Lot PD=16.00 minutes, and Lot PE=15.84 minutes.

(58) Vials of each lot were also retained for in vitro assays to measure biological function and release of pro-inflammatory cytokines from murine macrophages as well as for immunization of small animals.

(59) MALDI-TOF Assay for Characterizing and Differentiating Deacylated LPS and WT-LPS

(60) The deacylated LPS from the WR30 (msbB1/msbB2) has one acyl group missing on the Lipid A moiety. Comparing this deacylated LPS to WT-LPS isolated from 2457T (wild-type) is difficult because the change in mass (about 210 atomic mass units, or amu) is difficult to detect by using conventional means such as gel electrophoresis.

(61) A more sensitive assay was needed to differentiate between LPS produced from WR30 and wild-type LPS. To accomplish this task, the Lipid A from LPS samples isolated from wild-type 2457T and mutant WR30 was analyzed using MALDI-TOF mass spectrometry.

(62) The samples were prepared for MALDI by hydrolyzing the Lipid A from the LPS samples. The LPS samples were hydrolyzed by adding 20 l acetic acid to 180 l of the LPS samples. The samples were heated in a heat block at 100 C. for 1 hour. The samples were then centrifuged at 12,000g RCF for 4 minutes. The supernatant was discarded. The pellet containing the Lipid A was suspended in 5 l of matrix solution. The matrix solution was made by dissolving 10 mg of dihydroxybenzoic acid (DHB) in 500 l of a 1:1 ratio of WFI and HPLC grade acetonitrile. The samples were spotted on the MALDI targeting plate using the dried drop method.

(63) The samples were analyzed on a Bruker Microflex MALDI-TOF instrument in the linear negative ion mode. The major peak that is the focus on both the Lipid A spectrum is the parental mass peaks. Lipid A has a theoretical molecular mass of 1798 amu. The experimentally determined mass spectrum of Lipid A isolated from 2457T shows the parental peak at 1798 m/z and a dephosphorylation peak at 1716 m/z (FIG. 29, arrow). The deacylated Lipid A of WR30 has a theoretical molecular weight of 1588 amu. The Lipid A isolated from WR30 matches the theoretical molecular weight of a deacylated Lipid A with a de-phosphorylation peak at 1502 amu (FIG. 30) and confirms the identity of the product as deacylated LPS. The experimentally determined mass spectrum lacks a peak at 1798 m/z thereby indicating that the Lipid A produced by WR30 is deacylated (FIG. 30). Mass spectrum analysis may be used to distinguish deacylated LPS from WT-LPS and thus Invaplex.sub.AR-Detox from Invaplex.sub.AR-WT.

(64) Additionally, since each Invaplex formulation contains similar quantities of IpaB, IpaC, and LPS, the use of deacylated LPS does not significantly alter the mass of the IpaB/IpaC/LPS complex, thereby indicating that a similar quantity of protein and LPS are present in the Invaplex.sub.AR-WT and Invaplex.sub.AR-Detox.

(65) Cellular Uptake of Invaplex.sub.AR-Detox into Fibroblasts

(66) Since it is unknown whether Invaplex.sub.AR-Detox will stimulate or induce uptake by mammalian cells, the following experiment was conducted. Baby hamster fibroblast (BHK-12) cells were seeded in 8 well glass chamber slides (Nunc) and incubated overnight at 37 C. with 5% CO.sub.2. Cells were washed with serum-free culture medium and incubated for 60 minutes with Lot PE (wild-type), Lot PD (msbB1), Lot PB (msbB2), or Lot PC (msbB1/2). The Invaplex.sub.AR preparations were diluted to 2.5, 25, and 250 g/ml in serum-free culture medium and incubated in duplicate chamber slide wells. After a 60 minute incubation at 37 C., the wells were washed 5 with PBS and fixed with 10% formalin for 30 minutes at ambient temperature (about 23 C.).

(67) Internalized Invaplex.sub.AR was detected by incubating the chamber slide wells with an anti-S. flexneri 2a, 2457T polyclonal rabbit serum (WRAIR Rabbit #7 diluted 1:200 in 10% FCS containing 0.001% saponin). After incubation, unbound antibody was removed by washing. Bound rabbit antibodies were detected using goat anti-rabbit antibodies conjugated to Oregon Green 488 (Invitrogen; 0.5 g/ml) and the nucleus was stained with Dapi (0.1 g/ml).

(68) Cells were viewed at 60 magnification on a Nikon Optiphot fluorescent microscope. The integrity of the cells was evaluated using bright-field microscopy. The number of cells in five random fields per well were enumerated based on nuclear staining and the number of Invaplex positive cells (stained with Oregon Green) were enumerated. A minimum of 150 cells from each well were scored. The percentage of Invaplex.sub.AR positive cells was calculated by dividing the total number of Invaplex.sub.AR positive cells (Oregon Green stain) by the total number of cells (Dapi stain)

(69) Examination by bright-field microscopy did not reveal differences between cells treated with Invaplex.sub.AR-WT, Invaplex.sub.AR-Detox, and cells incubated with culture medium in terms of cellular damage, rounded cells, or loss of cells. The percentage of Invaplex positive cells after incubation with various concentrations of Invaplex preparations are shown in Table 3 as follows:

(70) TABLE-US-00003 TABLE 3 Percentage of Invaplex.sub.AR positive BHK-21 cells after incubation with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox diluted to: Treatment 0 g/ml 2.5 g/ml 25 g/ml 250 g/ml Invaplex.sub.AR-WT <1 9 44 93 (WT-LPS; Lot PE) Invaplex.sub.AR-Detox <1 31 30 74 (msbB1 LPS; Lot PD) Invaplex.sub.AR-Detox <1 23 15 84 (msbB2 LPS; Lot PB) Invaplex.sub.AR-Detox <1 9 62 99 (msbB1/2 LPS; Lot PC)

(71) Invaplex.sub.AR was found in a comparable percentage of cells incubated with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox (msbB1/2 LPS; Lot PC) across the various concentrations. At the 2.5 g/ml concentration, a >2-fold increase in the percentage of cells identified as Invaplex.sub.AR positive after treatment with Invaplex.sub.AR-Detox (msbB1 LPS; Lot PD) and Invaplex.sub.AR-Detox (msbB2 LPS; Lot PB) as compared to Invaplex.sub.AR-WT was seen. At the 250 g/ml concentration, 74% of BHK-21 cells were Invaplex.sub.AR-positive after treatment with the various Invaplex.sub.AR preparations.

(72) All three Invaplex.sub.AR-Detox preparations induce self-uptake in BHK-21 cells. Thus, the use of deacylated LPS to produce artificial Invaplex does not alter the native uptake activity of Invaplex.sub.AR and suggests Invaplex.sub.AR-Detox preparations may be suitable for use as vaccines, adjuvants and/or transporters.

(73) Pro-Inflammatory Cytokine Release from Macrophages Exposed to Invaplex

(74) Due to the known endotoxic properties of LPS from wild-type Shigella spp., it is probable that Invaplex made with Shigella WT-LPS will have endotoxic properties capable of inducing reactogenicity in parenterally immunized hosts. The potential toxicity of Invaplex products can be evaluated in vitro by using cultured cells that are sensitive to endotoxin and release pro-inflammatory cytokines such as TNF- upon exposure to LPS. Using cultured eukaryotic cells (mouse macrophages), the toxicity of each Invaplex.sub.AR preparation, and the LPS used to construct each preparation was determined.

(75) Murine macrophages (J77A.1; ATCC TIB-67) were seeded in 96 well tissue culture plates and incubated overnight at 37 C. Cells were washed with serum-free culture medium and incubated for 6 hours with LPS obtained from 2457T (wild-type), WR10, WR20, and WR30 at 1, 0.1, or 0.01 g/ml. Other wells were incubated with a given Invaplex.sub.AR preparation at 1, 0.1, or 0.01 g/ml. The LPS component to construct each Invaplex.sub.AR preparation was the same material used in the LPS-only experiments. After incubation for 2, 4, and 6 hours, supernatant was collected from triplicate wells and assayed by Luminex to determine TNF- concentration.

(76) As shown in FIG. 5, incubation of murine macrophages with WT-LPS (0.01 g/ml) resulted in the release of robust levels of TNF- at the 4 hour time point (mean=9507 pg/ml) and increased to 10,514 pg/ml after 6 hours. In contrast, TNF- was low (<10 pg/ml) in the macrophage supernatant after incubation with culture medium alone (DMEM). The concentration of TNF- in supernatants of cells incubated with LPS from any of the three msbB mutants was significantly lower as compared to TNF- concentrations after incubation with WT-LPS at the 4 hour time point. At the 6 hour time point, TNF- concentrations in the macrophage supernatants from cells incubated with LPS from the msbB1 mutants were comparable to the TNF- concentration in supernatant from cells incubated with WT-LPS whereas cells incubated with LPS from msbB2 or LPS from msbB1/2 were significantly lower levels of TNF- as compared to cells incubated with WT-LPS.

(77) Incubation of murine macrophages with 1, 0.1, and 0.01 g/ml of Invaplex.sub.AR-WT and each Invaplex.sub.AR-Detox resulted in release of significantly lower levels of TNF- as compared to the purified LPS preparations. Maximal TNF- release was 3097 pg/ml after incubation with 1 g/ml Invaplex.sub.AR-Detox assembled with msbB2 LPS. Incubation of macrophages with 0.1 or 0.01 g/ml of the Invaplex.sub.AR-Detox preparations caused the release of TNF- at low concentrations (<1000 pg/ml).

(78) Thus, murine macrophages incubated with deacylated LPS release significantly less TNF- as compared to WT-LPS. The lowest level of TNF- was observed in the supernatant of macrophages incubated with LPS from the double msbB mutant strain (msbB1/2). Incubation of macrophages with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox preparations significantly reduced the amount of TNF- released into the culture supernatant, as compared to LPS-treated cells, even when 10-fold more Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox was used for stimulation. Collectively, these data indicate that purified LPS from the double msbB mutant strain (msbB1/2) has the lowest potential to cause the release of pro-inflammatory cytokines. Formation of the Invaplex.sub.AR complex appears to further reduce the pro-inflammatory capacity of Shigella LPS.

(79) As the Invaplex.sub.AR-Detox preparations exhibit less toxicity in vitro, Invaplex.sub.AR-Detox preparations have advantages over Invaplex.sub.AR-WT, such as a reduced likelihood of inducing reactogenicity by parenteral (subcutaneous, intramuscular, intradermal, etc.) administration.

(80) Reactogenicity of Invaplex.sub.AR-Detox Preparations In Vivo

(81) The in vivo immunogenicity and reactogenicity of the Invaplex.sub.AR-WT and Invaplex.sub.AR-Detox preparations was evaluated. Groups of female CD-1 mice (5 mice/grp) were immunized intradermally (50 l total volume) on day 0 and 21 with dose amounts of 0.25, 2.5, or 25 g of the given Invaplex.sub.AR preparation described in Table 2. The control group received 0.9% sterile saline (negative control). Prior to immunization all mice were anesthetized with ketamine/rompun. At the site of immunization, each mouse was observed on days 2 and 3 after each immunization for edema, erythema, and induration. In addition, after the first immunization animal appearance and body weight were recorded as indicators of reactogenicity. On study day 35, all mice were euthanized and mucosal washes (lung and intestinal) were collected. Blood samples were collected from individual mice in each group on day 0 and 35. Blood and mucosal washes were assayed by ELISA for antigen-specific antibody endpoint titers.

(82) Individual mice were weighed with a calibrated scale prior to each immunization and then 24, 48, and 72 hours after each immunization. The percent weight change was calculated for individual animals at 24-hour intervals after the second immunization and the mean percent weight change was plotted for each dose group and the results are provided in FIG. 6 (0.25 g), FIG. 7 (2.5 g), and FIG. 8 (25 g). There were no statistically significant differences in the percent weight change between any of the groups immunized with 0.25 or 2.5 g of the Invaplex.sub.AR preparations and the control group immunized with saline (FIG. 6 and FIG. 7, respectively). In the 25 g dose arm (FIG. 8), all groups immunized with the Invaplex.sub.AR-Detox preparations had weight changes that were similar to the group immunized with Invaplex.sub.AR containing WT-LPS. All of the Invaplex.sub.AR-immunized groups had significantly more weight change (loss) than the group immunized with saline at 24 and 48 hours post immunization. Immunization of mice with Invaplex.sub.AR-Detox formulations did not result in a significant difference in terms of weight change as compared to groups immunized with Invaplex.sub.AR-WT.

(83) Individual mice were observed at 24, 48, and 72 hours post vaccine administration for ruffled fur and hunched posture, two physical signs of stress in the mouse model. Fur appearance was recorded as either normal, slightly ruffled, or ruffled and the posture was recorded as either normal, slightly hunched, or hunched. The observations were then assigned a numerical value based on severity on a scale of 1 to 3. A reactogenicity score was then calculated for each animal by summing the individual scores recorded for posture and physical appearance of the fur. Animals administered 0.9% saline did not exhibit fur ruffling or hunched posture at any time point. Similarly, there were minimal changes to fur and posture in groups immunized intradermally with either 0.25 g (FIG. 9) or 2.5 g (FIG. 10) of the Invaplex.sub.AR-Detox preparations. At the 25 g dose amount, mice immunized with Invaplex.sub.AR-WT had slightly ruffled fur and slightly hunched postures 24 hours post administration which resolved within 48 hours (FIG. 11). Moderate levels of reactogenicity were observed after immunization with Invaplex.sub.AR-Detox containing LPS from msbB1 but the reactogenicity was resolved by 72 hours post administration. Minimal levels of reactogenicity were observed in mice immunized with Invaplex.sub.AR-Detox containing LPS from the double msbB1/2 mutant and the levels were significantly lower (ANOVA; p<0.01) as compared to mice immunized with Invaplex.sub.AR-WT. The highest level of reactogenicity was observed in mice immunized with Invaplex.sub.AR-Detox containing LPS isolated from the msbB2 mutant. In summary, outward signs of stress (ruffled fur and hunched posture) were of minimal magnitude and duration in animals immunized with Invaplex.sub.AR-Detox assembled with LPS isolated from the msbB1/2 mutant.

(84) Vaccine administration sites were observed for erythema, edema, and induration prior to vaccination and three consecutive days post each vaccination. Administration sites were graded according to a modified Draize scale for administration site monitoring in Table 4:

(85) TABLE-US-00004 TABLE 4 Score Erythema No erythema 0 Very slight, barely perceptible erythema (pink/red to 1 red area) Well-defined erythema (easily identifiable area of 2 redness) Moderate to severe erythema (medium to dark red) 3 Edema formation No edema 0 Very slight, barely perceptible edema, no defined edges 1 (Raised circumference <5 mm) Moderate edema-area raised approximately 1 mm 2 (Raised circumference of 5-10 mm) Severe edema-area raised more than 1 mm, extending 3 beyond the area of exposure (Raised area >10 mm) Induration No induration 0 Identifiable area of firmness Measurements collected Unable to detect induration due to edema formation U

(86) Grading was conducted in a blinded manner, with the person assigning the grades unaware of the vaccine administered to the animals. Observations were conducted by a team of individuals trained on the scoring system and supervised by a board-certified veterinary pathologist. When induration was observed, measurements of the length and width of the affected tissue were recorded. The areas of induration, defined as a palpable, raised, hardened area at the administration site was calculated using the following formula:
Calculated induration=(0.5length)(0.5width)
The length and width were measured in millimeters using calibrated calibers and was defined as 3.14.

(87) Erythema was undetectable in groups immunized with saline or 0.25 g of the four Invaplex.sub.AR preparations used in the study (FIG. 12). Erythema was also absent in groups receiving 2.5 g of Invaplex.sub.AR-WT, Invaplex.sub.AR-Detox (msbB2 LPS), and Invaplex.sub.AR-Detox (msbB1/2 LPS) preparations. Low to moderate levels of erythema were observed after immunization with 25 g of all the Invaplex.sub.AR preparations.

(88) Similar to erythema results, edema was low or absent in mice immunized with saline or with 0.25 or 2.5 g of the four Invaplex preparations (FIG. 12). Edema was most prominent in groups immunized with 25 g dose amounts of the Invaplex.sub.AR preparations. In summary, mice immunized with Invaplex.sub.AR-Detox (msbB1/2 LPS) had the least amount of edema observed of all the formulations tested.

(89) Induration measurements were collected on the day of each immunization and for three days following each immunization for administration site 1 and 2 (FIG. 13). In most groups, induration was absent (scored as 0). In some groups, induration was undetectable or could not be measured accurately (scored as U) due to edema formation that hindered identifying clear perimeters for induration measurement. Due to the lack of clear induration data, induration was not considered when determining reactogenicity.

(90) Immunogenicity of Invaplex.sub.AR-Detox Preparations In Vivo

(91) Mice were immunized intradermally on days 0 and 21. Blood samples were collected from individual mice in each group on day 0 and 35. On study day 35, all mice were euthanized and mucosal washes (lung and intestinal) were collected. Blood and mucosal washes were assayed by ELISA for antigen-specific antibody endpoint titers. Serum for serology was collected and processed and the serum IgG and IgA endpoint titers specific for Shigella antigens (LPS, native Invaplex, IpaB, and IpaC) were determined using methods in the art.

(92) Blood collected on day 0 and 35 were analyzed by ELISA for serum IgG and IgA endpoint titers directed to S. flexneri 2a LPS, S. flexneri 2a Invaplex, IpaB, and IpaC. Antigen-specific serum IgG and IgA were undetectable in samples collected before immunization (day 0) from all mice in each treatment group. Similarly, mice immunized with saline did not have detectable (titer <180) Shigella-specific serum antibodies.

(93) Shigella LPS-specific serum IgG titers (FIG. 14, FIG. 15, and FIG. 16) were comparable across all groups immunized with comparable doses of the Invaplex.sub.AR preparations indicating the level of acylation of LPS did not significantly influence the anti-LPS response.

(94) Shigella Invaplex 24-specific serum IgG responses (FIG. 17, FIG. 18, and FIG. 19) followed a similar trend as described above for the anti-LPS serum IgG responses in that titers were similar across all groups immunized with comparable doses of the Invaplex.sub.AR preparations, reinforcing the observation that LPS acylation did not significantly influence the antigen-specific response.

(95) The serum IgA responses directed to LPS and Invaplex-24 after immunization with the Invaplex.sub.AR preparations (FIG. 20) were low across all groups with anti-LPS GMT 600 and anti-Invaplex GMTs 720. The LPS and Invaplex titers show dose dependency with groups immunized with 25 g having the highest titers and responder rates. In summary, immunization with Invaplex.sub.AR-Detox containing LPS isolated from msbB mutant Shigella strains elicited comparable levels of LPS-specific and Invaplex-specific serum IgG and IgA endpoint titers to immunization with Invaplex.sub.AR-WT. These results suggest that acylation of LPS does not significantly affect the anti-LPS serum antibody response when delivered in the context of Invaplex.sub.AR.

(96) Serum IgG and IgA endpoint titers directed to IpaB and IpaC (FIG. 20) largely followed a dose-dependent curve, with higher doses inducing higher antigen-specific titers. The dose-dependent trend was more evident with IgA titers as compared to IgG titers. Interestingly, the IpaB and IpaC-specific serum IgA titers were significantly lower in groups that were immunized with Invaplex.sub.AR-Detox assembled with LPS isolated from msbB mutant Shigella strains as compared to Invaplex.sub.AR-WT. A similar trend was seen in anti-IpaC serum IgG titers, albeit to a lesser extent. As these data suggest that the deacylated LPS may be less capable of enhancing the protein-specific antibody response as compared to Invaplex.sub.AR-WT, additional studies in guinea pigs were conducted to further elucidate the effect of deacylated LPS on the protein-specific response and the results are provided below.

(97) The IpaB and IpaC serum responses suggested that the deacylated LPS contained within the Invaplex.sub.AR-Detox preparations may affect the overall magnitude of the immune response. The phenotype of the immune response directed towards the IpaB protein was also investigated by determining serum IgG1 and IgG2a titers and calculating the IgG2a:IgG1 ratio. Groups immunized with Invaplex.sub.AR-WT induced a Th1-biased immune response (FIG. 21). Interestingly, groups immunized with any one of the three Invaplex.sub.AR-Detox preparations containing deacylated LPS had a more balanced Th1/Th2 immune response. These data suggest that the acylation state of the lipid A may contribute to the phenotype of the immune response. The immune response phenotype may be important when creating a vaccine designed to protect against invasive enteric bacterial pathogens, such as Shigella spp. For example, a Th2-dominant response will provide high levels of mucosal IgA potentially capable of neutralizing the bacteria's ability to transcytose from the intestinal lumen to the basolateral surface of the colonic epithelium where infection likely occurs. At the same time, Th1-dominated responses are capable of providing help to cell-mediated immunity to limit and clear intracellular pathogens. A balanced Th1/Th2 response could potentially offer two immunological opportunities to protect against shigellosis.

(98) Mucosal samples (intestinal and lung washes) were collected on day 35 (one week after the second and final immunization) and assayed by ELISA for antigen-specific IgA and IgG titers. Shigella antigen-specific intestinal IgA responses were low across all groups with all GMTs 35 (data not shown). Lung IgA responses were similarly low with all GMTS 10. Shigella antigen-specific lung IgG responses after immunization with the Invaplex preparations (FIG. 22) were higher than the mucosal IgA responses with 100% seroconversion in all the 25 g groups.

(99) Conclusions from Mouse Studies

(100) Evaluation of reactogenicity after intradermal immunization with Invaplex.sub.AR assembled using WT-LPS or deacylated LPS isolated from msbB mutant Shigella strains suggested that deacylated LPS from the msbB double mutant had reduced erythema and edema in the mouse model. There was no significant difference in percent weight change after immunization among the different groups in each dose category.

(101) Intradermal immunization with Invaplex.sub.AR assembled using WT-LPS or deacylated LPS isolated from msbB mutant Shigella strains resulted in comparable levels of LPS and Invaplex-specific immune responses. Immune responses generated to IpaB and IpaC were generally of comparable magnitude but there were several examples that suggested the Ipa-specific serum IgA may be impacted by the deacylated LPS. However, formulation optimizations may overcome these observations. In addition to the influence on the magnitude of the Ipa-specific responses, the immunophenotype was also different in groups immunized with Invaplex.sub.AR-Detox preparations as compared to Invaplex.sub.AR-WT, with a shift from a Th1-biased response to a balanced Th1/Th2 response.

(102) In Vivo Immunogenicity and Efficacy of Invaplex.sub.AR-Detox Preparations in Guinea Pigs

(103) The in vivo immunogenicity and efficacy of Invaplex.sub.AR-WT and Invaplex.sub.AR-Detox with LPS extracted from the msbB1/2 mutant strain of S. flexneri 2a was examined by using the guinea pig rectocolitis model. This model measures intestinal disease in the large intestine and is thought to be very similar to shigellosis in humans. Groups of male Hartley guinea pigs (5 guinea pigs/group) were immunized intranasally on days 0, 14, and 28 with dose amounts of 25 g or 100 g of each Invaplex.sub.AR preparation as outlined in Table 5 as follows:

(104) TABLE-US-00005 TABLE 5 Group n Vaccine Dose (g) 1 5 Invaplex.sub.AR-WT (WT-LPS) 25 3 5 Invaplex.sub.AR-WT (WT-LPS) 100 5 5 Invaplex.sub.AR-Detox (msbB1/2 LPS) 25 7 5 Invaplex.sub.AR-Detox (msbB1/2 LPS) 100 9 5 Saline

(105) The control group received 0.9% sterile saline (negative control). On study day 0 and 42 ocular wash samples were collected. Blood samples were collected on study days 0, 28, 42, and 58 and fecal samples were collected on study days 0 and 35. Blood, fecal, and ocular washes were assayed by ELISA for antigen-specific antibody endpoint titers. The efficacy of the Invaplex.sub.AR preparations was assessed on day 56 when animals were challenged intrarectally with S. flexneri 2a 2457T.

(106) Guinea pigs were challenged on day 56 (4 weeks after the third immunization) with 3.510.sup.10 cfu of virulent S. flexneri 2a 2457T. Guinea pigs were monitored daily for 48 hours post-infection. A composite disease score was calculated for each animal by adding the fecal, mucous, inflammation, and blood scores. Animals with disease scores of 8 or less were considered protected from challenge.

(107) As summarized in FIG. 23, animals immunized with saline (negative control) were not protected against rectal challenge with mean disease scores of 10.5. Animals immunized with Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox (25 g) were not significantly protected. However, animals immunized with 100 g Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox were significantly protected (p0.02). These results demonstrate that intranasal immunization with Invaplex.sub.AR-Detox induces an immune response that is protective against intrarectal challenge with wild-type Shigella spp. with an efficacy substantially similar to that of Invaplex.sub.AR-WT.

(108) Blood collected on day 0 and 42 was analyzed by ELISA for serum IgG and IgA endpoint titers directed to S. flexneri 2a LPS, S. flexneri 2a native Invaplex 24, IpaB, and IpaC. Antigen-specific serum IgG and IgA were undetectable in samples collected before immunization (Day 0) from all guinea pigs in each treatment group. Similarly, guinea pigs immunized with saline (group 9) did not have detectable responses (titer <180).

(109) As shown in FIG. 24, Shigella LPS-specific IgG titers were similar across all groups immunized with comparable doses of the various Invaplex.sub.AR preparations indicating that the acylation of LPS did not influence the anti-LPS response. Serum IgA responses directed to LPS were below detection (<90) in most groups. These results suggest that acylation of LPS does not affect the anti-LPS serum antibody response when delivered in the context of Invaplex.sub.AR.

(110) As shown in FIG. 25, S. flexneri 2a native Invaplex 24-specific serum IgA and IgG responses followed a similar trend as described above for the anti-LPS serum IgA and IgG responses in that titers were similar across all groups immunized with comparable doses of the Invaplex.sub.AR preparations. In summary, immunization with Invaplex.sub.AR-Detox containing LPS isolated from the msbB1/2 mutant strain elicited comparable levels of LPS and Invaplex-specific serum IgG and IgA endpoint titers as did immunization with Invaplex.sub.AR-WT. These results suggest that acylation of LPS does not significantly affect the anti-LPS serum antibody response when delivered in the context of Invaplex.sub.AR. Serum IgG and IgA endpoint titers directed to IpaB and IpaC were also comparable across all doses. These data suggest that deacylated LPS does not alter the anti-IpaB or IpaC serum antibody response.

(111) Antigen-specific mucosal IgA responses were undetectable in mucosal samples collected before immunization (day 0) from all guinea pigs in each treatment group. Similarly, guinea pigs immunized with saline (group 9) did not have detectable Shigella-specific mucosal antibodies.

(112) Eye-wash samples collected on day 0 and 42 were analyzed by ELISA for ocular IgA endpoint titers directed to S. flexneri 2a LPS, S. flexneri 2a native Invaplex 24, IpaB, and IpaC. Generally, guinea pigs immunized with Invaplex.sub.AR-Detox containing LPS isolated from the msbB1/2 mutant strain elicited similar levels of antigen-specific endpoint titers as compared to guinea pigs immunized with Invaplex.sub.AR-WT. As shown in FIG. 26, there was no dose dependent trend in antigen-specific titers. This suggests that acylation of LPS does not affect antigen-specific mucosal responses when delivered in the context of Invaplex.sub.AR.

(113) Fecal samples collected on day 0 and 35 were analyzed by ELISA for fecal IgA endpoint titers directed to S. flexneri 2a LPS and S. flexneri 2a native Invaplex 24. As shown in FIG. 27, anti-LPS and anti-Invaplex 24 titers were low across all groups (GMTs 20).

(114) In another series of experiments, groups of male Hartley guinea pigs (6-12 guinea pigs/group) were immunized on study days 0, 14, and 28 either intranasally, intramuscularly, or intradermally (100 l total volume) with varying doses of Invaplex.sub.AR-Detox with LPS extracted from the WR30 strain of S. flexneri 2a as outlined in Table 6.

(115) TABLE-US-00006 TABLE 6 Group N Vaccine (Dose g) Route of Immunization 1 6 Invaplex.sub.AR-Detox (25 g) Intranasal 2 6 Invaplex.sub.AR-Detox (25 g) Intramuscular 3 6 Invaplex.sub.AR-Detox (5 g) Intramuscular 4 6 Invaplex.sub.AR-Detox (25 g) Intradermal 5 6 Invaplex.sub.AR-Detox (5 g) Intradermal 6 12 Saline Intranasal

(116) The control group received 0.9% sterile saline (negative control). Guinea pigs were bled on study days 0, 28, 42, and 14 days after challenge (Day 63). Prior to immunization and bleeding, guinea pigs were anesthetized with a mixture of ketamine and xylazine. Blood was assayed by ELISA for antigen-specific antibody endpoint titers. The guinea pig keratoconjunctivitis model (Sereny test) was used for efficacy testing. Three weeks after the third intranasal, intradermal, or intramuscular immunization (Day 49) guinea pigs were challenged intraocularly with S. flexneri 2a strain 2457T (about 2.010.sup.8 cfu per eye) and observed daily for 5 days for the occurrence of keratoconjunctivitis. Animals in the negative control group were also challenged using identical procedures. The degree of inflammation and keratoconjunctivitis was scored using a scale of 0 to 3. Eyes with no inflammation (score of 0) or slight inflammation (score of 1) at Day 5 were considered protected. Eyes with scores of 2 (keratoconjunctivitis with no purulence) or 3 (fully developed keratoconjunctivitis with purulence) were considered not protected. Efficacy was calculated by the formula: [{% disease (controls)% disease (vaccines)}/% disease (controls)]100.

(117) As summarized in FIG. 28, guinea pigs immunized with saline (negative control) were not protected against keratoconjunctivitis with 100% disease in the controls. Guinea pigs immunized with 5 g of Invaplex.sub.AR-Detox intramuscularly were not significantly protected on Day 5 post challenge. However, guinea pigs immunized intradermally with 5 g of Invaplex.sub.AR-Detox were significantly protected on Day 5 (p=0.0373). All animals immunized with 25 g of Invaplex.sub.AR-Detox either intranasally, intramuscularly, or intradermally were significantly protected on Day 5 post challenge with a protective efficacy of 75%. These results demonstrate that parenteral immunization with Invaplex.sub.AR-Detox can induce an immune response that is as protective against ocular challenge with S. flexneri 2a strain 2457T as compared to immunization via a mucosal route.

(118) Conclusions from Guinea Pig Studies

(119) Intranasal immunization with Invaplex.sub.AR preparations assembled using WT-LPS or LPS isolated from the msbB1/2 mutant Shigella strain resulted in comparable levels of LPS, native Invaplex, IpaB, and IpaC-specific immune responses. Minimal levels of mucosal antibodies were detected in fecal and ocular washes.

(120) Animals immunized with 100 g Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox were significantly protected (p0.02) whereas animals immunized with the lower dose (25 g) of Invaplex.sub.AR-WT or Invaplex.sub.AR-Detox were not significantly protected. These results demonstrate that intranasal immunization with Invaplex.sub.AR-Detox or Invaplex.sub.AR-WT can induce immune responses that are protective against intrarectal challenge with virulent S. flexneri 2a 2457T. Therefore, in some embodiments, the present invention provides a method of immunizing a subject against one or more Shigella spp., which comprises administering to the subject an immunogenic amount of a Invaplex.sub.AR-Detox mucosally. Furthermore, parenteral immunization (e.g., intramuscular or intradermal) induced protection against ocular challenge with S. flexneri 2a, 2457T. Therefore, in some embodiments, the present invention provides a method of immunizing a subject against a mucosal challenge with one or more Shigella spp. which comprises administering to the subject an immunogenic amount of a Invaplex.sub.AR-Detox parenterally.

(121) All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

(122) As used herein, the terms subject, patient, and individual are used interchangeably to refer to humans and non-human animals. The term non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals. In some embodiments of the present invention, the subject is a mammal. In some embodiments of the present invention, the subject is a human.

(123) The use of the singular can include the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms a, an, and the can include plural referents unless the context clearly dictates otherwise. As used herein, and/or means and or or. For example, A and/or B means A, B, or both A and B and A, B, C, and/or D means A, B, C, D, or a combination thereof and said combination thereof means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).

(124) The phrase comprises or consists of is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue comprises something, and in some embodiments the given thing at issue consists of something. For example, the sentence In some embodiments, the composition comprises or consists of A is to be interpreted as if written as the following two separate sentences: In some embodiments, the composition comprises A. In some embodiments, the composition consists of A. Similarly, a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence In some embodiments, the composition comprises A, B, or C is to be interpreted as if written as the following three separate sentences: In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C.

(125) To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

(126) Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.