Abstract:
An artificial invasin complex is prepared from purified or recombinantly prepared invasins and gram negative bacteria lipopolysaccharides. Typically, IpaB is mixed with IpaC to form a IpaB:IpaC complex. This invasin protein complex is then mixed with the lipopolysaccharide to form an artificial invasin complex. Additional bioactive molecules can be incorporated into the complex during manufacture. This artificial invasin complex is similar in function to native Invaplex 24 or Invaplex 50. The artificial invasin complex has superior immunogenicity properties relative to the native complex and can be tailor made. Its method of preparation lends itself to scale up. The artificial invasin complex can facilitate transport of biomolecules, therapeutics and antibiotics across cell membranes in a manner similar to native  Shigella Invaplex.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of Invention 
     This invention provides a novel method for preparing an artificial Invaplex, an artificial invasin complex comprising at least two invasin proteins and a lipopolysaccharide from invasive gram negative bacteria. The artificial invasin complex of the present invention can be used as a vaccine, an adjuvant for vaccines, biochemical, or other substances, and as a diagnostic tool. 
     2. Description of Related Art 
     Shigellosis is a leading cause of human diarrheal disease particularly in developing countries where it is estimated that over 163 million cases, with 1 million fatal cases, occur annually [1]. The most common  Shigella  species causing disease worldwide are  S. flexneri  and  S. sonnei [ 1]. A lower incidence (1.5 million cases/yr) of shigellosis in industrialized countries [1] implies that the adult population is non-immune and susceptible. Even in areas relatively free of shigellosis, massive destruction due to war or natural disasters, such as the earthquake of 1999 in Kocaeli, Turkey, can suddenly give rise to multispecies, multi-focal increases in dysentery [2]. Similarly, when deployed troops, relief workers, or travelers from industrialized countries are in areas endemic for  Shigella  or in areas where the local infrastructure has collapsed, the threat of exposure to multiple  Shigella  spp. may be significant.  S. dysenteriae  1 causes the most severe form of shigellosis and is often associated with hemolytic uremic syndrome [3]. Although the incidence of  S. dysenteriae  1 is not as high as that of  S. flexneri  or  S. sonnei , epidemics caused by  S. dysenteriae  1 often occurs every 10 to 12 years [4]. The periodic occurrence is probably due to many factors, one of which is the likely waning of immunity to  S. dysenteriae  1. More important is the recent finding that antibiotic resistance to the flouroquinoline class of antibiotics has been demonstrated in  S. dysenteriae  1 isolated in recent outbreaks [5, 6]. The likelihood of another epidemic of  S. dysenteriae  1 is high and if it is caused by a strain with broad antibiotic resistance including the fluorquinolones, the consequences could be severe. 
       Shigella  species are one of the few proven agents for biological terrorism. The extremely low infectious dose (for  S. dysenteriae  1 it is reported to be 10 bacteria) [7] and the ease of growing the organism in simple bacteriological medium are suitable factors for considering  Shigella  spp as potent biological weapons. The proven effectiveness as a biological terrorism agent is a case in which employees in a hospital were deliberately infected with  S. dysenteriae  2 by a disgruntled coworker who had “painted” muffins with the organism [8]. Other food-borne outbreaks of  Shigella  infections are well documented—from cold food on airlines to salads on cruise ships [9, 10] demonstrating the ease by which shigellae can cause disease in a susceptible population. Prevention of  Shigella  infections is best accomplished by environmental controls to include good sanitation and clean water supplies. Such infrastructure improvements are easily overcome if shigellae are deliberately planted in food or drinks. Furthermore, the ease of isolating shigellae from nature [5] and the simplicity of developing antibiotic resistant  S. dysenteriae  1 strains in the laboratory validates this agent as a potential major bioterrorism threat. Although an initial outbreak might be difficult to completely stop in a non-immune population, it is important to limit secondary spread. An effective means of prevention are vaccines which are effective against shigellae regardless of antibiotic resistance. 
     The pathogenesis of  Shigella  spp. is attributed to the organism&#39;s ability to invade, replicate intracellularly, and spread intercellularly within the colonic epithelium. The initial entry of shigellae occurs through specialized intestinal epithelial cells called M cells. Several highly conserved, virulence plasmid-encoded proteins, called the invasion plasmid antigens (IpaA, IpaB, IpaC, and IpaD), are essential participants in the invasion process and are likely key to the efficiency of  Shigella  infections. Upon contact or attachment to host cells, the  Shigella  invasins are released [11] by a type III secretion apparatus [12] and induce a phagocytic event resulting in engulfment and internalization of the bacterium by the host cell [13]. The active components include an IpaB:IpaC complex that integrates into the host cell membrane, forming a channel by which other  Shigella  proteins gain entry into the host cell [14]. Recently, we have isolated from intact, virulent  Shigella  cells, an invasin protein-LPS complex which serves as an effective vaccine and adjuvant [15-19]. The  Shigella  invasin complex (Invaplex) is prepared by extracting intact virulent shigellae with water followed by anion-exchange chromatography of the water extract. Two fractions, called Invaplex 24 and Invaplex 50, contain the major antigens including LPS, IpaB and IpaC. The invasin complex (Invaplex) binds to surfaces of cultured epithelial cells whereas similar preparations from non-invasive shigellae do not exhibit this property. Shortly after attachment the Invaplex becomes localized inside the host cell via an acting-dependent endocytic event likely induced by the Invaplex. It appears that IpaC and IpaB have a significant role in the attachment and internalization process in that antibodies against IpaC or IpaB inhibit the binding of Invaplex to host cell membranes [20]. Antibodies to LPS do not alter the uptake event. Once internalized the Invaplex traffics through early endosomes, late endosomes, then the Golgi apparatus and eventually ends up free in the host-cell cytoplasm. The ability to bind to a eukaryotic host cell surface and stimulate endocytosis indicates that the Invaplex maintains an active, native virulence structure similar to that found in invasive  Shigella.    
     Historically, successful  Shigella  vaccines have emphasized presentation of LPS in an effective manner to elicit protection. Many approaches have been used for  Shigella  vaccines including live attenuated vaccines [21, 22], killed whole cell vaccines [23, 24], and delivery of  Shigella  LPS or O-polysaccharides with carriers such as proteosomes [25], tetanus toxoid [26], ribosomes [27] or Invaplex (17, 18; see below). Only the live attenuated vaccines utilize the native invasiveness of the shigellae to deliver the LPS and protein antigens to the mucosal immune system presumably via the follicle-associated epithelium [14]. The residual pathogenicity of the attenuated vaccine strains may limit this approach unless further attenuation is achieved [28]. 
     The ability to isolate a putative native surface structure such as Invaplex, which exhibits activities and immunogenicity similar to invasive shigellae, has significant implications in vaccine design and development. First, the isolated native complex may enhance delivery to the appropriate portal of entry (M-cells), similar to that targeted by live-attenuated vaccine strains. The attachment of Invaplex to host cells (and likely intestinal M cells or M-like cells in other mucosal tissues) allows the use of relatively low doses of this subcellular complex for immunization due to its delivery efficiency. Similar to live, attenuated vaccines Invaplex contains all major  Shigella  antigens (including IpaB, IpaC and LPS) and has the potential to stimulate an immune response equivalent to that produced during natural infection, including recognition of epitopes found only in native structures. Such epitopes may not be present in vaccines delivering only LPS or O-polysaccharide as the antigen. Furthermore, the conserved sequences and immunologic cross-reactivity of the Ipa proteins found in all  Shigella  species may enable a vaccine containing the invasins (or other conserved antigens) to be effective against more than one  Shigella  species. 
     The  Shigella  invasin complex (Invaplex) vaccine prepared from  Shigella flexneri  contains the major antigens LPS, IpaB and IpaC and is protective against homologous challenge in the guinea pig keratoconjunctivitis and mouse lung challenge models [17, 18]. After immunization, antibodies to LPS, IpaB and IpaC are produced which is similar to the antibody specificity observed after natural infection in humans [29]. Additional studies have shown that similar effective Invaplex vaccine products can be isolated from all species of  Shigella  and EIEC [15]. Although the Invaplex 24 and 50 preparations consist of many different proteins, the immunodominant antigens for Invaplex 24 are LPS, IpaB and IpaC and for Invaplex 50 the key antigens are LPS, IpaB, IpaC and the 84 kKa (EF-G) and 72 kKa (DnaK) protein antigens [18]. Other proteins within the Invaplex preparations are not immunogenic as determined by western blotting techniques using sera collected from Invaplex-immunized animals. Invaplex 24 and Invaplex 50 can be isolated from all wild-type  Shigella  species although often-times the Invaplex 24 form consistently contains higher quantities of IpaB, IpaC and LPS and fewer non-immunogenic proteins. With the Invaplex vaccine, the highest titers are often against the immunizing Invaplex (Invaplex 24 or Invaplex 50) when used as an ELISA antigen. This is thought to reflect a composite response to the Ipa proteins and LPS and to a set of conformational epitopes preserved by the invasin complex product when used as an ELISA antigen. 
     Efforts to identify and purify the active moiety within native Invaplex has identified a high molecular mass complex that was isolated by size exclusion chromatography from native Invaplex 24 preparations. This high molecular mass complex, referred to as “highly-purified” or HP Invaplex 24 consists of predominantly IpaB, IpaC and LPS. HP Invaplex 24 is immunogenic and protective at levels that are comparable to or exceed those exhibited by native Invaplex. No other fraction obtained by size-exclusion chromatography exhibited immunogenic or protective capacities comparable to native Invaplex and for this reason the HP Invaplex 24 was considered to be the active moiety within native Invaplex responsible for its immunogenicity and efficacy as a vaccine. 
     Virulent shigellae cause disease by invading, replicating, and spreading within the colonic epithelium by virtue of a complex series of cellular and molecular events orchestrated by an array of plasmid-encoded virulence factors among which are the Ipa proteins [49]. After invading the intestinal epithelial cells of the colonic mucosa a mucosal inflammatory response occurs characterized by an increase in proinflammatory cytokines that leads to recruitment of neutrophils and macrophages/monocytes. The resulting disease, shigellosis or bacillary dysentery, causes mild to severe diarrhea, fever and intestinal lesions. Since testing of the efficacy of vaccines in humans and nonhuman primates involves a post-immunization challenge with virulent shigellae, the use of small animal models for initial testing of vaccine candidates reduces the risk of illness to the human volunteers and primates. Small animal models such as the guinea pig keratoconjunctivitis model or the mouse lung model of experimental infection with shigellae are largely used in studies of pathogenesis and preclinical vaccine evaluation [18, 44, 50] with the mouse model often used in initial evaluations and the guinea pig model for testing vaccines that are protective in the mouse model. 
     The mouse intranasal challenge model of  Shigella  infection is useful to evaluate  Shigella  vaccines [18, 31, 51, 52]. The pathogenesis and immunobiology observed in the pulmonary model parallel those seen in the colon; that is, virulent  Shigella  strains invade, replicate, and spread within the epithelium and subsequently elicit antibody as well as cytokine responses [52]. After infection the mice lose weight and ultimately die unless protective immunity is present. The ability to measure a secretory antibody response, the cellular immune responses, and cytokine responses (largely due to the availability of commercial reagents) makes the mouse model highly attractive for studies on the immunobiology of shigellosis. 
     The guinea pig model is an accepted model that is useful for studying the virulence of both wild-type and attenuated  Shigella  strains, and for evaluating the efficacy of potential vaccine candidates [18, 44, 45, 53-56]. Several routes of immunization can be employed for immunization depending upon the vaccine: oral, intranasal, ocular, and parenteral immunizations have all been used to protect against ocular challenge. Immunogenicity and efficacy of  Shigella  vaccine in the guinea pig keratoconjunctivitis model is now used as a stepping-stone to phase 1 clinical studies. 
     There exists a need for a chemically defined, artificial Invaplex moiety, which is similar or superior in biological activity to a native Invaplex. There is also a need for an Invaplex manufacturing process which can be readily scaled up and results in a more specifically defined product dependent on the ratios of the individual component parts used. There is also a need for an Artificial Invaplex which is capable of being designed for specific applications or customized functionality. There is also a need for an Invaplex vaccine that can be manufactured quickly from its component parts which can be stockpiled in anticipation of future vaccine needs. 
     BRIEF SUMMARY OF THE INVENTION 
     The invention relates to an artificial Invaplex and a method for its manufacture. The artificial Invaplexes (InvaplexAR) are similar in composition to HP Invaplex 24. HP Invaplex 24 contains IpaB, IpaC and lipopolysaccharride. The Artificial Invaplex functions as an Invaplex obtained from a native source, though it may have superior activity. The Artificial Invaplex has defined components: a complex comprising the invasion proteins IpaB and IpaC, which complex is additionally complexed with a serotype-specific lipopolysaccharide component from a gram negative bacteria. The lipopolysaccharide is an immunogen as are IpaB and IpaC. InvaplexAR can function as a mucosal adjuvant and enhance both serum and mucosal antigen-specific antibody responses as well as cell-mediated immune responses. It is also possible to use other invasins from other bacteria species including SipB, SipC or viral (reo virus) proteins associated with endocytosis events or tissue tropisms. 
     The serotype associated lipopolysaccharides are obtained from gram negative bacteria such as  Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Shigella boydii, enteroinvasive E. coli, Yersinia  or  Salmonella . The more preferred species include  S. flexneri, S. sonnei, S. boydii, S. dysenteriae  or  enteroinvasive E. coli.    
     In preparing Artificial Invaplex, e.g. Invaplex AR , the invasin proteins used are either highly purified (hp-) or recombinantly (r-) prepared IpaB and IpaC and purified lipopolysaccharride are used as starting materials. The IpaB and IpaC proteins are mixed to form IpaB:IpaC complex. This complex is mixed with at least one lipopolysaccharide associated with a serotype of a gram negative bacteria to form an Artificial Invaplex. The artificial Invaplex is recovered from the mixture. Typically, the Artificial Invaplex is removed based on its charge. Other purification techniques can be used. The order of addition can be varied. The lipopolysaccharide can be complexed with either of the invasin proteins and then with the other invasin protein to form the Artificial Invaplex. 
     Typically, the amount of IpaC presented relative to IpaB present falls within a ratio range of 0.08:1 to 80:1, preferably 0.8:1 to 20:1, more preferably at least 8:1. It is even more preferred for the amount of IpaC in the first mixing step to be present in at least a ten fold excess relative to IpaB. In the second mixing step, the lipopolysaccharide and protein (IpaB and IpaC) are present at a ratio within the range 0.01:1 to 10:1, preferably at least 1:2. It is possible in the second mixing step to include two or more types of lipopolysacchides, which results in an artificial Invaplex being suitable for use in multivalent immunogenic compositions, as vaccines. It is also possible in addition to the lipopolysaccharide to include a detectant label, antibiotic, therapeutic or biomolecule comprising an enzyme, protein, polysaccharide, RNA or DNA, or derivatives thereof which one wants to incorporate within a cell. This gives rise to possible therapeutic and analytical/diagnostic uses. 
     The artificial Invaplexes of the invention include those where the IpaC and IpaB are present in a ratio selected from 0.08:1 to 80:1, preferably, 0.8:1 to 20:1, even more preferred approximately 8:1 and the lipopolysaccharride (LPS) is present at a ratio selected from the range of 0.01:1 to 10:1, preferably, from 0.5:1 to 5:1, even more preferred approximately 0.5:1 relative to the total protein (IpaB and IpaC) present. The preferred artificial Invaplexes include  S. flexneri  Invaplex AR   , S. dysenteriae  Invaplex AR  or  S. sonnei  Invaplex AR    
     The artificial Invaplexes of the invention can be formulated to form compositions suitable for use as immunogenic compositions or vaccines. The Invaplex can be used as the immunogen and/or as the adjuvant. The compositions would include in addition to the artificial Invaplex at least a pharmaceutically acceptable carrier. The amount of Invaplex present is an amount effective for the desired function and in at least the first instance, is empirically determined. This amount is expected to be similar to that of a native Invaplex, e.g. Invaplex 24, used for a similar function. 
     In addition the artificial invasin complexes facilitate the transport of a molecule and related materials across cell membranes. These molecules can be in close proximity to a cell of interest and the artificial Invaplex or can be present within and/or on the artificial Invaplex. The molecules can be detectant labels, antibiotics, therapeutics, and biomolecules including proteins, enzymes, RNA, DNA, lipopolysaccharides, polysaccharides and the like. 
     The artificial Invaplexes can be used in methodologies where native Invaplexes have been used, e.g. methods of immunization, methods for facilitating the transport of biomolecules, e.g. DNA, RNA, proteins, across the membranes of cells, etc. The artificial Invaplex can be designed to be multivalent or to have superior activity relative to the native Invaplex. This should improve the performance of the existing method or its utility. Invaplex AR  made from recombinant IpaB, IpaC and either  S. sonnei  LPS or  S. flexneri  2a LPS induced immune responses that are comparable or higher in magnitude to the immune response induced with native Invaplex. IpaB and IpaC responses after immunization with Invaplex AR  were consistently of higher magnitude than those induced after immunization with native Invaplex. Immunization with Invaplex AR  offers comparable or higher levels of protection against challenge in the mouse and guinea pig models as compared to immunization with native Invaplex. Immunization with Invaplex AR  induces cellular immunity which is of greater magnitude than the  Shigella -specific response induced with native Invaplex. See, for example, Tables 7 and 8. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows SDS-PAGE analysis of purified IpaB, IpaC and LPS used to assemble Invaplex AR . Purified recombinant IpaB (left) and IpaC (center) were separated by SDS-PAGE and blotted to nitrocellulose (left panel for IpaB and IpaC) or stained with coomassie blue (right panel for IpaB and IpaC). Proteins blotted to nitrocellulose were probed overnight with the anti-IpaB mAb 2F1 or anti-IpaC mAb2G2.  S. dysenteriae  1 LPS (right panel) was separated by SDS-PAGE. 
         FIG. 2  shows purification of  S. flexneri  2a (panel A),  S. sonnei  (panel B) and  S. dysenteriae  1 (Panel C) Invaplex AR  Purified IpaB, IpaC, and LPS from  Shigella  species (as indicated) were mixed together at 37° C. for 2 hrs in the presence of 0.5 M NaCl. Purification: after incubation, the mixture was diluted 1/5 and then applied to a HiTrap™ Q HP column and resolved with a NaCl step gradient as previously described [17]. Fractions within each peak were analyzed by 13% SDS-PAGE. The Invaplex AR  (red peak) eluted in the 0.5 M NaCl (50% Buffer B) step. Gels were either stained with Coomassie Blue (gel C), blotted to nitrocellulose and probed with mAbs specific for IpaB and IpaC (gel W) or reacted with silver (gel S). Purified LPS from the designated  Shigella species is in the left lane of the “S” panel. The 0.5M NaCl step contained the majority of IpaB, IpaC and LPS; the 0.24 M NaCl peak contained small quantities of IpaB, IpaC and LPS. 
         FIG. 3  shows SDS-PAGE (coomassie blue, western blots and silver stains) of components used to make InvaplexAR for  S. dysenteriae  1 and the final Invaplex AR  product. The gel on the left is a coomassie blue stained gel of purified IpaB, IpaC and two different lots of  S. dysenteriae  1 Invaplex AR . The prominence of IpaB and IpaC are evident in the Invaplex AR . The middle panel is a western blot that used mAbs to IpaB and IpaC to probe the purified proteins and the two different lots of  S. dysenteriae  1 InvaplexAR. The panel on the right is a silver-stained gel of the purified IpaB and IpaC proteins, purified LPS, and two lots of  S. dysenteriae  1 Invaplex AR  which show both the Ipa protein bands and the LPS bands in the InvaplexAR. 
         FIG. 4  shows detection of Internalized native and artificial  S. dysenteriae  1 Invaplex in mammalian cells. BHK-21 cells were incubated at 37° C. for 60 min with LPS (A), native Invaplex 24 (B), or Invaplex AR  (C) all produced from  S. dysenteriae -1. Fixed cells were probed with anti-S. dysenteriae 1 polyclonal sera followed by GAM-IgG-OG 488  to visualize internalized Invaplex (green). Cells were counterstained with DAPI to identify the nucleus (blue).  Shigella  LPS was not internalized by the host cells whereas both native Invaplex and artificial Invaplex were internalized by ˜70% of cells. 
         FIG. 5  shows Invaplex 50, Invaplex 24 and LPS-specific serum IgG and IgA end point titers on day 42 after intranasal immunization with  S. flexneri  2a native Invaplex 24, Invaplex AR , purified IpaB, IpaC or  S. flexneri  2a LPS. 
         FIG. 6  shows Anti-IpaB and IpaC serum IgG endpoint end point titers on day 24 after intranasal immunization with  S. flexneri  2a native Invaplex 24, Invaplex AR , purified IpaB, IpaC or  S. flexneri  2a LPS. 
         FIG. 7  shows antigen-specific IgA in intestinal washes collected on day 35 from mice intranasally immunized with native  S. flexneri  2a Invaplex or Invaplex AR . Data represents the mean endpoint titer plus one standard error of the mean for each group of mice (n=4 mice/grp). 
         FIG. 8  shows antigen-specific IgA in lung washes collected on day 35 from mice intranasally immunized with native  S. flexneri  2a Invaplex or Invaplex AR . Data represents the mean endpoint titer plus one standard error of the mean for each group of mice (n=4 mice/grp). 
         FIG. 9  shows Invaplex-specific serum IgG and IgA endpoint titers in mice after intranasal immunization with OVA alone, or OVA combined with InvaplexAR, native Invaplex, or cholera toxin. Groups of mice were intranasally inoculated on day 0, 14, and 28 with OVA or OVA combined with either Invaplex AR , Invaplex or CT. Blood collected on day 0, 28, and 42 were analyzed by ELISA for anti-Invaplex serum IgG and IgA endpoint titers. Data represents the mean (n=5 mice/grp) endpoint titer±1 SEM. 
         FIG. 10  shows  S. flexneri  2a LPS-specific serum IgG and IgA endpoint titers in mice intranasally immunized with OVA alone, OVA combined with InvaplexAR, native Invaplex, or cholera toxin. Groups of mice were intranasally inoculated on day 0, 14, and 28 with OVA or OVA combined with either Invaplex AR , Invaplex, or CT. Blood collected on day 0, 28, and 42 were analyzed by ELISA for anti-Invaplex serum IgG and IgA endpoint titers. Data represents the mean (n=5 mice/grp) endpoint titer±1 SEM. 
         FIG. 11  shows  Shigella  invasion-specific serum IgG and IgA endpoint titers in mice intranasally immunized with OVA alone, or OVA combined with InvaplexAR, native Invaplex or cholera toxin. Groups of mice were intranasally inoculated on day 0, 14, and 28 with OVA or OVA combined with either Invaplex AR , Invaplex or CT. Blood collected on day 0, 28, and 42 were analyzed by ELISA for anti-IpaB and IpaC serum IgG endpoint titers. Data represents the mean (n=5 mice/grp) endpoint titer±1 SEM. Comparisons between groups were accomplished using two-way ANOVA analysis of natural log-transformed endpoint titers. 
         FIG. 12  shows ovalbumin-specific serum IgG and IgA endpoint titers in mice intranasally immunized with OVA alone, or OVA combined with InvaplexAR, native Invaplex or cholera toxin. Groups of mice were intranasally inoculated on day 0, 14, and 28 with OVA or OVA combined with either Invaplex AR , Invaplex, or CT. Blood collected on day 0, 28, and 42 were analyzed by ELISA for anti-OVA serum IgG and IgA endpoint titers. Data represents the mean (n=5 mice/grp) endpoint titer±1 SEM. Comparisons between groups were accomplished using two-way ANOVA analysis of natural log-transformed endpoint titers. 
         FIG. 13  shows  S. flexneri  2a LPS, Invaplex 24 and ovalbumin-specific lung IgG and IgA endpoint titers in mice intranasally immunized with OVA alone, or OVA combined with InvaplexAR, native Invaplex or cholera toxin. Groups of mice were intranasally immunized on day 0, 14, and 28 with OVA or OVA combined with either InvaplexAR, Invaplex, or CT. Lung washes were collected from individual mice on day 42 and analyzed by ELISA for anti-LPS, anti-Invaplex 24, and anti-OVA IgG and IgA endpoint titers. Data represents the mean (n=5 mice/grp) endpoint titer±1 SEM. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Biochemistry of Artificial Invaplex Production and Purification of Recombinant IpaB and IpaC and native  S. flexneri, S. sonnei  and  S. dysenteriae  1 LPS 
     Purification of Ipa Proteins 
     The Ipa proteins are highly conserved in all  Shigella  spp [32, 33]. Recombinant  E. coli  expressing either IpaB or IpaC have been previously described. Two strategies are available for purifying recombinant Ipa proteins. Histidine-tagged (HisTag) recombinant IpaC is purified by affinity chromatography using nickel columns. The effect of the histidine residues on the Ipa protein&#39;s biological or immunological function or the ability of the proteins to form Invaplex is not known but HisTag-IpaC appears to maintain biological activity [30]. An alternative purification procedure has been used to produce recombinant IpaB. This method takes advantage of IpaB&#39;s native affinity for the chaperone IpgC [34, 35]. By co-expressing IpaB together with the HisTag-chaperone IpgC in the same recombinant organism, it will be possible to purify the IpaB/chaperone complex. After purification, the HisTag-IpaC/IpaB protein complex is denatured resulting in monomeric IpaB protein and HisTag-IpgC which allows the HisTagged chaperone to be selectively removed on a nickel column while the free Ipa protein runs through the column. 
     Purification of HisTag-IpaC 
     The recombinant IpaC was expressed in an  E. coli  background using an IPTG inducible expression system (pET plasmid). Specifically, the IpaC gene was cloned into the pET15b plasmid (Novagen) and expressed in the  E. coli  vector BL21(DE3)pLysS [30]. The recombinant IpaC protein has several histidine residues on the amino terminal end that allows subsequent purification on a nickel column [30]. The IpaC protein expressed in the recombinant is located in inclusion bodies and requires solubilization with urea. Removing the urea often leads to insolubility if the IpaC concentration is too high (&gt;1 mg/ml). Thus, IpaC eluted from the nickel column was maintained in urea to maintain solubility. Using small-scale cultures (3 L) it has been possible to produce approximately 75 mg of purified IpaC with a yield of approximately 8 mg/L. The product is greater than 90% pure and is reactive with anti-IpaC mAb 2G2 [32] ( FIG. 1 ). The purified protein is soluble and can be stored frozen. An example of purified IpaC is in  FIG. 1 . 
     Purification of IpaB 
     The recombinant organism expressing IpaB is in an  E. coli  BL21(DE3) background. The IpgC gene was cloned into pET15b; the IpaB gene was cloned separately into the pACYC-duet vector. After IPTG induction the HisTag IpgC/IpaB complex is solubilized and purified on a nickel affinity column. The HisTag-IpgC/IpaB complex is released with EDTA and prepared for second application to the nickel affinity column by removal of the EDTA and the addition of the non-ionic detergent OPOE (N-octyl-oligo-oxyethylene) to a final concentration of 1% v/v. The OPOE will disrupt the HisTag-IpgC/IpaB complex thereby allowing the free HisTag-IpgC protein to bind to the nickel (Ni-Sepharose) column and the untagged, free IpaB to flow through the column for collection. Fractions from the void volume were probed for IpaB with anti IpaB mAb 2F1. Positive fractions were analyzed by SDS-PAGE (stained) to determine if contaminating HisTag-IpgC was present. If so, the material was treated again with OPOE and reapplied to the nickel column to remove residual HisTag-IpgC. The resultant soluble IpaB product has little to no HisTag-IpgC “contamination”, is over 90% pure and reacts with anti IpaB mAb 2F1 [32] ( FIG. 1 ). The yield of IpaB per liter of starting culture was approximately 3.5 mg/L. 
     Purification of  S. flexneri  2a ,  S. sonnei , or  S. dysenteriae  1 LPS 
       S. flexneri  2a,  S. sonnei , and  S. dysenteriae  1 LPS were produced by the Westphal procedure [36] which involves a hot phenol/water extraction of the shigellae. Virulent or attenuated strains of  Shigella  can be used as source of LPS as long as the smooth LPS phenotype is expressed. In experiments described below, wild-type  S. flexneri  2a (strain 2457T) and  S. sonnei  (Mosely) were used. For  S. dysenteriae  1, the attenuated strain WRSd1 was used to minimize risk of infection to laboratory personnel. WRSd1 is a virG, stx knockout previously produced at WRAIR [37]. 
     Extraction, Purification and Characterization of LPS 
     LPS is extracted by the Westphal procedure [36]. Briefly, the bacterial cell pellets are suspended in hot (68° C.) distilled water (5 ml water for each gram of pellet). An equal amount of phenol, heated to 68° C., is added and the pellet solution is vigorously shaken for 15 minutes. The bottles are then cooled to approximately 10° C.±5° C. The samples are centrifuged, the aqueous phase removed and stored at 4° C. Extraction of the cell mass is performed a second time and all aqueous phases are pooled. The aqueous phase is dialyzed against distilled water for two days, and then centrifuged (8000×g, 30 min) to remove extraneous cellular debris. This supernatant is subjected to ultracentrifugation (90,000×g) for 2 hours and the pellet is saved. The pellet is rinsed with sterile distilled water, resuspended in sterile distilled water overnight at 4° C., pooled and lyophilized. The final lyophilized product is weighed and then a small portion (&lt;10 mg) is removed, dissolved in 1 ml of endotoxin-free water and characterized biochemically (see below). 
     Endotoxin content of the purified LPS is performed by the chromogenic LAL.  E. coli  endotoxin serves as a control reagent for this analysis. All results are reported in terms of international endotoxin units (EU). The purified LPS is also analyzed by SDS-PAGE with silver stain to determine if the typical multiple band profile of smooth LPS are present.  FIG. 1  shows a silver stained gel of the purified  S. dysenteriae  1 LPS demonstrating the typical multiple banding pattern of smooth LPS. The final LPS product has residual amounts of protein (&lt;5%, determined by Bradford assay) and DNA (&lt;5%, determined by Hoechst stain) and is reactive with LPS serotype specific antibodies (anti- S. dysenteriae  1 LPS mAb MAB753 (Chemicon International) for  S. dysenteriae  1 LPS; mAb 2E8 for  S. flexneri  2a LPS; MAB755 (Chemicon) for  S. sonnei  LPS) by western blot or ELISA. 
     Quantitation of IpaB and IpaC Content in Invaplex AR  by Immunoassay 
     The amount of IpaB and IpaC in Invaplex (artificial or native) was determined using a modified ELISA procedure. The ELISA used purified recombinant IpaB or IpaC proteins to generate standard curves for determination of the quantity of the antigens in the Invaplex preparations. Immulon 1B ELISA plates (ThermoLab Systems) were coated overnight at 4° C. with either 50 μl of recombinant IpaB, recombinant IpaC, or Invaplex AR . Antigen was titrated (in triplicate) using 2-fold serial dilutions in carbonate coating buffer (0.2 M carbonate, pH 9.8) with starting concentrations of 125 ng/ml (IpaB), 200 ng/ml (IpaC), and 10 ug/ml (Invaplex). After washing and blocking with casein, affinity-purified monoclonal antibodies specific for IpaB (2F1) or IpaC (2G2) [32] were incubated with the antigen-coated plates for 2 hours. After washing, antigen-specific antibody was detected using anti-mouse immunoglobulin G (IgG) conjugated with alkaline phosphatase (Kirkegaard &amp; Perry). Using the substrate para-nitrophenyl phosphate the optical density at 405 nm (OD 405 ) was measured using an ELISA plate reader (Molecular Devices, Menlo Park, Calif.) after a 60 minute incubation with substrate. Using the Softmax Pro 4.5 (Molecular Devices) program, a standard curve plotting OD 405  versus concentration (ng/ml) was determined. The concentration of the unknown samples were then interpolated from the standard curve. 
     Invaplex AR  is designed to have IpaB and IpaC concentrations and an IpaC/IpaB ratio that is similar to HP Invaplex 24. The ratio of the quantity of IpaC to IpaB was determined for the Invaplex AR  and compared to HP-Invaplex 24, the most pure form of Invaplex. The IpaC/IpaB molar ratio in HP Invaplex is approximately 8. This was determined by densitometry analysis of SDS-PAGE gels and quantitative antibody based assays for IpaB and IpaC. In addition the LPS content is expected to be at the same relative mass ratio (approximately 0.5 to 0.6 mg of LPS for every 1 mg of protein) that is found in HP-Invaplex. 
     Measurement of LPS in Invaplex 
     LPS content in Invaplex preparations was measured by determining the amount of 2-keto-3-deoxyoctonate (KDO) in each preparation [43] or by using the limulus amoebocyte lysate assay (Cambrex Inc.). 
     Method for Formation of Artificial Invaplex. Preparation of Invaplex AR  for  S. flexneri  2a,  S. sonnei  and  S. dysenteriae  1 
     The purified components were mixed at ratios similar to that found in highly purified native Invaplex to form the artificial Invaplex. Analysis of the  S. flexneri  2a HP Invaplex indicated that the IpaC/IpaB molar ratio was approximately 8.0 and the LPS to total protein ratio was approximately 0.56 mg LPS/mg total protein. Using these parameters as a guide for reconstituting Invaplex from purified IpaB, IpaC and LPS a series of experiments were conducted to create an Invaplex AR . Once formed, the artificial Invaplex was purified by ion-exchange FPLC. 
     Purified, soluble IpaB and IpaC, each in their respective final buffers were mixed together at an IpaC/IpaB molar ratio of 8. After the IpaB and IpaC were mixed, the solution was slowly added to dry LPS powder (ratio of LPS to total protein is 0.56). LPS from any  Shigella  species can be used; for the described experiments  S. flexneri  2a,  S. sonnei  or  S. dysenteriae  1 LPS was used. The mixture was incubated at 37° C. for approximately 2 hours with shaking. Afterwards the protein/LPS mixture was diluted to 20 mM Tris-HCl, 0.10 M NaCl and 1.2 M urea to reduce the NaCl concentration prior to ion-exchange chromatography. The diluted mixture was then purified on a HiTrap™ Q HP anion exchange column. 
     Assembly Experiments: Formation of a  S. flexneri  Invaplex AR    
     Preliminary assembly experiments accommodated the insolubility of IpaC by maintaining both IpaC and IpaB proteins in a buffer containing ≧4M urea. Once the IpaC/IpaB mixture was added to LPS, solubility was no longer an issue which permitted the eventual removal of the urea. Typically purified IpaC by itself will precipitate upon dilution. In preliminary mixing experiments the 8 IpaC/IpaB molar ratio was maintained by adding 8 μM HisTagIpaC to 1 μM IpaB in a final volume of 1 ml. Both proteins were initially prepared in 20 mM Tris-HCl, 0.5 M NaCl, 6 M urea, pH 7.9. The proteins were mixed in a glass test tube and incubated at 37° C. without shaking for 15 mins. Dry LPS (230 μg) in a separate glass tube was also incubated at 37° C. without shaking for 15 mins. After the 15 min incubation, the IpaB+IpaC mixture was used to solubilize the pre-warmed LPS by slowly adding the protein mixture down the side of the tube, followed by vortexing. No appreciable flocculation or precipitation was observed. The IpaB/IpaC/LPS mixture was then incubated at 37° C. with shaking (200 rpm) for 2 hrs. In preparation for ion exchange chromatography (IEC), the mixture was diluted five-fold with pre-warmed 20 mM Tris buffer, pH 9.0 to lower the salt concentration. No precipitation was observed as the mixture cooled to room temperature. For final purification, the diluted IpaB/IpaC/LPS mixture was applied to an anion exchange column (HiTrap™ Q HP) with Buffer A consisting of 20 mM Tris-HCl, pH 9.0 and Buffer B consisting of 1 M NaCl, 20 mM Tris-HCl, pH 9.0, and a step gradient of 0% (5 column volumes) to 24% (10 column volumes) to 50% (6 column volumes) to 100% Buffer B (5 column volumes). One ml fractions were collected from a 1 ml HiTrap™ Q HP column at an elution flow rate of 1 ml/min. Fractions from each step were analyzed for the presence of IpaB, IpaC and LPS by spotblot. The  S. flexneri  Invaplex AR  eluted in the 50% B step ( FIG. 2 ) which contained the greatest quantities of all three components (IpaB, IpaC and LPS) as determined by western blots and silver stained gels ( FIG. 2A ). When applied to a size exclusion column the IpaB/IpaC/LPS complex eluted at between the 1 MDa and 669 kKa standards which is the same size range as HP Invaplex. 
     Formation of  S. dysenteriae  1 Artificial Invaplex and Larger Scale Production 
     The above experiment was repeated but with a ten-fold increase in reactants to increase the Invaplex AR  yield. In addition  S. dysenteriae  1 LPS was used instead of  S. flexneri  LPS. As such, 3.28 mg of IpaC in 20 mM Tris-HCl, 0.5 M NaCl, 6 M Urea, pH 7.9, was mixed with 0.62 mg IpaB in 20 mM Tris-HCl, 0.5 M NaCl, pH 7.9, in a total volume of 5 mls. The protein mixture and a separate tube of LPS (2.1 mg) was pre-warmed to 37° C. The IpaB+IpaC mixture was added to the LPS tube and vortexed to solubilize the LPS. No precipitation was observed. The reaction mixture was incubated at 37° C. with shaking (200 rpm) for 2 hours. Next pre-warmed IEC Buffer A (20 mM Tris-HCl, pH 9.0) was added to dilute the salt concentration 1:5 yielding a final volume of 25 mls. After cooling to room temperature, the diluted reaction mixture was applied to a 1 ml HiTrap™ Q HP IEC column and subjected to an Invaplex purification step gradient (see above). The  S. dysenteriae  1 Invaplex AR  eluted in the 50% Buffer B step. It contained IpaB, IpaC and  S. dysenteriae  1 LPS. ( FIG. 2C ). Analysis of the 0.24 M and 1.0M NaCl peaks did not detect significant quantities of IpaB, IpaC or LPS. The yield of Invaplex AR  production was approximately 10 to 20% of the amount of total protein used to initiate the assembly process. 
     Similar assembly experiments have been conducted for  S. sonnei  resulting in an InvaplexAR for  S. sonnei  (see  FIG. 2B ). 
     Large Scale Production of Invaplex AR  of  Shigella flexneri  2a 
     A fifty-fold increase in reactants was used to produce more Invaplex AR  for advanced studies. Using an 8 IpaC/1 IpaB molar ratio, 16.4 mg of IpaC (in 20 mM Tris-HCl, 0.5 M NaCl, 6 M Urea, pH 7.9) was mixed with 3.1 mg IpaB (in 20 mM Tris-HCl, 0.5 M NaCl, pH 7.9) in a total volume of 20 mls. The protein mixture and a separate tube of  Shigella flexneri  2a LPS (11.4 mg) were pre-warmed to 37° C. The IpaB+IpaC mixture was added to the LPS tube and vortexed to solubilize the LPS. The reaction mixture was incubated at 37° C. with shaking (200 rpm) for 2 hours. Next pre-warmed IEC Buffer A (20 mM Tris-HCl, pH 9.0) was added to dilute the salt concentration 1:5 yielding a final volume of 100 mls. After cooling to room temperature, the diluted reaction mixture was applied to a 5 ml HiTrap™ Q HP IEC column and subjected to an Invaplex purification step gradient (see above). The  S. flexneri  2a Invaplex AR  eluted in the 50% Buffer B step. It contained IpaB, IpaC and  S. flexneri  2a LPS. Analysis of the 0.24 M and 1.0M NaCl peaks did not detect significant quantities of IpaB, IpaC or LPS. The yield of Invaplex AR  production was approximately 9% of the amount of total protein used to initiate the assembly process. 
     Analysis of Invaplex AR  Preparations by Electrophoresis and Western Blots for IpaB and IpaC 
     The total protein composition of the Invaplex AR  preparations was determined by stained SDS-PAGE gels. Western blots were used to determine the presence, size and relative concentrations of IpaB and IpaC. Silver stained gels were used to assess the LPS profile [18]. Monoclonal antibodies specific for either the IpaB or IpaC proteins were used to probe the nitrocellulose blots [32]. After probing with alkaline phosphatase-conjugated protein A, blots are developed with ASMX phosphate (Sigma) and Fast Red TR salt (Sigma). Video images of the blots are displayed on a Macintosh computer using a CCD video camera (Cohu) and captured with NIH Image software (version 1.62). Densitometry analysis of the digital images were performed with GelPro Image analysis software (version 2.0.10, Media Cybernetics, Inc.). The total protein gel profile and the IpaB and IpaC content should resemble that found in HP-Invaplex 24 or HP-Invaplex 50. 
     Size Exclusion Chromatography 
     Size-exclusion chromatography was used to determine if the components of Invaplex AR  found in the ion-exchange peak are truly complexed. This is the same method used to produce HP-Invaplex. A Superose 6 HR 10/30 column (Amersham Pharmacia), calibrated with Blue Dextran  200  (2 MDa), thyroglobulin (669 kKa),and ovalbumin (43 kKa), was loaded with Invaplex AR  (approximately 3 mg). Fractions (0.5 ml, flow rate 0.25 ml/min) were collected and analyzed by immuno-spot blot for the presence of IpaB, IpaC and LPS. All three Invaplex AR  components (IpaB, IpaC and LPS) eluted in fractions between 2 MDa and 669 kKa indicating they are associated in a complex as they eluted at a size much greater than the individual components (43 kKa for IpaC; 62 kKa for IpaB). The SEC size of Invaplex AR  is similar to the size where HP-Invaplex elutes. 
     Evaluation of  S. dysenteriae  1 Invaplex AR  in Tissue Culture model of Internalization 
     Invaplex binds to and stimulates endocytosis in cultured mammalian cells. This property is dependent on the presence of the invasins IpaB and IpaC and utilizes host-cell acting during the endocytic process [38]. The Invaplex internalization assay is an in vitro model used to measure the functional activity of Invaplex preparations. The assay is modeled after the  Shigella  plaque assay [39]and  Shigella  uptake assays [40, 41]. The assay involves incubating Invaplex AR  with BHK-21 fibroblast cells (60-70% confluent) in chamber slides. After incubation for 5, 15, 30, or 60 minutes the cells were washed 3× with PBS and formalin-fixed. Fixed cells were permeabilized with PBS supplemented with 0.1% Triton X-100 and 0.1% BSA (wash buffer) and probed with polyclonal mouse serum with specificity for LPS, IpaB, and IpaC. Bound polyclonal mouse antibodies were detected with GAM-IgG-Oregon Green 488 (Molecular Probes) followed by counterstaining with DAPI to identify the nuclei. The Invaplex treated cells were examined by fluorescent microscopy with digital image capturing. Both native and artificial  S. dysenteriae  1 Invaplex were internalized by BHK cells with similar cytoplasmic staining pattern for both preparations indicating that the Invaplex AR  maintains the capacity to stimulate internalization by mammalian cells ( FIG. 4 ). This indicates that the assembly of recombinant IpaB and IpaC with  S. dysenteriae  1 LPS into a complex isolated by ion-exchange FPLC has resulted in a synthetic product which maintains the same biological activity as the native complex. 
     Immunogenicity, Adjuvanticity and Protective Efficacy of Native and Artificial Invaplex Vaccines 
     The mouse lung-infection [31] and the guinea pig keratoconjunctivits models [44] were used to determine whether intranasal immunization with  S. flexneri  2a Invaplex AR  stimulates a protective immune response that is effective against a homologous  S. flexneri  2a challenge. Separate mouse studies were used to determine whether immunization with  S. sonnei  Invaplex AR  could protect against a homologous  S. sonnei  challenge and against a heterologous challenge with  S. flexneri  2a. Additional studies were conducted to assess the adjuvant activity of Invaplex AR  using ovalbumin (OVA) as an immunogen. In the adjuvanticity experiments, immune responses induced after immunization with OVA alone were compared to immune responses induced with OVA combined with either native Invaplex, Invaplex AR , or cholera toxin, an established, potent mucosal adjuvant. The guinea pig and mouse experiments were conducted at WRAIR under IACUC approved protocols. 
     Mouse Experiments 
     Four separate animal experiments have been completed to evaluate the immunogenicity, adjuvanticity, and protective efficacy of artificial Invaplex. Common methods were used in each experiment and are outlined in the following paragraph. Methods and materials specific to each study are indicated in separate sections. 
     Immunizations and Challenge 
     Mice were immunized on days 0, 14 and 28. A total antigen volume of 25 ul was delivered in 5 to 6 small drops applied to the external nares with a micropipette. Blood taken by tail bleed from all mice at days 0, 28 and 42 and 2 weeks post-challenge (day 63). On day 35 or 42 mice were euthanized followed by immediate collection of mucosal washes and removal spleen for in vitro proliferation and cytokine analysis  S. flexneri  2a Invaplex AR . Three weeks after the final immunization mice (15 per group) were challenged intranasally with a lethal dose (1.6×10 7  cfu) of  S. flexneri  2a (2457T) or with  S. sonnei  (Mosely, 8×10 6  cfu) as described for the mouse lung model [34]. Prior to intranasal immunization or challenge, mice are anesthetized with a mixture of ketamine hydrochloride (40 mg/kg) and xylazine (12 mg/kg). Challenged mice were monitored daily for two weeks for weight loss, fur ruffling and lethargy, with death used as an endpoint. Surviving mice were bled 14 days post-challenge (day 63). 
       S. flexneri  2a Invaplex AR  Immunogenicity and Protection Study I 
     Separate groups of female Balb/c mice (10-15 mice/group) were immunized intranasally on day 0, 14, and 28 with 5 ug of either the native  S. flexneri  2a Invaplex (lot JWJX) or 2.5 μg of artificial  S. flexneri  2a Invaplex AR  (Lot KF-D3D4) vaccines. Control animals were inoculated with saline. Blood was collected on day 0, 28, 42, and 63. Animals were challenged intranasally on day 49. 
       S. flexneri  2a Invaplex AR  Immunogenicity and Protection Study II 
     Separate groups of female Balb/c mice (15-20 mice/group) were immunized intranasally on day 0, 14, and 28 with 5 ug of either the native  S. flexneri  2a Invaplex (Lot JWJX) or 2.5 μg of artificial  S. flexneri  2a Invaplex AR  (lot KR-C5) vaccines. Control animals were inoculated with saline. Blood was collected on day 0, 28, 42, and 63. Lung and intestinal washes were collected on day 42 from 4 animals/group. Remaining animals were challenged intranasally on day 49. 
       S. flexneri  2a Invaplex AR  Adjuvanticity Study 
     The ability of Invaplex AR  to function as a mucosal adjuvant was determined in mice using ovalbumin (OVA) as a model protein antigen. Balb/cByJ mice (5 mice/group) were intranasally immunized on day 0, 14, and 28 with OVA (5 μg) alone, or OVA (5 μg) combined with either  S. flexneri  2a Invaplex AR  (lot KF-D3D4; 2.5 μg), native  S. flexneri  2a Invaplex (Lot JWJX; 5 μg), or cholera toxin (CT; 5 μg). Blood was collected on day 0, 28, and 42. Control animals were inoculated with saline. Lung and intestinal washes were collected on day 42. Serum endpoint titers on day 0, 28 and 42 with specificity for Invaplex 24, Invaplex 50, LPS, OVA, IpaB and IpaC were determined by ELISA. Antigen-specific antibodies in mucosal washes were also assessed. 
       S. sonnei  Invaplex AR  Immunogenicity and Protection Study 
     The immunogencity and protective efficacy of  S. sonnei  Invaplex AR  was determined in mice using a homologous ( S. sonnei  53G) and heterologous ( S. flexneri  2a 2457T) challenge. Balb/cByJ mice (10-15 mice/group) were intranasally immunized on day 0, 14, and 28 with  S. sonnei  Invaplex AR  (lot KJ-D3D6; 2.5 μg), native  S. sonnei  Invaplex (Lot JOJP; 5 μg), or native  S. flexneri  2a Invaplex (Lot JWJX; 5 μg). Control animals were inoculated with saline. Blood was collected on day 0, 28, and 42. 
     Guinea Pig Experiments 
     Guinea pigs (Hartley strain, 6 to 10 per group) were immunized intranasally with the artificial or native Invaplex vaccines (25 ug/dose). The dose volume (100 ul) was split equally between each nostril. Saline was used to immunize control animals. Guinea pigs were immunized on days 0, 14, and 28 and bled from the lateral ear vein on days 0, 28, 42, and 14 days after challenge. Prior to intranasal immunization, guinea pigs were anesthetized with a mixture of ketamine (40 mg/kg) and xylazine (4 mg/kg). Three weeks after the third immunization guinea pigs were challenged intraocularly with  S. flexneri  2a (strain 2457T) and observed daily for 5 days to assess the degree of inflammation and keratoconjunctivitis as previously described [44]. 
     ELISA 
     ELISA assays are used to measure antigen specific IgA and IgG antibodies in sera and mucosal secretions, including lung and intestinal lavages and stool extracts from immunized and/or challenged mice and guinea pigs [18, 29, 46]. Antigens used in ELISAs, including purified  S. flexneri  2a LPS,  S. sonnei  LPS, water-extracted  Shigella  antigens, purified IpaB and IpaC proteins, and  S. flexneri  2a native Invaplex and OVA were coated onto ImmunIon IB 96-well ELISA plates (ThermoLab Systems) overnight at 4° C. After blocking with 2% casein (in Tris-saline buffer, pH 7.5) sera and mucosal washes were serially diluted in duplicate and incubated with the antigen-coated plates for 4 hours at 25° C. After washing with PBS containing 0.05% Tween 20, antigen-specific antibody is probed with alkaline phosphatase conjugated anti-mouse IgG or anti-mouse IgA (Kirkegaard &amp; Perry). Alkaline phosphatase activity was detected with p-nitrophenyl phosphate (1 mg/ml). After a 30-min incubation in substrate, the optical density (OD 405 ) was measured using an ELISA plate reader (Molecular Devices, Menlo Park, Calif.). Endpoint titers were determined for each animal and was used to calculate geometric mean titers for each group at each time point. Typically animals intranasally immunized with Invaplex respond with a 4 to 8-fold higher serum (IgG and IgA) and mucosal (lung and intestine) IgA titers to  Shigella  antigens as compared to preimmune samples or saline control animals. 
     Total IgA Assay 
     A capture enzyme immunoassay was used to determine total IgA concentrations in mucosal samples. Sample concentrations were determined from standard curves, using purified mouse IgA assayed in parallel. Specific mucosal IgA activities were calculated by dividing the endpoint titer for each individual mucosal sample by the concentration of total IgA within the same sample [47]. 
     Antigen Stimulation of Cultured Lymphocytes from Mice Immunized with Invaplex AR    
     Lymphocyte proliferation upon antigen stimulation was determined using splenocytes collected from Invaplex-immunized mice and naive mice. Mononuclear cells (2×10 5  per well) were incubated with Invaplex, IpaB, or IpaC or  S. flexneri  2a LPS preparations. Simultaneously in separate microtiter wells, splenocytes were stimulated with concanavalin A to provide positive controls. Negative controls included cells incubated with complete medium alone and cells from naive mice stimulated with antigen. After incubation with antigen for 4 to 7 days, cell proliferation was measured by a non-radioactive assessment of dehydrogenase activity using MTS (Promega) [48]. There is a strong correlation with increasing optical density readouts in this assay and the number of viable cells in a well. Prior to measuring proliferation, cell culture supernatants were collected on days 3 and 5 for cytokine measurements (see below). Stimulation indices (SI) were calculated by dividing the mean optical density of antigen-stimulated cells by the mean optical density of medium-only stimulated cells. The SI of cells from mice immunized with Invaplex was compared to the SI of cells from non-immunized mice. 
     Antigen-Specific Systemic Antibody Response after Intranasal Immunization with Invaplex AR    
     The immunogenicity of artificial  S. flexneri  2a Invaplex (Invaplex AR ), manufactured from individual purified components rather than the virulent organism (native Invaplex) was evaluated in mice. Groups of mice (n=6-10) were intranasally immunized on day 0, 14, and 28 with native  S. flexneri  2a Invaplex 24 (5 or 10 μg), Invaplex AR  (2.5 μg), purified IpaB (2.5 μg), purified IpaC (2.5 μg), LPS (2.5 μg), or saline. Three weeks after the third immunization (day 49), the mice were challenged with  Shigella flexneri  2a (2457T). Blood collected on day 0, 28, 42, and 63 was analyzed by ELISA for serum IgG and IgA responses to Invaplex 50, Invaplex 24, purified LPS, IpaB and IpaC.  FIGS. 5 and 6  outline the serum IgG and IgA endpoint titers determined in blood collected on day 42 (two weeks after the third immunization). 
     Saline-inoculated mice and preimmune sera from immunized mice did not have detectable levels of antigen-specific antibodies (data not shown). Immunization with  S. flexneri  2a Invaplex AR  induced serum IgG and IgA responses to Invaplex 50 and Invaplex 24 of comparable magnitude to those induced with native Invaplex 24 ( FIG. 5 ), and significantly higher (p&lt;0.001) than those induced after inoculation with saline. A two-fold increase in the amount of native Invaplex (lot JWJX) used for immunization did not result in an increase in the magnitude of the Invaplex 50, LPS, or Invaplex 24-specific serum IgG or IgA responses measured on day 42 ( FIG. 5 ). Immunization with purified IpaB resulted in a strong IpaB-specific ( FIG. 6 ) and Invaplex 24-specific response (GMT&gt;5760 and 3800, respectively) and a moderate response to Invaplex 50 (GMT 950). Immunization with purified LPS did not induce a detectable serum IgG or IgA response to any of the antigens used in ELISA in the majority (5/6) of mice in the vaccine group. Similarly, immunization with purified IpaC also did not induce a detectable immune response to any of the antigens assayed, including a IpaC-specific ELISA. Interestingly, the mice immunized with Invaplex AR  had among the highest IpaC-specific endpoint titers ( FIGS. 6 and 8 , significantly higher (p&lt;0.001) than those induced after immunization with native Invaplex. 
     Protection of Mice from a Lethal Challenge of  S. flexneri  after Intranasal Immunization with Invaplex AR    
     The lethal lung model entails intranasal inoculation of mice with a lethal dose of shigellae which establish an infection of the lungs leading to severe weight loss, pneumonia and ultimately death over the observation period of two weeks. Three weeks after intranasal immunization with  S. flexneri  2a Invaplex AR  or native Invaplex mice were challenged with a lethal dose of  S. flexneri  2a (strain 2457T). In naive mice (treated with saline) 11 of 13 mice died with a mean maximum weight loss of 31.4% of the pre-challenge weight (see Table 1). All mice immunized with  S. flexneri  2a Invaplex AR  (p&lt;0.001) or native Invaplex (p&lt;0.001) survived the lethal challenge with  S. flexneri  2a. With respect to weight loss (which is likely a more sensitive indicator of protective immunity) mice immunized with Invaplex AR  lost less of their pre-challenge weight (21.3%) as compared to 23.8% to 26.0% weight loss in the native Invaplex-immunized mice. Furthermore the day of weight rebound (an indication of recovery from the challenge) was day 7 for Invaplex AR  and day 13 (native Invaplex, 5 μg) or day 7 (native Invaplex, 10 μg). 
     In addition the protective capacity of the individual components (IpaB, IpaC and LPS) used to construct Invaplex AR  were also evaluated in the mouse lethal lung model (see Table 1). Immunization with purified LPS and purified IpaC did not protect mice from death (both P&gt;0.05) and although immunization with IpaB protected 5 of 6 mice from a lethal challenge the mice never regained weight and remained symptomatic (low weight, ruffled fur) through the end of the observation period. The mean maximum weight loss after challenge was 29.9% for LPS, 31.0% for IpaB and 33.9% for IpaC all of which are very close to the naïve mice weight loss value of 31.4%. 
     The results of the challenge of Invaplex AR  mice with a lethal dose of  S. flexneri  2a indicate that Invaplex AR  stimulates a level of protection that is comparable to or exceeds that of native Invaplex. Furthermore, it appears that the complex of IpaB, IpaC and LPS is required in that individual components are incapable of stimulating a fully effective protective immune response. 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Lethal Challenge of mice immunized with  S. flexneri  2a InvaplexAR or native Invaplex 1 . 
               
             
          
           
               
                   
                   
                 Day of 
                   
                   
                   
                   
                   
               
               
                   
                 Maximum 
                 50% 
                   
                   
                 Total 
               
               
                 Immunizing 
                 Wgt 
                 Wgt 
                 No. 
                 No. 
                 No. of 
                   
                 P 
               
               
                 Antigen 
                 Loss (%) 
                 recovery 
                 Survivors 
                 Dead 
                 Animals 
                 % Protection† 
                 Value* 
               
               
                   
               
             
          
           
               
                   S. flexneri  2a 
                 21.32 
                 7 
                 15 
                 0 
                 15 
                 100% 
                 &lt;0.001 
               
               
                 Invaplex AR  , 2.5 μg 
               
               
                 Invaplex 24 JWJX, 
                 26.02 
                 13 
                 15 
                 0 
                 15 
                 100% 
                 &lt;0.001 
               
               
                 5 μg 
               
               
                 Invaplex 24 JWJX, 
                 23.81 
                 7 
                 12 
                 0 
                 12 
                 100% 
                 &lt;0.001 
               
               
                 10 μg 
               
               
                   S. flexneri  2a LPS, 
                 29.95 
                 8 
                 3 
                 3 
                 6 
                 40.9%  
                 0.262 
               
               
                 2.5 μg 
               
               
                 Purified 1paB, 
                 30.97 
                 &gt;14 
                 5 
                 1 
                 6 
                 80.3%  
                 0.010 
               
               
                 2.5 μg 
               
               
                 Purified 
                 33.91 
                 &gt;14 
                 1 
                 5 
                 6 
                  1.5% 
                 1.000 
               
               
                 HisTagIpaC, 2.5 μg 
               
               
                 0.9% Saline 
                 31.35 
                 10 
                 2 
                 11 
                 13 
                  0% 
                 — 
               
               
                   
               
               
                   1 Three weeks after the final immunization, mice were intranasally challenged with 1.5 × 10 7  cfu of  S. flexneri  2a,. Weight loss and symptoms were monitored daily for 14 days. 
               
               
                 †% Protection = [(%Death Control  - % Death Vaccinees )/% Death Control ] × 100 
               
               
                 *Fisher Exact Test 
               
             
          
         
       
     
     Antigen-Specific Mucosal Antibody Response After Intranasal Immunization with Invaplex AR    
     Intestinal and lung washes collected on day 35 from mice immunized with  S. flexneri  2a Invaplex AR  or native Invaplex were assessed by ELISA for antigen-specific IgA levels. Immunization with Invaplex AR  induced levels of LPS and Invaplex-specific intestinal IgA that were comparable to levels induced by immunization with native Invaplex ( FIG. 7 ) and significantly higher levels (p&lt;0.001) of IpaB-specific IgA. Minimal IpaC-specific intestinal IgA was elicited after immunization with any of the Invaplex vaccine preparations (Tables 7 and 8). 
     Intranasal immunization with Invaplex AR  elicited strong antibody responses in the lung, directed to LPS, Invaplex 50, IpaB and IpaC ( FIG. 8  and Tables 7 and 8). Minimal levels of LPS-specific IgA were induced in the lung after immunization with native Invaplex with undetectable levels of antibodies specific for Invaplex 50, IpaB and IpaC. 
     Antigen-Specific Cellular Proliferative Response and Secreted Cytokine Profiles after Intranasal Immunization with Invaplex AR    
     Splenocytes collected from immunized mice on day 35 were stimulated in vitro with Invaplex 24, IpaB, or IpaC to assess induction of antigen-specific cell-mediated responses. Proliferation of cells after incubation with antigen indicates prior exposure and immunological memory. Concavalin A (ConA), which non-specifically activates T cell proliferation, was used as a positive control to demonstrate viable cell levels. Stimulation indices (Sis) after stimulation with ConA ranged from 13.8 to 15.9 (Table 2). Cells from saline inoculated animals did not proliferate after incubation with Invaplex, IpaB, or IpaC. Splenocytes from animals (4/4) immunized with Invaplex AR  proliferated after incubation with Invaplex (SI=10.2), IpaB (SI=8.7), and IpaC (SI=6.9) indicating immunological memory to these antigens was present. The IpaB and IpaC-specific proliferative responses in groups immunized with Invaplex AR  were significantly higher (P&lt;0.01) than the proliferative responses in groups immunized with native Invaplex. Splenocytes from 4/4 animals immunized with native Invaplex proliferated after incubation with Invaplex (SI=6.8). Low to undetectable levels of proliferation occurred in splenocytes from mice immunized with native Invaplex after ex vivo incubation with IpaB (1/4 mice) or IpaC (0/4 mice). 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Antigen-specific cellular proliferation of splenocytes from mice intranasally 
               
               
                 immunized with  S. flexneri  2a Invaplex AR  or native Invaplex after in vitro stimulation. 
               
             
          
           
               
                   
                 Cellular proliferative responses after in vitro stimulation a  with: 
               
             
          
           
               
                   
                   
                   S. flexneri  2a 
                   
                   
                   
               
               
                   
                   
                 Invaplex 24 
                 IpaB 
                 IpaC 
                 ConA 
               
               
                 Grp. 
                 Treatment 
                 (1 μg/ml) 
                 (5 μg/ml) 
                 (5 μg/ml) 
                 (5 μg/ml) 
               
               
                   
               
               
                 31 
                   S. flexneri  2a 
                 10.2 ± 3.4 c *, ** 
                 8.7 ± 2.2*, ** 
                 6.9 ± 3.9*, ** 
                 14.2 ± 4.5*, ** 
               
               
                   
                 Invaplex AR   
                 (4/4) 
                 (4/4) 
                 (4/4) 
                 (4/4) 
               
               
                   
                 (KR-C5; 2.5 μg) b   
               
               
                 32 
                   S. flexneri  2a 
                  6.8 ± 1.5* 
                 2.2 ± 1.4 
                 1.6 ± 0.9 
                 15.9 ± 2.3 
               
               
                   
                 native Invaplex 
                 (4/4) 
                 (1/4) 
                 (0/4) 
                 (4/4) 
               
               
                   
                 24 
               
               
                   
                 (JWJX; 2.5 μg) 
               
               
                 33 
                 0.9% saline 
                  1.1 ± 0.4 
                 1.2 ± 0.8 
                 1.8 ± 1.1 
                 13.8 ± 3.3 
               
               
                   
                   
                 (0/4) 
                 (0/4) 
                 (0/4) 
                 (4/4) 
               
               
                   
               
               
                   a proliferative responses in splenocytes were determined after stimulation in vitro for 3 days with ConA and 5 days with  S. flexneri  2a Invaplex 24 (lot JWJX), IpaB or IpaC. 
               
               
                   b (lot number; immunization dose) 
               
               
                   c Mean stimulation index ± ISD (number of responders/total number in group) 
               
               
                 *P &lt; 0.05 as compared to saline-inoculated group (unpaired t test). 
               
               
                 **P &lt; 0.01 as compared to native Invaplex-immunized group (unpaired t test). 
               
             
          
         
       
     
     Confirmation of Protection of Mice from a Lethal Challenge of  S. flexneri  after Intranasal Immunization with Invaplex AR  using a Different Lot of Invaplex AR    
     The experiment describing protection with Invaplex AR  in the mouse lethal lung model was repeated with a different lot of  S. flexneri  2a Invaplex AR . In addition the evaluation of the purified components was repeated along with mice immunized with mixtures of two purified components (IpaB+IpaC; IpaB+LPS; IpaC+LPS). For this challenge, mice were inoculated intranasally with a slightly higher challenge dose (1.6×10 6  cfu) of  S. flexneri  2a (strain 23457T). In naive mice (treated with saline) 14 of 14 mice died with a mean maximum weight loss of 34.5% of the pre-challenge weight (see Table 3). Mice immunized with  S. flexneri  2a Invaplex AR  (13 of 14 survived; p&lt;0.001) survived the lethal challenge with  S. flexneri  whereas mice immunized with native Invaplex had a much lower survival rate for the challenge dose used in this experiment (See Table 3). With respect to weight loss mice immunized with Invaplex AR  lost less of their pre-challenge weight (26.6%) as compared to 31.6% weight loss in the native Invaplex-immunized mice and 34.5% in the mice inoculated with saline. 
     The protective capacity of the individual components (IpaB, IpaC and LPS) or pairs of purified components used to construct Invaplex AR  confirmed previous results in that IpaC and LPS are not protective. Purified IpaB was not protective either. (see Table 3). Mixtures of two of the purified components resulted in protection in the IpaB+LPS combination and the IpaC+LPS combination whereas the IpaB+IpaC combination was not fully protective. 
     The results of the second experiment evaluating the protective capacity of Invaplex AR  clearly shows that Invaplex AR  stimulates a level of protection that exceeds that of native Invaplex. Furthermore, it appears that the individual components (IpaB, IpaC or LPS) are incapable of stimulating a fully effective protective immune response. 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Lethal Challenge of mica immunized with  S. flexneri  2a InvaplexAR or native Invaplex 1   
               
             
          
           
               
                   
                 Maximum 
                 Day of 50% 
                   
                   
                   
                   
                   
               
               
                   
                 Wgt 
                 Wgt 
                 No. 
                   
                 Total No. of 
               
               
                 Antigen 
                 Loss (%) 
                 Recovery 
                 Survivors 
                 No. Dead 1   
                 Animals 
                 % Protection 2   
                 P Value 3   
               
               
                   
               
             
          
           
               
                 IVP AR  S.flex 2a 
                 26.6 
                 10  
                 13 
                 1 
                 14 
                 92.9 
                 &lt;0.001 
               
               
                 KR-C5, 2.5 μg 
               
               
                 IVP 24  S.flex2a 
                 31.6 
                 — 
                 4 
                 10 
                 14 
                 28.6 
                 0.098 
               
               
                 JWJX,5 μg 
               
               
                 0.9% Saline 
                 34.5 
                 — 
                 0 
                 14 
                 14 
                 0 
               
               
                 S.flex2a LPS, 
                 32.7 
                 — 
                 0 
                 14 
                 14 
                 0 
                 NS 
               
               
                 2.5 μg 
               
               
                 IpaB, 2.5 μg 
                 29.8 
                 13  
                 1 
                 13 
                 14 
                 7.1 
                 NS 
               
               
                 HisTag IpaC, 
                 38 
                 — 
                 0 
                 14 
                 14 
                 0 
                 NS 
               
               
                 2.5 μg 
               
               
                 S.flex2a LPS/ 
                 24 
                 8 
                 14 
                 0 
                 14 
                 100 
                 &lt;0.001 
               
               
                 IpaB, 2.5 μg each 
               
               
                 S.flex2a LPS/ 
                 25 
                 8 
                 13 
                 1 
                 14 
                 92.9 
                 &lt;0.001 
               
               
                 HisTag IpaC, 
               
               
                 2.5 μg each 
               
               
                 IpaB/HisTag 
                 26.7 
                 8 
                 5 
                 9 
                 14 
                 37.7 
                 0.041 
               
               
                 IpaC, 2.5 μg each 
               
               
                   
               
               
                   1 Three weeks after the final immunization, mice were intranasally challenged with 1.6 × 10 7  cfu of  S. flexneri  2a,. Weight loss and symptoms were monitored daily for 14 days. 
               
               
                   2 % Protection = [(% Death Control  - % Death Vaccinees )/% Death Control] × 100   
               
               
                   3 Fisher Exact Test 
               
             
          
         
       
     
       S. sonnei  Invaplex AR  Murine Immunogenicity and Protection Study 
     Serum antibody responses directed to  S. sonnei  Invaplex 50,  S. sonnei  LPS, IpaB and IpaC were determined by ELISA. Mice inoculated with saline (negative control) did not have detectable levels of antigen-specific serum IgG or IgA on day 42 (Table 4). Similar  S. sonnei  Invaplex 50 and LPS-specific serum IgG and IgA endpoint titers were achieved after immunization with  S. sonnei  Invaplex AR  and native Invaplex (Table 4). Groups of mice immunized with  S. sonnei  Invaplex AR  had higher levels of IpaB-specific serum IgG (GMT&gt;5760, P&lt;0.001) as compared to groups of mice immunized with native  S. sonnei  Invaplex (GMT 546). Animals immunized with Invaplex AR  had an anti-IpaC serum IgG mean titer of 1091 (P&lt;0.001) whereas animals immunized with native Invaplex or saline had undetectable levels of IpaC-specific IgG. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Antigen-specific serum IgG and IgA endpoint titers on day 42 in mice after 
               
               
                 intranasal immunization with  S. sonnei  Invaplex AR  or native  S. sonnei  Invaplex 50 
               
             
          
           
               
                   
                   S.sonnei  Invaplex 50 
                   S. sonnei  LPS 
                 IpaB 
                 IpaC 
               
             
          
           
               
                   
                 IgG 
                 IgA 
                 IgG 
                 IgA 
                 IgG 
                 IgG 
               
               
                   
                   
               
             
          
           
               
                 Saline 
                 90 ± 0 c,d   
                 45 ± 0  
                 90 ± 0 
                 45 ± 0 
                 90 ± 0  
                 90 ± 0 
               
               
                 Native 
                 2183 ± 789   
                 1254 ± 322  
                 103 ± 40 
                 157 ± 40 
                 546 ± 483 
                 90 ± 0 
               
               
                 
                   S. sonnei 
                 
               
               
                 Invaplex 50 
               
               
                 (5 μg) a   
               
               
                 Artificial 
                 5014 ± 1288** 
                 2864 ± 36** 
                  136 ± 117 
                  313 ± 224* 
                 &gt;5760** 
                  1091 ± 1172** 
               
               
                 
                   S. sonnei 
                 
               
               
                 Invaplex AR   
               
               
                 (2.5 μg) b   
               
               
                   
               
               
                   a Group 6 
               
               
                   b Group 3 
               
               
                   c Geometric mean endpoint titer ± 1 standard deviation 
               
               
                   d Comparisons between groups were accomplished using two-way ANOVA analysis of natural log-transformed endpoint titers. 
               
               
                 *p &lt; 0.05 as compared with native  S. sonnei  Invaplex 50 
               
               
                 **p &lt; 0.001 as compared with native  S. sonnei  Invaplex 50 
               
             
          
         
       
     
     Protection of Mice from a Lethal Challenge of  S. sonnei  or Heterologous  S. flexneri  after Intranasal Immunization with  S. sonnei  Invaplex AR    
     The protective capacity of InvaplexAR was also evaluated for an InvaplexAR manufactured from purified  S. sonnei  LPS and recombinant IpaB and IpaC using the mouse lethal lung model. It was compared to native  S. sonnei  Invaplex. In addition the capacity of  S. sonnei  Invaplex AR  to protect against a heterologous  S. flexneri  challenge was also evaluated. In nave mice (treated with saline) 15 of 15 mice died with a mean maximum weight loss of 23.4% of the pre-challenge weight (see Table 5). Mice immunized with  S. sonnei  Invaplex AR  survived (15 of 15 survived; p&lt;0.001) the lethal challenge with  S. sonnei  whereas mice immunized with native Invaplex also exhibited solid protection (14 of 14 survived, P&lt;0.001) (See Table 5). With respect to weight loss, mice immunized with  S. sonnei  Invaplex AR  lost 7.7% of their pre-challenge weight as compared to 9.2% weight loss in the native  S. sonnei  Invaplex-immunized mice and 23.4% in the mice inoculated with saline. 
     Interestingly, the  S. sonnei  Invaplex AR  also provided significant protection (15 or 15 challenged mice survived, P&lt;0.001)) against a heterologous  S. flexneri  2a challenge suggesting that the immune response to either IpaB or IpaC may contribute significantly to the protective immune response. 
     The results of this experiment evaluating the protective capacity of  S. sonnei  Invaplex AR  clearly shows that Invaplex AR  from another  Shigella  species stimulates a level of protection that is comparable to that of native Invaplex. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Lethal Challenge of mica immunized with  S. sonnei  Invaplex AR  or native  S. sonnei  Invaplex 1 . 
               
               
                 Stimulation of homologous and heterologous immunity 
               
             
          
           
               
                   
                   
                   
                 Day of 
                   
                   
                   
                   
                   
               
               
                   
                   
                 Maximum 
                 50% 
               
               
                   
                 Challenge 
                 Wgt Loss 
                 Wgt 
                 No. 
                 No. 
                 Total No. of 
                 % 
                 P 
               
               
                 Antigen 
                 Agent 
                 (%) 
                 Recovery 
                 Survivors 
                 Dead 1   
                 Animals 
                 Protection 2   
                 Value 3   
               
               
                   
               
             
          
           
               
                 Saline 
                 
                   S. sonnei 
                 
                 23.4 
                 — 
                 0 
                 15 
                 15 
                 0 
                 — 
               
               
                   Sonnei  IVP- 
                 
                   S. sonnei 
                 
                 7.7 
                 3 
                 15 
                 0 
                 15 
                 100 
                 &lt;0.001 
               
               
                 AR-KJD3D6 
               
               
                 2.5 ug 
               
               
                 IVP50 JOJP 
                 
                   S. sonnei 
                 
                 9.2 
                 7 
                 14 
                 0 
                 14 
                 100 
                 &lt;0.001 
               
               
                 5 ug 3x 
               
               
                 Saline 
                 
                   S. flexneri 
                 
                 35.1 
                 — 
                 0 
                 15 
                 15 
                 — 
                 — 
               
               
                   
                 2a 
               
               
                   Sonnei  IVP- 
                 
                   S. flexneri 
                 
                 31.0 
                 9 
                 15 
                 0 
                 15 
                 100 
                 &lt;0.001 
               
               
                 AR 2.5 ug 
                 2a 
               
               
                 S. flex 2a 
                 
                   S. flexneri 
                 
                 25.0 
                 — 
                 11 
                 3 
                 14 
                 78.6 
                 &lt;0.001 
               
               
                 JWJX 5 ug 3x 
                 2a 
               
               
                 (Nasal) 
               
               
                   
               
               
                   1 Three weeks after the final immunization, mice were intranasally challenged with 1.6 × 10 7  cfu of  S. flexneri  2a or 8 × 10 6  cfu  S. sonnei , as indicated. Weight loss and symptoms were monitored daily for 14 days. 
               
               
                   2 % Protection = [(% Death Control  - % Death Vaccinees )/% Death Control ] × 100 
               
               
                   3 Fisher Exact Test 
               
             
          
         
       
     
     Protection of Guinea Pigs with  S. flexneri  2a Invaplex AR  using the Guinea Pig Keratoconjunctivitis Model 
     The guinea pig keratoconjunctivitis model is used to evaluate  Shigella  vaccines and is often used as the precursor to human trials. The model involves infection of the guinea pig eyes with  Shigella  which establish infection in the corneal epithelium. This outcome (severe keratoconjunctivitis) is a result of invasion of the corneal epitheilium by the shigellae and the subsequent inflammatory response by the host not unlike that observed in the human intestinal tract. 
     In this experiment guinea pigs were immunized three time intranasally with either  S. flexneri  2a InvaplexAR or native Invaplex. A saline control group was also utilized. Three weeks after the final immunization animals were challenged ocularly with  S. flexneri  2a. Both the InvaplexAR (90% protection, P&lt;0.001) and native Invaplex (100% protection, P&lt;0.001) provided solid protection (See Table 6) indicating that the Invaplex AR  is comparable to the native Invaplex. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Protection against  Shigella  infection in guinea pigs with  S. flexneri   
               
               
                 2a Invaplex AR  using the keratoconjunctivitis model. 
               
             
          
           
               
                   
                   
                   
                 % 
                   
               
               
                 Treatment 
                 # positive 1   
                 # Negative 
                 protection 2   
                 P value 3   
               
               
                   
               
             
          
           
               
                   S. flexneri  2a native 
                 0 
                 10 
                 100%  
                 &lt;0.001 
               
               
                 Invaplex 50, 25 
               
               
                 μg/dose 
               
               
                   S. flexneri  2a 
                 1 
                 9 
                 90% 
                 &lt;0.001 
               
               
                 Invaplex AR , 25 
               
               
                 μg/dose 
               
               
                 IpaB (25 μg) + IpaC 
                 4 
                 6 
                 40% 
                 0.01 
               
               
                 (25 μg) per dose 
               
               
                 IpaB (25 μg) + IpaC 
                 0 
                 10 
                 100%  
                 &lt;0.001 
               
               
                 (25 μg) + LPS (25 μg) 
               
               
                 per dose 
               
               
                 (0.9% Saline) 
                 10 
                 0 
                  0% 
                 — 
               
               
                   
               
               
                   1 Guinea pigs were challenged intraocularily with 1 × 10 8  cfu of  S. flexneri  2a. Eyes were evaluated on day 5 post-infection for keratoconjunctivitis as described by Hartman et al. [44] 
               
               
                   2 % Protection = [(%Disease Control  − % Disease Vaccinees )/% Disease Control ] × 100 
               
               
                   3 Fisher Exact Test 
               
             
          
         
       
     
       S. flexneri  2a Invaplex AR  Murine Adjuvanticity Study 
     Intranasal immunization with OVA alone or OVA combined with CT or pre-immune samples from immunized animals (day 0) did not induce detectable levels of serum IgG or IgA with specificity to  S. flexneri  2a Invaplex 50,  S. flexneri  2a Invaplex 24 ( FIG. 9 ),  S. flexneri  2a LPS ( FIG. 10 ), IpaB or IpaC ( FIG. 11 ). Immunization with OVA combined with either Invaplex AR  or native Invaplex induced similar Invaplex 50, Invaplex 24 and LPS-specific serum IgG and IgA endpoint titers ( FIGS. 9 and 10 . Endpoint titers against purified IpaB (p&lt;0.05) and IpaC (p≦0.001) were higher in mice immunized with OVA+Invaplex AR  as compared to mice immunized with OVA+native Invaplex ( FIG. 11 ). Comparable levels of OVA-specific serum IgG and IgA were induced in mice immunized with OVA combined with Invaplex AR , native Invaplex, or CT on day 42 and were significantly higher (p≦0.001) than the responses induced in mice after immunization with OVA alone ( FIG. 12 ). 
     Antigen-specific antibodies in lung washes were also assessed by ELISA to investigate the mucosal immune responses induced after immunization ( FIG. 13 ). Immunization with OVA combined with Invaplex AR  induced similar levels of LPS and Invaplex 24-specific IgG and IgA in lung washes as compared to responses after immunization with OVA combined with native Invaplex and higher than the levels induced after immunization with OVA alone, or OVA combined with CT. Similar levels of OVA-specific lung IgG and IgA were induced after immunization with OVA combined with Invaplex AR , native Invaplex, or CT which were significantly (p&lt;0.001) higher than those induced after immunization with OVA alone. Moderate levels of LPS and Invaplex-specific IgA in intestinal washes (data not shown) were detected in washes from mice immunized with OVA combined with Invaplex AR  or native Invaplex and were undetectable in mice immunized with OVA alone or OVA combined with CT. OVA-specific intestinal IgA responses were below levels of detection in all samples assayed. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Anti-IpaB and anti-IpaC serum (day 42) and mucosal (day 35) antibody levels after 
               
               
                 intranasal immunization of mice with  S. flexneri  2a Invaplex or Invaplex AR   
               
             
          
           
               
                   
                 Anti-IpaB 
                 Anti-IpaC 
               
             
          
           
               
                 Study 
                   
                 Serum 
                 Lung 
                 Intestinal 
                 Serum 
                 Lung 
                 Intestinal 
               
               
                 Name 
                 Treatment 
                 IgG a   
                 IgA 
                 IgA 
                 IgG 
                 IgA 
                 IgA 
               
               
                   
               
               
                   S. flexneri  2a 
                 Invaplex 
                 &gt;5760 
                 ND 
                 ND 
                 136 ± 117 
                 ND 
                 ND 
               
               
                 Invaplex AR   
                 (5 μg) 
                 (&gt;5760) 
                   
                   
                 (90–360) 
               
               
                 Mouse Study I 
                 Invaplex AR   
                 &gt;5760 
                 ND 
                 ND 
                 1440 ± 4593* 
                 ND 
                 ND 
               
               
                   
                 (2.5 μg) 
                 (&gt;5760) 
                 ND 
                 ND 
                 (90–11520) 
                 ND 
                 ND 
               
               
                   
                 Saline 
                 90 ± 0 
                   
                   
                 90 ± 90) 
               
               
                   
                   
                 (90 − 90  
                   
                   
                 (90 − 90  
               
               
                   S. flexneri  2a 
                 Invaplex 
                 TBD 
                 2 ± 0 
                 3 ± 1 
                 TBD 
                 2 ± 0 
                 2 ± 0 
               
               
                 Invaplex AR   
                 (5 μg) 
                   
                 (2 − 2) 
                 (2 − 4) 
                   
                 (2 − 2) 
                 (2 − 2) 
               
               
                 Mouse Study II 
                 Invaplex AR   
                 TBD 
                 54 ± 58* 
                 64 ± 54* 
                 TBD 
                 5 ± 15 
                 2 ± 0 
               
               
                   
                 (2.5 μg) 
                   
                 (8 − 128) 
                 (16 − 128) 
                   
                 (2 − 32) 
                 (2 − 2) 
               
               
                   
                 Saline 
                 TBD 
                 2 ± 0 
                 2 ± 0 
                 TBD 
                 2 ± 0 
                 2 ± 0 
               
               
                   
                   
                   
                 (2 − 2) 
                 (2 − 2) 
                   
                 (2 − 2) 
                 (2 − 2) 
               
               
                   S. flexneri  2a 
                 Invaplex + 
                 2507 ± 2366 
                 ND 
                 ND 
                 157 ± 148 
                 ND 
                 ND 
               
               
                 Invaplex AR   
                 OVA 
                 (90 − 90) 
                   
                   
                 (90 − 360) 
               
               
                 Adjuvanticity 
                 Invalpex AR  + 
                 &gt;5760* 
                 ND 
                 ND 
                 950 ± 2318* 
                 ND 
                 ND 
               
               
                 Study 
                 OVA 
                 (&gt;5760) 
                   
                   
                 (90 ± 5760) 
               
               
                   
                 OVA 
                 90 ± 0 
                 ND 
                 ND 
                 90 ± 0 
                 ND 
                 ND 
               
               
                   
                   
                 (90 − 90) 
                   
                   
                 (90 − 90) 
               
               
                   
               
               
                   a Serum responses in the  S. flexneri  2a Invaplex AR  Mouse Study I are bllod collected on day 42. Serum responses in the  S. flexneri  2a Invaplex AR  Adjuvanticity Study are from blood collected on day 35. 
               
               
                   b Geomean ± 1 standard deviation from the mean (n = 5 mice/group) (range of endpoint titers) 
               
               
                 *Significantly higher endpoint titers (one way ANOVA of log transformed endpoint titers; p ≦ 0.001) as compared to endpoint titers of animals immunized with native Invaplex. 
               
               
                 ND; not determined, 
               
               
                 TBD; to be determined 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Antigen-specific serum IgG and IgA endpoint titers on day 42 in guinea pigs 
               
               
                 intranasally immunized with native Invaplex, Invaplex AR , or mixtures of IpaB and IpaC. 
               
             
          
           
               
                   
                 ELISA Antigen 
               
             
          
           
               
                   
                   S. flexneri  2a Invaplex 50 
                   S. flexneri  2a LPS 
                 IpaB 
                 IpaC 
               
             
          
           
               
                 Treatment 
                 IgG 
                 IgA 
                 IgG 
                 IgA 
                 IgG 
                 IgG 
               
               
                   
               
               
                   S. flexneri  2a 
                 827 ± 1046 b   
                 475 ± 1148 
                 1091 ± 2873 
                 827 ± 1018 
                 1712 ± 2305 
                 90 ± 0 
               
               
                 Invaplex 
                 (180 − 2880) 
                 (180 − 2880) 
                 (180 − 5760) 
                 (360 − 2880) 
                 (720 −≧ 5760) 
                 (90 − 90) 
               
               
                 (lot 1307; 25 μg) 
               
               
                   S. flexneri  2a 
                 4073 ± 2160* 
                 856 ± 360 
                 2880 ± 2700 
                 827 ± 1046 
                 All &gt; 5760* 
                 All &gt; 5760* 
               
               
                 Invaplex AR   
                 (1440 −≧ 5760) 
                 (720 − 2880) 
                 (360 −≧ 5760) 
                 (720 − 2880) 
                 (&gt;5760) 
                 (&gt;5760) 
               
               
                 (lot KR-C5; 25 μg) 
               
               
                 IpaB + IpaC 
                 1211 ± 1034 
                 255 ± 104 
                 90 ± 0 
                 45 ± 0 
                 All &gt;5760* 
                 3629 ± 2494* 
               
               
                 (25 μg + 25 μg) 
                 (360 − 2880) 
                 (180 − 360) 
                 (90 − 90) 
                 (45 − 45) 
                 (&gt;5760) 
                 (1440 −≧ 5760) 
               
               
                 Saline 
                 90 ± 0 
                 45 ± 0 
                 90 ± 0 
                 45 ± 0 
                 90 ± 0 
                 90 ± 0 
               
               
                   
                 (90 − 90) 
                 (45 − 45) 
                 (90 − 90) 
                 (45 − 45) 
                 (90 − 90) 
                 (90 − 90) 
               
               
                   
               
               
                   a Serum responses in the  S. flexneri  2a GMP Stability and Invaplex AR  guinea pig study are from blood collected on day 42. 
               
             
          
         
       
     
     Method for Formation and Evaluation of Artificial Invaplex Complexed with an Antibiotic or other Therapeutic Molecule for Intracellular Delivery 
     Many therapeutic biochemicals, including antibiotics, are often ineffective against intracellular target because they are unable to cross mammalian cell membranes or because they require high extracellular concentrations to achieve therapeutic concentrations inside of mammalian cells. The artificial Invaplex provides a mechanism to create a complex of invasins, LPS and antibiotic by mixing the components during the assembly stage for creating artificial Invaplex. Once assembled, the native properties of Invaplex allow it to transport the complexed antibiotic or therapeutic molecule into mammalian cells. 
     Purified, soluble IpaB and IpaC, each in their respective final buffers are mixed together at an IpaC/IpaB molar ratio of 8. After the IpaB and IpaC are mixed, a solution of antibiotic, for example gentamicin at 5 to 100 μg/ml, is added to the mixture. Next the IpaB, IpaC and antibiotic solution is slowly added to dry LPS powder (ratio of LPS to total protein is 0.56). LPS from any  Shigella  species can be used; for the described experiments  S. flexneri  2a,  S. sonnei  or  S. dysenteriae  1 LPS was used. The mixture is incubated at 37° C. for approximately 2 hours with shaking. Afterwards the protein/LPS/antibiotic mixture is diluted to 20 mM Tris-HCl, 0.10 M NaCl and 1.2 M urea to reduce the NaCl concentration prior to ion-exchange chromatography. For final purification, the diluted IpaB/IpaC/LPS/antibiotic mixture is applied to an anion exchange column (HiTrap™ Q HP) with Buffer A consisting of 20 mM Tris-HCl, pH 9.0 and Buffer B consisting of 1 M NaCl, 20 mM Tris-HCl, pH 9.0, and a step gradient of 0% (5 column volumes) to 24% (10 column volumes) to 50% (6 column volumes) to 100% Buffer B (5 column volumes). One ml fractions were collected from a 1 ml HiTrap™ Q HP column at an elution flow rate of 1 ml/min. Fractions from each step were analyzed for the presence of IpaB, IpaC and LPS by spotblot. The fractions containing IpaB, IpaC and LPS are the artificial Invaplex fractions. 
     The effectiveness of the artificial Invaplex-antibiotic complex will be evaluated in its ability to kill intracellular microorganisms such as shigellae growing in tissue culture cells. The artificial Invaplex-antibiotic complex will be incubated with  Shigella -infected tissue culture cells. After 2 to 24 hours the number of intracellular  Shigella  will be determined by plating lysates of the treated cells (and untreated control cells) on trypticase soy agar plates to determine the level intracellular killing in the cells treated with the artificial Invaplex-antibiotic complex. 
     Further variations and modification of the foregoing will be apparent to those skilled in the art and are intended to be encompassed by the claims appended thereto. 
     REFERENCES 
     The references which follow are identified by number within the specification. The contents of each of these documents are expressly incorporated herein to the degree necessary to understand the invention.
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