RU2440136C2 - Synthetic invaplex - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to pharmaceutics. Elaborated is chemically standardised synthetic three-component complex Invaplex, including invasive proteins IpaB and IpaC and lipolysaccharide from Gram-negative bacteria. Sertotype-specific lipopolysaccharide component can include two or more lipopolysaccharides, selected from, at least, two different serotypes of Gram-negative bacteria. Elaborated is method of obtaining complex, which includes combination of IpaB and IpaC, with, at least, one lipopolysaccharide, bound with serotype of Gram-negative bacteria, stage A) being carried out as two separate mixing stages, including mixing of IpaB and IpaC with formation of complex IpaBTpaC and mixing complex IpaB:IpaC with, at least, one lipopolysaccharide (LPS) with formation of synthetic Invaplex and its isolation.

EFFECT: elaborated is chemically standardised synthetic three-component complex Invaplex for immunisation of subject, transportation of molecule through cell membrane, possessing improved properties due to stability of composition and biological activity.

34 cl, 2 ex, 8 tbl, 13 dwg

Description

FIELD OF THE INVENTION

The present invention provides a new method for preparing a synthetic Invaplex invasive complex comprising at least two invasive proteins and a lipopolysaccharide from invasive gram-negative bacteria. The synthetic invasive complex of the present invention can be used as vaccines, adjuvant for vaccines, biochemical or other substances and as a diagnostic tool.

State of the art

Shigellosis is the main cause of acute intestinal infection in humans, especially in developing countries, where it is estimated that more than 163 million infections occur annually, with 1 million deaths [1]. The most common Shigella species causing the disease are S. flexneri and S.sonnei [1]. The presence of an even lower number of cases (1.5 million cases / year) of shigellosis in industrialized countries [1] also shows that the adult population is not immunologically protected and susceptible. Even in areas relatively free of shigellosis, massive destruction from war or natural disasters, such as the 1999 earthquake in Kocaeli, Turkey, can quickly cause an outbreak of multi-species, multifocal dysentery [2]. Similarly, when deploying troops, relief workers or travelers from developed countries are in areas of the Shigella epidemic or in areas where local infrastructure is destroyed, then treatment for an outbreak of 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 disease is not as high as with S.flexneri or S.sonnei, epidemics caused by S.dysenteriae 1 occur every 10–12 years [4]. The periodic occurrence is a consequence of many factors, one of which, apparently, is associated with a decrease in immunity to S. dysenteriae 1. The most important is the recent discovery that antibiotic resistance of the fluoroquinolone series is observed with S. dysenteriae 1, isolated during recent outbreaks [5 , 6]. The likelihood of the next S.dysenteriae 1 epidemic is high, and if it is caused by strains with resistance to broad-spectrum antibiotics, including fluoroquinolones, the consequences can be severe.

Shigella species are some of the proven agents of biological terrorism. A very low infectious dose (for S.dysenteriae 1 it can be 10 bacteria) [7] and the simplicity of growing an organism in a simple bacteriological environment are suitable factors for considering Shigella spp as a powerful biological weapon. Efficiency as an agent of biological terrorism is confirmed by the case when hospital staff were deliberately infected with S.dysenteriae 2 by a disgruntled employee who had "painted" breasts on his body [8]. Other foodborne outbreaks of Shigella infection are well known, from cold food on planes to salads on cruise ships [9, 10], which demonstrates the ease with which shigellae can cause disease in a vulnerable population. Shigella infection protection is best done through environmental monitoring, which includes good sanitation and the provision of clean drinking water. Such infrastructure improvements are not difficult to cope with if shigellae is intentionally introduced into food or drinks. In addition, the ease of isolation of shigellae from nature [5] and the simplicity of the development of antibiotic resistance of S.dysenteriae 1 strains in the laboratory confirm that these agents are a possible major threat of terrorism. Although it may be difficult to completely stop the onset of an outbreak, limiting secondary distribution is important. Effective preventive routes are vaccines that are effective against shigellae, regardless of antibiotic resistance.

Pathogenesis of Shigella spp. lies in the body’s ability to introduce, intracellular replication and intracellular distribution in the epithelium of the large intestine. The initial introduction of shigellae occurs through specialized intestinal epithelial cells called M cells. Some highly conserved, virulent plasmid encoded proteins, called plasmid invasion antigens (IpaA, IpaB, IpaC and IpaD), are key players in the invasion process and are likely to be key to the effectiveness of Shigella infections. Upon contact or attachment to host cells, Shigella invasives are released [11] by type III secretion apparatus [12] and induce a phagocytosis process, which leads to the capture and absorption of bacteria by the host cell [13]. Active components include the IpaB: IpaC complex, which is distributed in the membrane of the host cell, forming a channel through which other Shigella proteins enter the host cell [14]. Recently, we isolated an invasive protein-LPS complex from intact, virulent Shigella cells, which serves as an effective vaccine and adjuvant [15-19]. The invasive Shigella complex (Invaplex) was obtained by extraction of the virulent shigella with water, followed by anion exchange chromatography and aqueous extraction. Two fractions, called Invaplex 24 and Invaplex 50, contained major antigens, including LPS, IpaB and IpaC. An invasive complex (Invaplex) was attached to the surfaces of cultured epithelial cells, while similar preparations from non-invasive Shigella did not provide this property. Immediately after attachment, Invaplex became localized within the host cell by actin-dependent endocytosis, apparently induced by Invaplex. It appears that IpaC and IpaB play a significant role in attachment and uptake, in which antibodies against IpaC or IpaB interfere with the attachment of Invaplex to the membranes of the host cell [20]. Antibodies to LPS do not alter the absorption process. As soon as the absorbed Invaplex moves through the early endosomes, late endosomes, then the Golgi apparatus is eventually free in the cytoplasm of the host cell. The ability to attach to the surface of a eukaryotic host cell and stimulate endocytosis indicates that Invaplex maintains an active, native virulent structure similar to that found for invasive Shigella.

Historically, successful Shigella vaccines reinforce the notion of the effective role of LPS in defense development. Many approaches are used for Shigella vaccines, including live attenuated vaccines [21, 22], inactivated vaccines [23, 24] and the 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 live attenuated vaccines use the native invasiveness of Shigella to deliver LPS and protein antigens to the mucosal immune system, probably through follicle-associated epithelium [14]. The residual pathogenicity of attenuated vaccine strains may limit this approach, although additional attenuation is achieved [28].

The ability to isolate a putative native surface structure such as Invaplex, which exhibits activities and immunogenicity similar to invasive shigella, has significant difficulties in creating and developing vaccines. First, an isolated native complex can enhance delivery to suitable entry gates (M cells), similar to that directed by live attenuated vaccine strains. Attachment of Invaplex to host cells (and possibly intestinal M cells or M-like cells in other mucous tissues) allows relatively low doses of this subcellular complex to be used for immunization due to their delivery efficiency. Similarly to live, attenuated vaccines, Invaplex contains all of the major Shigella antigens (including IpaB, IpaC and LPS) and has the potential to stimulate an immune response equivalent to that obtained during a 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 antigen. In addition, the conserved sequences and immunological cross-reactivity of Ipa proteins found in all Shigella species can provide the efficacy of a vaccine containing invasives (or other conserved antigens) against more than one Shigella species.

A vaccine from the Shigella invasive complex (Invaplex) prepared from Shigella flexneri contains the main antigens LPS, IpaB, and IpaC and is protective against homologous infection in models of keratoconjunctivitis in guinea pigs and lung infection in mice [17, 18]. After immunization, antibodies to LPS, IpaB, and IpaC are produced, which are similar to the specificity of antibodies that are observed after a natural infection in humans [29]. Additional studies have shown that Invaplex vaccine products of similar efficacy can be isolated from Shigella and EIEC species [15]. Although Invaplex 24 and 50 are composed 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 84 kDa (EF-G) and 72 kDa (DnaK) protein antigens [18]. The other proteins in Invaplex preparations are not immunogenic, as determined by Western blotting methods using serum collected from animals immunized with Invaplex. Invaplex 24 and Invaplex 50 can be isolated from wild Shigella species, although often the Invaplex 24 form will constantly contain higher amounts of IpaB, IpaC and LPS and some non-immunogenic proteins. With the Invaplex vaccine, high titers are often used against Invaplex immunization (Invaplex 24 or Invaplex 50) when used as an antigen for ELISA. It is believed that this reflects a combined response to Ira and LPS proteins and a number of co-formation epitopes preserved by the complex product of invasives when used as an antigen for ELISA.

As a result of identification and purification of the active part in the native Invaplex, a high molecular weight complex was identified that was isolated by gel filtration from preparations of the native Invaplex 24. This high molecular weight complex, referred to as “highly purified” or HP-Invaplex 24, consists mainly of IpaB, IpaC and LPS. HP-Invaplex 24 is immunogenic and protective at levels that are comparable to or greater than those found in native Invaplex. No other gel filtration fractions had immunogenicity and protective ability comparable to that of native Invaplex, and for this reason HP-Invaplex 24 is considered as the active part in the native Invaplex, responsible for its immunogenicity and effectiveness as a vaccine.

Virulent Shigella cause the disease by penetration, replication and spread in the epithelium of the colon through a complex series of cellular and molecular events controlled by a number of plasmid-coding virulent factors, among which are Ira proteins [49]. After penetration into intestinal mucosal intestinal epithelial cells, an inflammatory response occurs in the mucosa, characterized by an increase in pro-inflammatory cytokines, which leads to stimulation of neutrophils and macrophages / monocytes. As a result, a disease, shigellosis or bacterial dysentery, causes the development of moderate to severe diarrhea, fever and intestinal damage. Since human and primate vaccine efficacy testing involves infection with virulent shigella after immunization, the use of small experimental models for initial testing of vaccine candidates reduces the risk of volunteers and primates. Small experimental models, such as the guinea pig keratoconjunctivitis model or the pulmonary mouse model with experimental Shigella infection, are widely used to study the pathogenesis and preclinical evaluation of the vaccine [18, 44, 50], while the mouse model is often used in initial assessments and the guinea pig model is used for testing vaccines that are protective using the mouse model as an example.

A model of intranasal infection of mice with Shigella infection is suitable for evaluating Shigella vaccines [18, 31, 51, 52]. Pathogenesis and immunobiology were observed in the pulmonary model in parallel with observations in the colon; in this case, virulent Shigella strains penetrate, replicate and spread in the epithelium and subsequently cause an immune response, as well as a cytokine response [52]. After infection, the mice lose weight and eventually die, unless they develop protective immunity. The ability to measure the response of secretory antibodies, the cellular immune response and the cytokine response (mainly due to the availability of commercial reagents) makes the mouse model very attractive for studies of shigellosis immunobiology.

The guinea pig model is a suitable model that is used to study the virulence of both wild and attenuated Shigella strains and to evaluate the effectiveness of potential vaccine candidates [18, 44, 45, 53-56]. Several immunization routes can be used for immunization depending on the vaccine: oral, intranasal, ocular and parenteral immunizations, all of which are used to protect against ocular infection. The immunogenicity and effectiveness of the Shigella vaccine in the guinea pig model of keratoconjunctivitis is currently used as a springboard in the first phase of clinical trials.

There is a need for a chemically defined, synthetic part of Invaplex that is similar or superior in biological activity to native Invaplex. There is also a need for the Invaplex manufacturing process, which can be quickly put into serial production and result in a more specific product, depending on the ratios of the parts of the individual components that are used. There is also a need for a synthetic Invaplex, which is provided for specific applications or has special functions. There is also a need for an Invaplex vaccine that can be prepared quickly from its constituents, which can accumulate in anticipation of future needs for the use of the vaccine.

SUMMARY OF THE INVENTION

The invention relates to a synthetic Invaplex and a method for its preparation. Synthetic Invaplex (Invaplex _AR ) is similar to HP Invaplex 24. HP Invaplex 24 contains IpaB, IpaC and lipopolysaccharide. Synthetic Invaplex acts as an Invaplex obtained from a native source, although it may be more active. Synthetic Invaplex contains certain components: a complex containing the invasive proteins IpaB and IpaC, which additionally includes a serotype-specific lipopolysaccharide component from gram-negative bacteria. Lipopolysaccharide is an immunogen, as are IpaB and IpaC. Invaplex _AR can function as a mucosal adjuvant and elicit both serum and mucosal antigen-specific immune responses as well as cell-mediated immune responses. It is also possible to use other invasives from other types of bacteria, including SipB, SipC or viral (reoviral) proteins associated with endocytosis or tissue tropism.

Serotype-linked lipopolysaccharides are derived from gram-negative bacteria such as Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Shigella boydii, enteroinvasive E. coli, Yersinia or Salmonella. More preferred species include S. flexneri, S.sonnei, S.boydii, S.dysenteriae, or enteroinvasive E. coli.

To prepare a synthetic Invaplex, for example Invaplex _AR , either highly purified (hp-) or recombinant (r-) invasive IpaB and IpaC proteins are used, and the purified lipopolysaccharide is used as the starting material. The IpaB and IpaC proteins are mixed to form the IpaB: IpaC complex. This complex is mixed with at least one lipopolysaccharide associated with a serotype of gram-negative bacteria to form a synthetic Invaplex. Synthetic Invaplex isolated from the mixture. Usually, a synthetic Invaplex is removed based on its charge. Other cleaning techniques are also used. The order of addition is subject to change. The lipopolysaccharide can complex with any invasive protein and then with another invasive protein to form a synthetic Invaplex.

Typically, the amount of IpaC present relative to the presence of IpaB falls within the range of ratios from 0.08: 1 to 80: 1, preferably from 0.8: 1 to 20: 1, more preferably at least 8: 1. Most preferred for the amount of IpaC in the first mixing step is its presence of at least ten times more with respect to IpaB. In a second mixing step, the lipopolysaccharide and protein (IpaB and IpaC) are present in a ratio of 0.01: 1 to 10: 1, preferably at least 1: 2. In the second mixing step, it is possible to include two or more types of lipopolysaccharides, which lead to the production of a synthetic Invaplex, which is suitable for use in multivalent immune compositions, in the form of vaccines. It is also possible, in addition to the lipopolysaccharide, to include a detecting label, antibiotic, drug or biomolecule containing an enzyme, protein, polysaccharide, RNA or DNA, or derivatives thereof, one of which is able to combine with the cell. This helps to increase the possibilities of therapeutic and analytical / diagnostic applications.

Synthetic Invaplex inventions include those in which IpaC and IpaB are present in a ratio selected from 0.08: 1 to 80: 1, preferably from 0.8: 1 to 20: 1, more preferably about 8: 1, and a lipopolysaccharide (LPS ) is present in a ratio selected from the range from 0.01: 1 to 10: 1, preferably from 0.5: 1 to 5: 1, more preferably about 0.5: 1 relative to the total protein content (IpaB and IpaC). Preferred synthetic Invaplex include Invaplex _AR S.flexneri, Invaplex _AR S.dysenteriae or Invaplex _AR S.sonnei.

The synthetic Invaplex of the invention can be formulated to formulate compositions suitable for use as immunogenic compositions or vaccines. Invaplex can be used as an immunogen and / or as an adjuvant. The compositions will include, in addition to the synthetic Invaplex, at least a pharmaceutically acceptable carrier. Invaplex is present in an amount effective for the desired action and, at least in the first place, is determined empirically. This amount is assumed to be similar to that of the native Invaplex, for example Invaplex 24, used for a similar purpose.

In addition, synthetic invasive complexes facilitate the transport of molecules and related materials across the cell membrane. These molecules may be located in close proximity to the cell of interest and the synthetic Invaplex, or may be present in and / or on the synthetic Invaplex. Molecules can be detecting tags, antibiotics, drugs or biomolecules containing proteins, enzymes, RNA or DNA, lipopolysaccharides, polysaccharides and the like.

Synthetic Invaplex can be used in methods that use native Invaplex, for example, immunization methods, methods for improving the transport of biomolecules, for example DNA, RNA, proteins, through cell membranes, etc. Synthetic Invaplex can be created as multivalent or have a high activity relative to the native Invaplex. This should increase the efficiency of the existing method and its applicability. Invaplex _AR , made from recombinant IpaB, IpaC and either LPS S.sonnei or LPS S.flexneri 2a, elicited immune responses that are comparable to or higher than the immune response elicited by the native Invaplex. The IpaB and IpaC responses to Immunization Invaplex _AR were consistently higher than those that were caused after immunization with native Invaplex. Immunization Invaplex _AR provides comparable or higher levels of protection against infection on models of mice and guinea pigs compared with immunization with native Invaplex. Immunizations Invaplex _AR induces cellular immunity greater than the Shigella-specific response caused by native Invaplex. See, for example, tables 7 and 8.

Brief Description of the Drawings

Figure 1 shows an analysis by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) of purified IpaB, IpaC and LPS used to recruit Invaplex _AR ;

Figure 2 shows the cleaning of Invaplex _AR S.flexneri 2a (panel A), S.sonnei (panel B) and S.dysenteriae 1 (panel C);

Figure 3 shows the SDS-PAGE (Coomassie Blue, Western Blots, and Silver stains) of the components used to prepare Invaplex _AR for S.dysenteriae 1 and the final Invaplex _AR product;

figure 4 shows the definition of the internalized native and synthetic Invaplex S.dysenteriae 1 in mammalian cells;

5 shows titers of Invaplex 50, Invaplex 24 and LPS-specific serum IgG and IgA at the endpoints on day 42 after intranasal immunization with native Invaplex 24 S.flexneri 2a, Invaplex _AR , purified IpaB, IpaC or LPS S.flexneri 2a;

Fig.6 shows titers of anti-IpaB and IpaC serum IgG at the end points on day 24 after intranasal immunization with Invaplex 24 S. flexneri 2a, Invaplex _AR , purified IpaB, IpaC or LPS S.flexneri 2a;

Fig.7 shows the level of antigen-specific IgA in intestinal swabs collected on day 35 from mice immunized intranasally with native Invaplex S.flexneri 2a or Invaplex _AR ;

Fig. 8 shows the level of antigen-specific IgA in lung swabs collected on day 35 from mice immunized intranasally with native Invaplex S.flexneri 2a or Invaplex _AR ;

Fig.9 shows titers of Invaplex-specific serum IgG and IgA at the endpoint in mice after intranasal immunization with only OVA or OVA in combination with Invaplex _AR , native Invaplex or cholera toxin;

figure 10 shows the titers of LPS-specific serum IgG and IgA S.flexneri 2a at the endpoint in mice immunized intranasally with OVA, OVA only in combination with Invaplex _AR , native Invaplex or cholera toxin;

11 shows titers of invasive specific serum IgG and Shigella IgA at the endpoint in mice immunized intranasally with OVA or OVA only in combination with Invaplex _AR , native Invaplex or cholera toxin;

12 shows titers of ovalbumin-specific serum IgG and IgA at the endpoint in mice immunized intranasally with only OVA or OVA in combination with Invaplex _AR , native Invaplex or cholera toxin;

Fig. 13 shows LPS, Invaplex 24 and ovalbumin-specific pulmonary IgG and IgA S.flexneri 2a titers at the endpoint in mice immunized intranasally with OVA or OVA only in combination with Invaplex _AR , native Invaplex or cholera toxin.

DETAILED DESCRIPTION OF THE INVENTION

Biochemistry Synthetic Invaplex

Preparation and purification of recombinant IpaB and IpaC and native LPS S.flexneri, S.sonnei and S.dysenteriae 1

Purification of Ipa Proteins

Ipa proteins are highly stable in all Shigella spp [32, 33]. Recombinant E. coli expressing either IpaB or IpaC has been previously described. There are two approaches to the purification of recombinant Ipa proteins. Histidine-labeled (HisTag) recombinant IpaC was purified by affinity chromatography using nickel columns. The effect of histidine residues on Ipa protein biological and immunological functions or the ability of proteins to form Invaplex has not been studied, but HisTag-IpaC is believed to retain biological activity [30]. An alternative purification procedure is used to obtain recombinant IpaB. This method takes advantage of the native affinity of IpaB for the chaperone IpgC [34, 35]. By coexpressing IpaB together with HisTag chaperone IpgC in the same recombinant organism, it is possible to purify the IraB / chaperone complex. After purification, the HisTag-IpaC / IpaB protein complex is denatured, resulting in the formation of the monomeric IpaB and HisTag-IpgC protein, which allows histidine-labeled chaperone to be selectively removed on a nickel column while the free Ipa protein passes through the column.

Cleaning HisTag-IpaC

Recombinant IpaC was expressed in E. coli using an inducible IPTG expression system (pET plasmid). The ipaC gene was cloned into the pET15b plasmid (Novagen) and expressed in the E. coli BL21 (DE3) pLysS vector [30]. The recombinant IpaC protein contained several histidine residues at the N-terminus, which allows purification on a nickel column [30]. The expressed IpaC protein is located in inclusion bodies and requires urea solubilization. Urea removal often leads to loss of solubility if the concentration of IpaC is too high (> 1 mg / ml). Thus, IpaC eluted from the nickel column was maintained in urea while maintaining solubility. Using a small scale culture (3 L), it is possible to obtain approximately 75 mg of purified IpaC with a yield of approximately 8 mg / L. The product is more than 90% pure and active with anti-IpaC mAb 2G2 [32] (FIG. 1). The cleaned block is soluble and can be stored frozen. An example of purified IpaC is shown in FIG.

IpaB Cleaning

Recombinant IpaB was expressed in E. coli BL21 (DE3). The ipgC gene was cloned into pET15b; The ipaB gene was cloned separately into pACYC, a duet vector. After IPTG administration, the HisTag IpgC / IpaB complex was dissolved and purified on a nickel affinity column. The HisTag-IpgC / IpaB complex was eluted in the presence of EDTA. For re-purification on a nickel affinity column, EDTA was removed and the non-ionic detergent OPOE (N-octyl-oligo-hydroxyethylene) was added to a final concentration of 1% v / v. OPOE destroys the HisTag-IpgC / IpaB complex, thereby allowing the free HisTag-IpgC protein to bind to a nickel (Ni-Sepharose) column, and to pass through the column free of the IpaB tag. Free volume fractions were examined for the presence of IpaB using anti-IpaB mAb 2F1. Positive fractions were analyzed by SDS-PAGE (stained) to determine the presence of contaminated HisTag-IpgC. In case of contamination, the material is again treated with OPOE and re-applied to a nickel column to remove residual HisTag-IpgC. The final soluble product of IpaB is practically not contaminated with HisTag-IpgC, approximately 90% pure and interacts with anti-IpaB mAb 2F1 [32] (Fig. 1). The yield of IpaB per liter of starting culture was approximately 3.5 mg / L.

Cleaning LPS S.flexneri 2a, S.sonnei or S.dysenteriae 1

LPS S.flexneri 2a, S.sonnei and S.dysenteriae 1 was obtained according to the Westfall procedure [36], which involves the extraction of shigella with hot phenol / water. Virulent or attenuated Shigella strains can be used as the source of LPS, as the smooth LPS phenotype is expressed. In the above experiments, wild types S.flexneri 2a (strain 2457T) and S.sonnei (Mosely) were used. For S.dysenteriae 1, a weakened strain of WRSdI was used to reduce the risk of infection in laboratory personnel. WRSdI is virG, a stx knockout previously obtained at WRAIR [37].

Extraction, purification and characterization of LPS

LPS was extracted using the Westfall method [36]. Briefly, bacterial cell pellets are suspended in hot (68 ° C) distilled water (5 ml of water per gram of pellet). The same amount of phenol is added, heated to 68 ° C, and the solution is thoroughly shaken for 15 minutes. The tubes are then cooled to approximately 10 ° C ± 5 ° C. Samples are centrifuged, the aqueous phase is removed and stored at 4 ° C. Cell mass extraction is carried out twice and all aqueous phases are collected. The aqueous phase is dialyzed against distilled water for two days and then centrifuged (8000 × g, 30 min) to remove extraneous cell breakdown products. This supernatant was ultra-centrifuged (90,000 x g) for 2 hours and the pellet was retained. The precipitate is washed with sterile distilled water, resuspended in sterile distilled water at 4 ° C overnight, combined and lyophilized. The final lyophilized product is weighed and then a small portion (<10 mg) is taken, dissolved in 1 ml of toxin-free water, and a biochemical analysis is performed (see below).

Determination of purified LPS endotoxin is performed using a chromogenic horseshoe crab amoebocyte lysate (LAL). E. coli endotoxin serves as a control reagent for this assay. All results were presented in the international endotoxin unit (EU). Purified LPS was also analyzed by SDS-PAGE with silver staining to determine the presence of a typical multiband smooth LPS profile. Figure 1 shows a silver stained gel of purified LPS S.dysenteriae 1, revealing the typical multiband character of smooth LPS. The final LPS product has a trace protein content (<5% determined by Bradford analysis) and DNA (<5% determined by Hoechst stain) and reacts with serotype-specific antibodies to LPS (anti-S.dysenteriae 1 LPS mAb MAB753 (Chemicon International) for LPS S.dysenteriae 1; mAb 2E8 for S.flexneri 2a LPS; MAB755 (Chemicon) for LPS S.sonnei), determined by Western blotting or ELISA.

Quantification of IpaB and IpaC Content in Invaplex _AR Using Immunological Assay

The amount of IpaB and IpaC in Invaplex (synthetic or native) was determined using a modified ELISA procedure. The ELISA used purified recombinant IpaB or IpaC proteins to obtain standard graphs for determining the amount of antigens in Invaplex preparations. Immulon IB ELISA plates (ThermoLab Systems) were sensitized overnight at 4 ° C or 50 μl of recombinant IpaB, recombinant IpaC or Invaplex _AR . The antigen was titrated (thrice) using 2-fold serial dilutions in a carbonate sensitizing buffer (0.2 M carbonate, pH 9.8) at initial concentrations of 125 ng / ml (IpaB), 200 ng / ml (IpaC) and 10 μg / ml (Invaplex). After washing and blocking with casein, affinity-purified monoclonal antibodies specific for IpaB (2F1) or IpaC (2G2) [32] were incubated with antigen sensitized plates for 2 hours. After washing, an antigen-specific antibody was detected using anti-mouse immunoglobulin G (IgG) conjugated to alkaline phosphatase (Kirkegaard & Perry). Para-nitrophenyl phosphate was used as the substrate and absorbance was measured at a wavelength of 405 nm (OD405) using an ELISA plate reader (Molecular Devices, Menlo Park, CA) 60 minutes after incubation with the substrate. Using the program Softmax Pro 4.5 (Molecular Devices), standard curves were plotted by applying OD405 against concentration (ng / ml). The concentration of unknown samples was then interpolated from the standard curve.

Invaplex _AR was designed to have IpaB and IpaC concentrations and an IpaC / IpaB ratio similar to Invaplex 24 HP. The ratio of IpaC to IpaB was determined for Invaplex _AR and compared with HP-Invaplex 24, the purest form of Invaplex. The molar ratio of IpaC / IpaB in the HP-Invaplex was approximately 8. This was determined by densitometry of SDS-PAGE gels and antibody determination assays for IpaB and IpaC. In addition, the LPS content was assumed to correspond to the relative weight (approximately 0.5-0.6 mg LPS for every 1 mg protein) that was found for HP-Invaplex.

Invaplex LPS Measurements

Invaplex LPS was measured by determining the amount of 2-keto-3-deoxyoctonate (KDO) in each preparation [43] or by using the limulus amoebocytic lysate assay (Cambrex Inc.).

The method of forming a synthetic Invaplex. Cooking Invaplex _AR for S.flexneri 2a, S.sonnet and S.dysenteriae 1

The purified components were mixed in a ratio similar to the highly purified native Invaplex to form a synthetic Invaplex. HP-Invaplex S.flexneri 2a analysis showed 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 criterion for recovering Invaplex from purified IpaB, IpaC, and LPS, a series of experiments were performed to create Invaplex _AR . Synthetic Invaplex was purified immediately after formation by flash resolution chromatography (FPLC).

Purified, soluble IpaB and IpaC, each in their respective final buffer, were mixed with each other in a molar ratio of IpaC / IpaB 8. After mixing IpaB and IpaC, the solution was slowly added to the dry LPS powder (LPS to total protein ratio was 0.56). LPS from any Shigella species can be used; for the experiments described, LPS S.flexneri 2a, S.sonnei or S.dysenteriae 1 was used. The mixture was incubated at 37 ° C for approximately 2 hours with shaking. The protein / LPS mixture was then diluted with 20 mM Tris-HCl, 0.10 M NaCl and 1.2 M urea to reduce the concentration of NaCl before performing ion exchange chromatography. The diluted mixture was then purified on a HiTrap (TM) Q HP anion exchange column.

Assembly experiments: formation of Invaplex _AR S.flexneri

Preliminary assembly experiments compensated for the insolubility of IpaC by storing both IpaC and IpaB proteins in a buffer containing 4 4M urea. Once the IpaC / IpaB mixture was added to the LPS, solubility was not more problematic and allowed the urea to be removed. The purified typical IpaC itself precipitates upon dilution. In preliminary experiments, the molar ratio of IpaC / IpaB 8 was maintained by adding 8 μM HisTaglpaC 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. Proteins were mixed in a glass tube and incubated at 37 ° C without shaking for 15 minutes. Dry LPS (230 μg) in a separate glass tube was also incubated at 37 ° C without stirring for 15 minutes. After 15 minutes of incubation, the IpaB + IpaC mixture was used to solubilize the preheated LPS by slowly adding the protein mixture over the wall of the tube, followed by vigorous stirring. No noticeable lump formation and precipitation were observed. The IpaB / IpaC / LPS mixture was then incubated at 37 ° C with shaking (200 rpm) for 2 hours. For ion exchange chromatography (IEC), the mixture was diluted five times with pre-heated 20 mM Tris buffer, pH 9.0, to reduce the salt concentration. Precipitation of the mixture upon cooling to room temperature was not observed. For final purification, a diluted mixture of IpaB / IpaC / LPS was applied to an anion exchange column (HiTrap (TM) Q HP), buffer A containing 20 mM Tris-HCl, pH 9.0, and buffer B containing 1 M NaCl were used for chromatography. 20 mM Tris-HCl, pH 9.0, with a stepwise gradient elution from 0% (5 column volumes) to 24% (10 column volumes) to 50% (6 column volumes) to 100% Buffer B (5 column volumes). 1 ml fractions were collected from a 1 ml HiTrap (TM) Q HP column at an elution rate of 1 ml / min. Fractions were analyzed for the presence of IpaB, IpaC and LPS using spot blotting. Invaplex _AR S.flexneri eluted at 50% B (FIG. 2) contained the highest amount of all three components (IpaB, IpaC and LPS) as determined by western blotting and silver stained gels (FIG. 2A). The IpaB / IpaC / LPS complex was eluted between the 1 MDa and 669 kDa standards, i.e., it had the same size range as the HP-Invaplex.

Large-scale production of synthetic Invaplex S.dysenteriae 1

The above experiment was repeated, with only a ten-fold increase in the number of reagents to increase the yield of Invaplex _AR . In addition, LPS S.dysenteriae 1 was used instead of S.flexneri LPS. Also, 3.28 mg of IpaC in 20 mM Tris-HCl, 0.5 M NaCl, 6 M Urea, pH 7.9, were mixed with 0.62 mg of IpaB in 20 mM Tris-HCl, 0.5 M NaCl, pH 7.9, in a total volume of 5 ml. The protein mixture and the LPS hotel tube (2.1 mg) were preheated to 37 ° C. A mixture of IpaB + IpaC was added to the LPS tube and mixed thoroughly to dissolve the LPS. No precipitation was observed. The reaction mixture was incubated at 37 ° C with shaking (200 rpm) for 2 hours. Then, pre-heated IEC A Buffer A (20 mM Tris-HCl, pH 9.0) was added to dilute the salt 1: 5, giving a final volume of 25 ml. After cooling to room temperature, the diluted reaction mixture was placed on a 1 ml HiTrap (TM) Q HP IEC column and was eluted with a step gradient for Invaplex purification (see above). Invaplex _AR S.dysenteriae 1 was eluted in 50% Buffer B. It contained IpaB, IpaC and LPS S.dysenteriae 1 (FIG. 2C). Peak analysis of 0.24 M and 1.0 M NaCl did not determine significant amounts of IpaB, IpaC or LPS. The yield of Invaplex _AR obtained was approximately 10-20% of the total amount of protein used to produce the complex.

Similar assembly experiments were conducted for S.sonnei, resulting in an Invaplex _AR S.sonnei (see FIG. 2B).

Large-scale production of Invaplex _AR Shigella flexneri 2a

A fifteen-fold increase in reagent content was used to produce more Invaplex _AR for special studies. Using a molar ratio of 8 IpaC / 1 IpaB, 16.4 mg IpaC (in 20 mM Tris-HCl, 0.5 M NaCl, 6 M Urea, pH 7.9) were mixed with 3.1 mg IpaB (in 20 mM Tris- HCl, 0.5 M NaCl, pH 7.9) in a total volume of 20 ml. The protein mixture and a separate tube with Shigella flexneri 2a LPS (11.4 mg) were pre-heated to 37 ° C. A mixture of IpaB + IpaC was added to the LPS tube and mixed thoroughly to dissolve the LPS. The reaction mixture was incubated at 37 ° C with shaking (200 rpm) for 2 hours. Then, pre-heated IEC A Buffer A (20 mM Tris-HCl, pH 9.0) was added to dilute the salt 1: 5, giving a final volume of 100 ml. After cooling to room temperature, the diluted reaction mixture was loaded onto a 5 ml HiTrap (TM) Q HP column for IEC and was eluted with a step gradient for Invaplex purification (see above). Invaplex _AR S.flexneri 2a was eluted in 50% Buffer B. It contained IpaB, IpaC and LPS S.flexneri 2a. Peak analysis of 0.24 M and 1.0 M NaCl did not determine significant amounts of IpaB, IpaC or LPS. The yield of Invaplex _AR obtained was approximately 9% of the total amount of protein used to produce the complex.

Analysis of Invaplex _AR preparations by electrophoresis and Western blotting for IpaB and IpaC

Total proteins of Invaplex _AR preparations were determined using SDS-PAGE stained gels. Western blots were used to determine the presence, size, and relative concentrations of IpaB and IpaC. Silver stained gels were used to evaluate the LPS profile [18]. Monoclonal antibodies specific for either the IpaB protein or IpaC were used to analyze nitrocellulose blots [32]. After incubation with protein A conjugated with alkaline phosphatase, the blots were treated with ASMX phosphate (Sigma) and Fast red TR dye (Sigma). The video images of the blots were analyzed on a Macintosh computer using a CCD video camera (Cohu) and NIH Image software (version 1.62). Densitometry of digital images was carried out using the analytical program GelPro Image (version 2.0.10, Media Cybernetics, Inc.). The profile of the gel containing the total protein, and the contents of IpaB and IpaC should be the same as those for HP-Invaplex 24 or HP-Invaplex 50.

Gel filtration

Gel filtration was used to determine if the Invaplex _AR components detected by ion exchange chromatography really form a complex. This is the same method used to produce HP-Invaplex. Invaplex _AR (approximately 3 mg) was applied to a Superose 6 HR 10/30 column (Amersham Pharmacia) calibrated with blue dextran 200 (2 MDa), thyroglobulin (669 kDa) and ovalbumin (43 kDa). Fractions (0.5 ml, at a flow rate of 0.25 ml / min) were collected and analyzed by immuno-spot blotting for IpaB, IpaC and LPS. All three Invaplex _AR components (IpaB, IpaC, and LPS) eluted in the fraction between 2 MDa and 669 kDa, demonstrating that they were bound together, as they eluted with a size much larger than the individual components (43 kDa for IpaC; 62 kDa for IpaB). The SEC Invaplex _AR size is similar to the size at which HP-Invaplex was eluted.

Assessment of Invaplex _AR S.dysenteriae 1 in tissue absorption model

Invaplex attaches to and stimulates endocytosis in cultured mammalian cells. This property depends on the presence of IpaB and IpaC invasives and the use of host cell actin during the endocytosis process [38]. Invaplex Absorption Analysis is an in vitro model used to measure the functional activity of Invaplex preparations. The analysis was modeled after analysis of Shigella plaque formation [39] and Shigella absorption analysis [40, 41]. The analysis included incubation of Invaplex _AR with BHK-21 fibroblasts (60-70% adhesion) on a glass slide. After incubation for 5, 15, 30 or 60 minutes, the cells were washed three times with PBS and fixed with formalin. The permeability of the membranes of fixed PBS cells supplemented with 0.1% Triton X-100 and 0.1% BSA (wash buffer) was impaired and examined using polyclonal murine serum antibodies specific for LPS, IpaB and IpaC. Bound polyclonal murine antibodies were detected using GAM-IgG-Oregon Green 488 (Molecular Probes), followed by contrast staining with DAPI to identify the nucleus. Invaplex-treated cells were examined under a fluorescence microscope with digital imaging. Both native and synthetic Invaplex S.dysenteriae 1 were absorbed by BHK cells with the same type of stained cytoplasm for both preparations, confirming that Invaplex _AR retains the ability to stimulate uptake by mammalian cells (Figure 4). This shows that combining the recombinant IpaB and IpaC with S. dysenteriae 1 LPS into a complex isolated by ion exchange FPLC results in a synthetic product that retains the same biological activity as the native complex.

Immunogenicity, adjuvance and protective efficacy of vaccines containing native and synthetic Invaplex

Models of mouse lung infection [31] and guinea pig keratoconjunctivitis [44] were used to determine whether intranasal immunization with Invaplex _AR S.flexneri 2a stimulates a protective immune response that is effective against infection with homologous S.flexneri 2a. Separate studies in mice were used to determine if immunization with Invaplex _AR S.sonnei could protect against infection with homologous S.sonnei and against infection with heterologous S.flexneri 2a. Additional studies were performed to evaluate the adjuvant activity of Invaplex _AR using ovalbumin (OVA) as an immunogen. In adjuvant experiments, the immune responses elicited after OVA immunization alone were compared with the immune responses elicited by OVA in combination with or with native Invaplex, Invaplex _AR, or with the cholera toxin of a potent mucosal adjuvant established. Guinea pig and mouse experiments were performed in WRAIR using the IACUC protocol.

Mouse experiments

Four separate animal experiments were performed to evaluate the immunogenicity, adjuvance, and protective efficacy of the synthetic Invaplex. Traditional methods were used in each experiment and described in the following paragraphs. Methods and materials specific to each study are indicated in separate sections.

Immunization and infection

Mice were immunized on days 0, 14 and 28. A total volume of antigen of 25 μl was injected in 5-6 small drops, which were instilled into the external nostrils using a micropipette. Blood was taken from the tail, taken from mice on days 0, 28 and 42 and 2 weeks after infection (day 63). On day 35 or 42, the mice were euthanized, followed by immediate collection of mucosal swabs, and the spleen was removed to analyze proliferation and cytokine formation in vitro of Invaplex _AR S.flexneri 2a. Three weeks after the final immunization, mice (15 per group) were infected with an intranasally lethal dose (1.6 × 10 ⁷ cfu) S.flexneri 2a (2457T) or S.sonnei (Mosely, 8 × 10 ⁶ cfu), as described for the model infection of the lungs of mice [34]. Before intranasal immunization or infection, mice were anesthetized with a mixture of ketamine hydrochloride (40 mg / kg) and xylazine (12 mg / kg). Infected mice were monitored daily for two weeks to determine weight loss, tousled hair and drowsiness, as well as death used as an endpoint. Surviving mice bled 14 days after infection (day 63).

Study of the immunogenicity and protection of Invaplex _AR S.flexneri 2a

Separate groups of Balb / c (10-15 mice / group) female mice were immunized intranasally on days 0, 14 and 28 either 5 ug of native Invaplex S.flexneri 2a (series JWJX) or 2.5 ug synthetic Invaplex _AR S.flexneri 2a (KF-D3D4 Series). Control animals were animals inoculated with saline. Blood was collected on day 0, 28, 42 and 63. Animals were infected intranasally on day 49.

Study of the immunogenicity and protection of Invaplex _AR S.flexneri 2a II

Separate groups of Balb / c (15-20 mice / group) female mice were immunized intranasally on days 0, 14 and 28 either 5 ug of native Invaplex S.flexneri 2a (series JWJX) or 2.5 ug synthetic Invaplex _AR S.flexneri 2a (KR-C5 series). Control animals were animals inoculated with saline. Blood was collected on day 0, 28, 42 and 63. Lavage from the lungs and intestines was collected on day 42 in 4 animals from the group. The remaining animals were intranasally infected on day 49.

Invaplex _AR S.flexneri 2a adjuvant study

The ability of Invaplex _{AR to} function as a mucosal adjuvant was determined in mice using ovalbumin (OVA) as a model of protein antigen. Balb / cByJ mice (5 mice / group) were intranasally immunized on day 0, 14 and 28 with only OVA (5 μg) or OVA (5 μg) in combination with either Invaplex _AR S.flexneri 2a (KF-D3D4 series; 2, 5 μg), native Invaplex S.flexneri 2a (JWJX Series; 5 μg) or with cholera toxin (CT; 5 μg). Blood was collected on day 0, 28 and 42. Control animals were inoculated with saline. Lavage from the lungs and intestines were collected on day 42. Endpoint serum 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 swabs from the mucous membranes were also evaluated.

Immunogenicity and protection study InvapIex _AR S.sonnet

The immunogenicity and protective efficacy of Invaplex _AR S.sonnei was determined in mice using homologous (S.sonnei 53G) and heterologous (S.flexneri 2a 2457T) infections. Balb / cByJ mice (10-15 mice / group) were intranasally immunized on days 0, 14 and 28 of Invaplex _AR S.sonnei (KJ-D3D6 series; 2.5 μg), native Invaplex S.sonnei (JOJP series; 5 μg) or native Invaplex S.flexneri 2a (JWJX Series; 5 mcg). Control animals were inoculated with saline. Blood was collected on day 0, 28, and 42.

Guinea pig experiments

Guinea pigs (Hartley species, 6-10 per group) were immunized intranasally with vaccines containing a synthetic or native Invaplex (25 μg / dose). A dose volume (100 μl) was distributed evenly between each nostril. Saline was used to immunize control animals. Guinea pigs were immunized on days 0, 14, and 28, and blood from the lateral ear vein was bleeding on days 0, 28, 42, and within 14 days after infection. Before intranasal immunization, guinea pigs were anesthetized with a mixture of ketamine (40 mg / mg) and xylazine (4 mg / kg). Three weeks after the third immunization, guinea pigs were intraocularly infected with S. flexneri 2a (strain 2457T) and they were monitored daily for 5 days to assess the degree of inflammation and keratoconjunctivitis, as previously described [44].

ELISA

Antigen-specific IgA and IgG antibodies in serum and mucosal secretions, including pulmonary and intestinal lavages and feces from immunized and / or infected mice and guinea pigs [18, 29, 46], were determined using ELISA. Immunlon TJB 96-well ELISA plates (ThermoLab Systems) were sensitized with antigens, including purified LPS S.flexneri 2a, LPS S.sonnei, water-extracted Shigella antigens, purified IpaB and IpaC proteins and native Invaplex S.flexneri 2a and OVA for nights at 4 ° C. The plates were blocked with 2% casein (in Tris-physiological saline buffer, pH 7.5) and a series of two dilutions of serum and mucosal washings were incubated with antigen sensitized plates for 4 hours at 25 ° C. After washing with PBS containing 0.05% Tween 20, antigen-specific antibody binding was determined using anti-mouse IgG or anti-mouse IgA antibodies conjugated with alkaline phosphatase (Kirkegaard & Perry). Alkaline phosphatase activity was determined using p-nitrophenyl phosphate as a substrate (1 mg / ml). After 30 minutes of incubation with the substrate, the optical density (OD405) was measured using an ELISA plate reader (Molecular Devices, Menlo Park, CA). For each animal, titers at the end point were determined and used to calculate geometric mean titers for each group at each time point. Typically, animals intranasally immunized with Invaplex responded with a 4–8-fold increase in serum (IgG and IgA) and mucosal (lungs and intestines) IgA thirates to Shigella antigens compared to preimmune samples or control animals immunized with saline.

Total IgA Analysis

The concentration of total IgA in mucosal samples was determined using enzyme-linked immunosorbent assay. Concentrations of samples were determined from standard curves using purified murine IgA assayed in parallel. The specific activity of mucosal IgA was calculated by dividing the titer value at the end point for each individual mucosal sample by the concentration value of total IgA in the same sample [47].

Antigenic stimulation of cultured lymphocytes from mice immunized with Invaplex _AR

Lymphocyte proliferation during antigen stimulation was determined using splenocytes isolated from Invaplex-immunized mice and native mice. Mononuclear cells (2 × 10 ⁵ per well) were incubated with Invaplex, IpaB or IpaC or S.flexneri 2a LPS preparations. At the same time, in separate wells, splenocytes were stimulated with concanavalin A as a positive control. Negative controls included cells incubated in complete medium and cells from native mice stimulated with antigen. After antigen incubation for 4-7 days, cell proliferation was measured by non-radioactive determination of dehydrogenase activity using MTS (Promega) [48]. A strong correlation was observed with an increase in the optical density of indicators and the number of viable cells per well. To determine the cytokines before measuring proliferation on days 3 and 5, cell culture supernatants were collected (see below). Stimulation indices (SI) were calculated by dividing the average optical density of antigen-stimulated cells by the average optical density of cells stimulated only by medium. SI cells from Invaplex immunized mice were compared with SI cells from non-immunized mice.

Antigen-specific systemic immune response after intranasal immunization with Invaplex _AR

The immunogenicity of the synthetic Invaplex (Invaplex _AR ) S.flexneri 2a made from separate purified components rather than a 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 Invaplex 24 S.flexneri 2a (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 infected with Shigella flexneri 2a (2457T). Blood sampled on day 0, 28, 42, and 63 was analyzed by ELISA to determine serum IgG and IgA responses to Invaplex 50, Invaplex 24, purified LPS, IpaB, and IpaC. Figures 5 and 6 depict serum IgG and IgA titers at the endpoint, determined in blood sampled on day 42 (two weeks after the third immunization).

Inoculated with saline mouse and pre-immune serum from immunized mice did not contain detectable levels of antigen-specific antibodies (data not shown). Immunization of Invaplex _AR S.flexneri 2a elicited immune responses, determined by serum IgG and IgA, on Invaplex 50 and Invaplex 24 at a comparable level with that which was induced by native Invaplex 24 (Fig. 5), and were significantly higher (P < 0.001) compared with those that were caused by saline. A twofold increase in the amount of native Invaplex (JWJX series) used for immunization did not increase the level of Invaplex 50, LPS, or Invaplex 24-specific serum IgG or IgA responses measured on day 42 (Figure 5). Immunization with purified IpaB elicited a strong IpaB-specific (FIG. 6) and an Invaplex 24-specific response (GMT> 5760 and 3800, respectively) and a moderate response to Invaplex 50 (GMT 950). Immunization with purified LPS did not elicit a serum IgG or IgA response to any antigen used in the ELISA in most (5/6) mice in the vaccinated group. Similarly, immunization with purified IpaC also did not elicit a detectable immune response to any analyzed antigen, including the IpaC-specific ELISA. Remarkably, Invaplex _AR immunized mice had, among others, the highest IpaC-specific titers at the endpoint (FIGS. 6 and 8), significantly higher (p <0.001) than those induced after immunization with the native Invaplex.

Protecting mice from lethal S.flexneri infection after intranasal immunization with Invaplex _AR

A lethal lung model includes an intranasal vaccination in mice of a lethal dose of Shigellae, which causes infection in the lungs, resulting in severe weight loss, pneumonia, and ultimately death after a two-week observation period. Three weeks after intranasal immunization with Invaplex _AR S.flexneri 2a or native Invaplex, mice were infected with a lethal dose of S.flexneri 2a (strain 2457T). In native mice (which were injected with saline), 11 out of 13 mice died with a maximum weight loss of an average of 31.4% of the weight before infection (see table 1). All mice immunized with Invaplex _AR S.flexneri 2a (p <0.001) or native Invaplex (p <0.001) survived lethal infection with S.flexneri 2a. Regarding weight loss (which is the most sensitive indicator of protective immunity), Invaplex _AR mice lost less than their weight measured before infection (21.3%) compared with 23.8% -26.0 weight loss % in native Invaplex-immunized mice. In addition, the day of weight recovery (recovery rate after infection) was day 7 for Invaplex _AR and day 13 (native Invaplex, 5 μg) or day 7 (native Invaplex, 10 μg).

In addition, the protective ability of the individual components (IpaB, IpaC, and LPS) used to construct Invaplex _AR was also evaluated in a murine lethal lung model (see Table 1). Immunization with purified LPS and purified IpaC did not protect mice from death (both P> 0.05) and although immunization with IpaB protected 5 of 6 mice from lethal infection, the mice never regained weight and remained symptomatic (low weight, tousled hair) until the end observation period. The maximum weight loss after infection averaged 29.9% for LPS, 31.0% for IpaB and 33.9% for IpaC, all of which were very close to the weight loss in native mice - 31.4%.

The results of infection of mice immunized with Invaplex _AR with a lethal dose of S.flexneri 2a show that Invaplex _AR stimulates a degree of protection that is comparable to or greater than that for native Invaplex. In addition, it seems that a complex of IpaB, IpaC and LPS is necessary for such individual components that are not able to stimulate a fully effective protective immune response.

Table 1 Lethal infection of mice immunized with Invaplex _AR S.flexneri 2a or native Invaplex ¹ . Immunizing antigen Maximum weight loss (%) Day 50% weight recovery Number of survivors Number of dead Total number of animals % Protection † P value * Invaplex _AR S.flexneri 2a, 2.5 mcg 21.32 7 fifteen 0 fifteen one hundred% <0.001 Invaplex 24 JWJX, 5 mcg 26.02 13 fifteen 0 fifteen one hundred% <0.001 Invaplex 24 JWJX, 10 mcg 23.81 7 fifteen 0 fifteen one hundred% <0.001 LPS S.flexneri 2a, 2.5 mcg 29.95 8 3 3 12 40.9% 0.262 IpaB S.flexneri 2a, 2.5 mcg 30.97 > 14 5 one 6 80.3% 0.010 Purified HisTagIpaC, 2.5 mcg 33.92 > 14 one 5 6 1.5% 1,000 0.9% saline 31.35 10 2 eleven 13 0% - 1. Three weeks after the last immunization, mice were intranasally infected with 1.5 x 10 CFU S.flexneri 2a. Weight loss and symptoms were monitored daily for 14 days. †% 3 protection = [(% Death _control -% Death _vaccine ) /% Death _control ] × 100 * Fisher's exact test

Antigen-specific mucosal immune response after intranasal immunization with Invaplex _AR

Lavage from the intestinal and lung mucosa collected on day 35 from mice immunized with Invaplex _AR S.flexneri 2a or native Invaplex were analyzed by ELISA to determine antigen-specific IgA levels. Immunization with Invaplex _AR induced levels of LPS and Invaplex-specific intestinal IgA, which were comparable to levels induced by immunization with native Invaplex (Fig. 7), and were significantly higher than the levels (p <0.001) of IpaB-specific IgA. The minimum level of IpaC-specific intestinal IgA was established after immunization with any Invaplex vaccine preparations (Tables 7 and 8).

Invaplex _AR intranasal immunization caused a strong immune response in the lungs aimed at LPS, Invaplex 50, IpaB and IpaC (Fig. 8 and tables 7 and 8). Minimum levels of LPS-specific IgA were induced in the lungs after immunization with native Invaplex with undetectable levels of antibodies specific for Invaplex 50, IpaB and IpaC.

Antigen-specific cell proliferative response and secreted cytokine profiles after intranasal immunization with Invaplex _AR

Splenocytes collected from immunized mice on day 35 were stimulated in vitro using Invaplex 24, IpaB or IpaC to evaluate the induction of antigen-specific cell-mediated responses. Proliferation of cells after incubation with antigen indicated preliminary memory of exposure and immunological memory. Concavalin A (ConA), which non-specifically activates T cell proliferation, was used as a positive control to demonstrate levels of viable cells. Stimulation indices (SIs) after ConA stimulation ranged from 13.8 to 15.9 (Table 2). 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), showing immunological memory for the antigens present . IpaB and IpaC-specific proliferative responses in groups immunized with Invaplex _AR were significantly higher (P <0.01) than proliferative responses in groups immunized with native Invaplex. Splenocytes from 4/4 of animals immunized with native Invaplex proliferated after incubation with Invaplex (SI = 6.8). Low to undetectable proliferation levels 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 cell proliferation of splenocytes from mice intranasally immunized with Invaplex _AR S.flexneri 2a or native Invaplex, after in vitro stimulation Group Treatment Cell proliferative responses after stimulation ^a in vitro with: Invaplex 24 S.flexneri 2a (1 mcg / ml) IpaB (5 μg / ml) IpaC (5 μg / ml) ConA (5 μg / ml) 31 InvapIexar S.flexneri 2a (KR-C5; 2.5 mcg) ^b 10.2 ± 3.4 ^{s *, **} (4/4) 8.7 ± 2.2 ^{*, **} (4/4) 6.9 ± 3.9 ^{*, **} (4/4) 14.2 ± 4.5 ^{*, **} (4/4) 32 Native Invaplex 24 S.flexneri 2a (JWJX; 2.5 mcg) 6.8 ± 1.5 * (4/4) 2.2 ± 1.4 (1/4) 1.6 ± 0.9 (0/4) 15.9 ± 2.3 (4/4) 33 0.9% saline 1.1 ± 0.4 (0/4) 1.2 ± 0.8 (0/4) 1.8 ± 1.1 (0/4) 13.8 ± 3.3 (4/4) ^{: a} proliferative responses in splenocytes were determined after in vitro stimulation for 3 days with ConA and 5 days with Invaplex 24 S.flexneri 2a (JWJX series), IpaB or IpaC. ^b (batch number; immunization dose) ^c Average stimulation index ± ISD (number of responders / total number in the group) ^* P <0.05 compared with the group inoculated with saline (t-test for one sample). ^** P <0.01 compared with the group immunized with native Invaplex (t-test for one sample).

Confirmation of protection of mice against lethal infection with S.flexneri after intranasal immunization with Invaplex _AR using another Invaplex _AR series

An experiment evaluating the protective ability of Invaplex _AR in a murine lethal lung model was repeated using a different Invaplex _AR S.flexneri 2a series. In addition, the potential assessment of the purified components was repeated on mice immunized with a mixture of two purified components (IpaB + IpaC; IpaB + LPS; IpaC + LPS). For this infection, mice were inoculated intranasally with a slightly higher infection dose (1.6 × 10 ⁶ CFU) of S. flexneri 2a (strain 23457T). In native mice (which were injected with saline), 14 out of 14 mice died with a maximum weight loss of an average of 34.5% of the weight before infection (see table 3). Mice immunized with Invaplex _AR S.flexneri 2a (13 of 14 survivors; p <0.001) survived lethal S.flexneri infection, while mice immunized with native Invaplex had a significantly lower survival rate for the infection dose used in this experiment ( see table 3). As for weight loss in mice immunized with Invaplex _AR , they lost less from their weight before infection (26.6%) compared to 31.6% of weight loss in mice immunized with native Invaplex and 34.5% in mice inoculated with saline.

The protective ability of individual components (IpaB, IpaC, and LPS) or the pair of purified components used to construct Invaplex _AR confirmed the previous results that IpaC and LPS are not protective. Purified IpaB was also not protective (see table 3). Mixtures of two purified components led to protection in the combination of IpaB + LPS and the combination of IpaC + LPS, while the combination of IpaB + IpaC was not completely protective.

The results of a second experiment evaluating the protective ability of Invaplex _AR clearly show that Invaplex _AR stimulates a degree of protection that exceeds that of native Invaplex. In addition, it appears that the individual components (IpaB, IpaC or LPS) are unable to stimulate a fully effective protective immune response.

Invaplex _AR S.sonnei Immunogenicity Study and Mouse Protection

Serum immune responses against Invaplex 50 S.sonnei, LPS S.sonnei, 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). Similarly, titers of serum IgG and IgA specific for Invaplex 50 S.sonnei and LPS reached the endpoint after immunization with Invaplex _AR S.sonnei and native Invaplex (Table 4). Groups of mice immunized with Invaplex _AR S.sonnei had higher levels of IpaB-specific serum IgG (GMT> 5760, P <0.001) compared with groups of mice immunized with native Invaplex S.sonnei (GMT 546). Animals immunized with Invaplex _AR had an average titer of anti-IpaC serum IgG of 1091 (P <0.001), while animals immunized with native Invaplex or saline had undetectable levels of IraC-specific IgG.

Table 4 End-point titers of antigen-specific serum IgG and IgA on day 42 in mice after intranasal immunization with Invaplex _AR S.sonnei or native Invaplex 50 S.sonnei Invaplex 50 S.sonnei LPS S.sonnei Ipab IpaC IgG IgA IgG IgA IgG IgA Saline 90 ± 0 ^c'd 45 ± 0 90 ± 0 45 ± 0 90 ± 0 90 ± 0 Native Invaplex 50 S.sonnei (5 mcg) ^a 2183 ± 789 1254 ± 322 103 ± 40 157 ± 40 546 ± 483 90 ± 0 Synthetic Invaplex _AR S.sonnei (5 mcg) ^b 5014 ± 1288 ** 2864 ± 36 ** 136 ± 117 313 ± 224 * > 5760 ** 1091 ± 1172 ** ^a Group 6 ^b Group 3 ^c Geometrical mean titer at endpoint ± 1 standard deviation ^d Intercomparison comparisons were performed using ANOVA analysis of natural log transformed endpoint titers * P <0.05 compared to native Invaplex 50 S.sonnei ** P <0.001 compared to native Invaplex 50 S.sonnei

Protection of mice from lethal infection with S.sonnei or heterologous S.flexneri after intranasal immunization with Invaplex _AR S.sonnei

The protective ability of Invaplex _AR was also evaluated for Invaplex _AR made from purified S.PSNEYI LPS and recombinant IpaB and IpaC using a mouse lethal lung model. It was compared with the protective ability of the native Invaplex S.sonnei. In addition, the ability of Invaplex _AR S.sonnei to protect against heterologous S.flexneri infection was also evaluated. In native mice (which were injected with saline) 15 out of 15 mice died with a maximum weight loss of 23.4% on average weight before infection (see table 5). Mice immunized with Invaplex _AR S.sonnei survived (15 of 15 survivors; p <0.001) lethal S.sonnei infection, while mice immunized with native Invaplex also showed solid protection (14 of 14 survivors, P <0.001) (see table 5). Regarding weight loss, mice immunized with Invaplex _AR S. sonnei lost 7.7% of their weight before infection, compared with 9.2% weight loss in mice immunized with native Invaplex S.sonnei and 23.4 % in mice inoculated with saline.

Notably, Invaplex _AR S.sonnei also provided significant protection (15 of the 15 infected mice survived, (P <0.001)) against infection with heterologous S. flexneri 2a, suggesting that the immune response to either IpaB or IpaC may reflect a significant degree of protective immune response.

The results of these experiments evaluating the protective ability of Invaplex _AR S.sonnei clearly show that Invaplex _AR, among other Shigella species, stimulates a degree of protection that is comparable to that of the native Invaplex.

Guinea pig guinea pigs Invaplex _AR S.flexneri 2a using a guinea pig model of keratoconjunctivitis

Guinea pigs keratoconjunctivitis model is used to evaluate Shigella vaccines and is often used as precursors of human clinical trials. The model includes Shigella guinea pig eye infection, which causes infection in the corneal epithelium. This complication (severe keratoconjunctivitis) is the result of invasion of the corneal epithelium by shigella and the subsequent inflammatory response of the host, almost the same as that observed in the human intestinal tract.

In this experiment, guinea pigs were immunized three times intranasally with either Invaplex _AR S.flexneri 2a or native Invaplex. A control group that received saline was also used. Three weeks after the last immunization, animals were infected ocularly with S.flexneri 2a. Both Invaplex _AR (90% protection, P <0.001) and native Invaplex (100% protection, P <0.001) provided solid protection (see Table 6), demonstrating that Invaplex _AR is comparable to native Invaplex.

Table 6 Protection against Shigella infection in guinea pigs immunized with Invaplex _AR S.flexneri 2a using the keratoconjunctivitis model Immunization Number of positives ¹ Number of negative % protection ² P value ³ Native Invaplex 50 S.flexneri 2a, 25 mcg / dose 0 10 one hundred% <0.001 Invaplex _AR S.flexneri 2a, 25 mcg / dose one 9 90% <0.001 IpaB (25 mcg) + IpaC (25 mcg) per dose four 6 40% 0.01 IpaB (25 μg) + IpaC (25 μg) + LPS (25 μg) per dose 0 10 one hundred% <0.001 0.9% saline 10 0 0% - 1. Guinea pigs were infected intraocularly with 1 × 10 ⁸ coe S.flexneri 2a. Eyes were evaluated on day 5 after infection for the development of keratoconjunctivitis, as described by Hartman et al. [44] 2.% Protection = [(% Death _control -% Death _vaccinated ) /% Death _control ] × 100 3. Fisher's exact test

The study of adjuvance (immunogenicity) Invaplex _AR S.flexneri 2a in mice

Intranasal immunization with only OVA or OVA combined with CT, or pre-immunized samples from immunized animals (day 0) did not produce detectable serum IgG or IgA levels with specificity for Invaplex 50 S.flexneri 2a, Invaplex 24 S.flexneri 2a (FIG. .9), LPS S.flexneri 2a (FIG. 10), IpaB or IpaC (FIG. 11). Immunization with OVA combined with either Invaplex _AR or native Invaplex caused similar titers of serum IgG and IgA specific for Invaplex 50, Invaplex 24 and LPS at the endpoint (Figures 9 and 10). Endpoint titers against purified IpaB (p <0.05) and IpaC (p <0.001) were higher in mice immunized with OVA + Invaplex _AR compared with mice immunized with OVA + native Invaplex (FIG. 11). Comparable levels of OVA-specific serum IgG and IgA were induced in mice immunized with OVA in combination with InvaplexAR, native Invaplex or CT, at day 42 and were significantly higher (p <0.001) than the responses elicited in mice after immunization with OVA only (Fig. 12).

Antigen-specific antibodies in lung washes were also analyzed by ELISA to study mucosal immune responses induced after immunization (FIG. 13). Immunization with OVA in combination with Invaplex _AR caused similar levels of LPS- and Invaplex of 24-specific IgG and IgA in washouts from the lungs compared with responses after immunization with OVA in combination with native Invaplex and higher than the levels induced after immunization with only OVA or OVA in combination with CT. Similar levels of OVA-specific pulmonary IgG and IgA were caused after immunization with OVA in combination with Invaplex _AR , native Invaplex or CT, which were significantly (P <0.001) higher than those that were caused after immunization with OVA only. Average levels of LPS- and Invaplex-specific IgA in intestinal washes (data not shown) were found in washes from mice immunized with OVA in combination with Invaplex _AR or native Invaplex, and were undetectable in mice immunized with OVA or OVA in combination with CT . OVA-specific intestinal IgA responses were lower than detection levels in all test samples.

Table 8 Endpoint titers of antigen-specific serum IgG and IgA on day 42 in guinea pigs intranasally immunized with native Invaplex, Invaplex _AR, or a mixture of IpaB and IpaC Immunization Antigen ELISA Invaplex 50 S.flexneri 2a LPS S.flexneri 2a Ipab IpaC IgG IgA IgG IgA IgG IgA Invaplex S.flexneri 2a (1307 series, 25 mcg) 827 ± 1046 ^b (180-2880) 475 ± 1148 (180-2880) 1091 ± 2873 (180-5760) 827 ± 1018 (360-2880) 1712 ± 2305 (720-> 5760) 90 ± 0 (90-90) Invaplex _AR S.flexneri 2a (KR-C5 series; 25 mcg) 4073 ± 2160 * (1440-≥5760) 856 ± 360 (720-2880) 2880 ± 2700 (360-≥5760) 827 ± 1046 (720-2880) All> 5760 * (> 5760 *) All> 5760 * (> 5760 *) IpaB + IpaC (25 mcg + 25 mcg) 1211 ± 1034 (360-2880) 255 ± 104 (180-360) 90 ± 0 (90-90) 45 ± 0 (45-45) All> 5760 * (> 5760 *) 3629 ± 2494 * (1440-≥5760) Saline 90 ± 0 (90-90) 45 ± 0 (45-45) 90 ± 0 (90-90) 45 ± 0 (45-45) 90 ± 0 (90-90) 90 ± 0 (90-90) ^a Serum reactions in the stability study of GMP S.flexneri 2a and Invaplex _AR in guinea pigs were determined by blood taken on day 42

Method for the formation and evaluation of a synthetic Invaplex coupled to an antibiotic or other drug molecule for intracellular delivery

Many drugs, including antibiotics, are often ineffective against an intracellular target because they are unable to penetrate the membranes of mammalian cells or because they require high extracellular concentrations to achieve therapeutic concentrations within mammalian cells. Synthetic Invaplex provides a mechanism for creating a complex of invasives, LPS and antibiotic by mixing the components during the assembly stage to create a synthetic Invaplex. Upon completion of assembly, the native properties of Invaplex allow it to transport an attached antibiotic or therapeutic molecule to mammalian cells.

Purified, dissolved IpaB and IpaC, each in its respective final buffer, were mixed with each other in a molar ratio of IpaC / IpaB 8. After mixing IpaB and IpaC, an antibiotic solution, for example gentamicin at 5-100 μg / ml, was added to the mixture. Then IpaB, IpaC and the antibiotic solution were slowly added to the dry LPS powder (LPS to total protein ratio was 0.56). LPS from any type of Shigella can be used; LPS S. flexneri 2a, S. sonnei or S. dysenteriae 1 was used for the described experiments. The mixture was incubated at 37 ° C for approximately 2 hours with shaking. The protein / LPS / antibiotic mixture was then diluted with 20 mM Tris-HCl, 0.10 M NaCl and 1.2 M urea to reduce the concentration of NaCl before performing ion exchange chromatography. For final purification, the diluted IpaB / IpaC / LPS / antibiotic mixture was purified on an anion exchange column (HiTrap (TM) Q HP) using 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 from 0% (5 column volumes) to 24% (10 column volumes) to 50% (6 column volumes) to 100% Buffer B (5 column volumes ) 1 ml fractions were collected from a 1 ml HiTrap (TM) Q HP column at an elution rate of 1 ml / min. Fractions from each step were analyzed for IpaB, IpaC and LPS using spot blotting. Fractions containing IpaB, IpaC, and LPS were synthetic Invaplex fractions.

The effectiveness of the synthetic Invaplex-antibiotic complex will be evaluated by its ability to kill intracellular microorganisms, such as Shigella, in cultured cells. The synthetic Invaplex-antibiotic complex was incubated with Shigella-infected cultured cells. After 2-24 hours, the amount of intracellular Shigella is determined by applying lysates of the treated cells (and untreated control cells) to trypticase-soy agar plates to determine the level of intracellular lysis in cells that were injected with a synthetic Invaplex-antibiotic complex.

Additional options and modifications of the above will be apparent to a person skilled in the art and are covered by the claimed claims.

Claims (34)

1. Synthetic Invaplex, comprising a complex of invasive proteins, consisting of a complex IraB: IraC associated with a serotype-specific lipopolysaccharide component from gram-negative bacteria, in which an invasive protein complex is obtained by mixing highly purified or recombinant IpaB and IpaC to form IraB: IraC complex wherein IpaC and IpaB are present in a ratio of 0.08: 1 to 80: 1, obtained by mixing the IraB: IraC complex with at least one lipopolysaccharide (LPS), where LPS is present relative to the total protein (IpaB and IpaC) in a ratio of from 0.01: 1 to 10: 1. 2. The synthetic Invaplex according to claim 1, wherein the serotype-specific lipopolysaccharide component is a serotype-determining gram-negative bacteria selected from S. flexneri, S.sonnei, S.dysenteriae, S.boydii, enteroinvasive E. coli, Yersinia or Salmonella. 3. The synthetic Invaplex according to claim 2, in which the serotype-specific lipopolysaccharide component is the determining serotype of gram-negative bacteria selected from S.flexneri, S.sonnei, S.boydii, S.dysenteriae or enteroinvasive E.coli. 4. The synthetic Invaplex according to claim 3, in which gram-negative bacteria are selected from S.flexneri, S.sonnei, S.boydii or S.dysenteriae. 5. Synthetic Invaplex no. 1, in which LPS is associated with the Shigella serotype. 6. Synthetic Invaplex no. 1, wherein the IpaB is a purified native IpaB or recombinant IpaB. 7. The synthetic Invaplex of claim 1, wherein the IpaC is a purified native IpaC or recombinant IpaC. 8. The synthetic Invaplex according to claim 1, wherein the synthetic Invaplex is Invaplex _AR . 9. The synthetic Invaplex of claim 8, wherein Invaplex _AR is Invaplex _AR S.flexneri, Invaplex _AR S.dysenteriae or Invaplex _AR S.sonnet. 10. The synthetic Invaplex of claim 8, in which the content of invasives and lipopolysaccharide in Invaplex _AR corresponds to the content of highly purified from native Invaplex 24. 11. Synthetic Invaplex no. 8, in which the serotype-specific lipopolysaccharide component includes two or more lipopolysaccharides selected from at least two different serotypes of gram-negative bacteria. 12. The synthetic Invaplex according to claim 1, in which the ratio is from 0.8: 1 to 20: 1. 13. The synthetic Invaplex of claim 12, wherein the ratio is approximately 8: 1. 14. The synthetic Invaplex according to claim 1, in which the ratio is from 0.5: 1 to 5: 1. 15. The synthetic Invaplex of claim 14, wherein the ratio is from about 0.5: 1. 16. The synthetic Invaplex of claim 1, further comprising a label, antibiotic, drug, or biomolecule. 17. The synthetic Invaplex according to clause 16, in which the biomolecule includes proteins, enzymes, polysaccharide or nucleic acids. 18. A method of obtaining a synthetic Invaplex, including
A) combining IpaB and IpaC with at least one lipopolysaccharide associated with a serotype of gram-negative bacteria, step A) being performed as two separate mixing steps, including mixing IpaB and IpaC with the formation of the IraB: IraC complex and mixing the IpaB: IpaC complex, at least one lipopolysaccharide (LPS) to form a synthetic Invaplex; and B) extracting synthetic Invaplex. 19. The method of claim 18, wherein the IpaB is r-IpaB and IpaC is r-IraC. 20. The method according to p, in which IpaC and IpaB are present in a ratio selected from the range from 0.08: 1 to 80: 1. 21. The method according to p, in which the LPS relative to the amount of total protein (IpaB and IpaC) is present in a ratio of from 0.01: 1 to 10: 1. 22. A composition comprising the synthetic Invaplex according to claim 1 and a pharmaceutically acceptable carrier. 23. The composition of claim 22, further comprising an immunogen. 24. The composition of claim 22, wherein the synthetic Invaplex is present in sufficient quantity to enhance or develop an immune response to the immunogen. 25. The composition according to item 22, in which the amount of synthetic Invaplex present is sufficient to induce a protective immune response. 26. The composition of claim 22, wherein the synthetic Invaplex further comprises a labeled detector, antibiotic, drug, or biomolecule. 27. The composition according to p, in which the biomolecule comprises proteins, enzymes, polysaccharide or nucleic acids. 28. A composition comprising the synthetic Invaplex according to claim 11 and a pharmaceutically acceptable carrier. 29. The use of the synthetic Invaplex according to claim 1 for immunization of a subject. 30. The use of synthetic Invaplex according to claim 11 for immunization of a subject. 31. The use of the synthetic Invaplex according to claim 1 for transporting a molecule through a cell membrane. 32. The application of clause 31, in which the molecule is a labeled detector, antibiotic, drug or biomolecule. 33. The use of the synthetic Invaplex according to clause 16 for transporting labels, antibiotics, drugs or biomolecules through the cell membrane. 34. The use of claim 33, wherein detecting the presence of a label, antibiotic, drug, or biomolecule in the cell.

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