Patent Grant
9492523
This application includes as the Sequence Listing the complete contents of the accompanying text file “Sequence.txt”, created Dec. 7, 2011, containing 17,471 bytes, hereby incorporated by reference.
The invention generally relates to broad based, cross-protective, O-serotype independent vaccines against Shigella. In particular, the invention provides broadly protective Shigella vaccines comprising one or more of the type III secretion (TTS) apparatus needle proteins IpaD and IpaB, and/or the IpaB cognate chaperone protein IpgC, and/or chimeric proteins thereof.
Shigellosis is a severe diarrheal disease caused by infection by bacteria of the genus Shigella. Shigellosis is associated with high morbidity and mortality rates, particularly in the developing world. It is also responsible for long-term effects on cognitive and physical development in children. The global burden has been estimated at more than 160 million cases per year; the most affected being children under 5 years of age living in endemic areas. The causative agent can be transmitted from person to person through fecal-oral contact or through contaminated fomites; ingestion of as few as 10 organisms can cause illness in adult volunteers. In industrialized countries, Shigella is known to be responsible for cases of pediatric diarrhea and to cause occasional foodborne outbreaks. Other susceptible groups include travelers, military personnel and refugees. Additionally, the Centers for Disease Control and Prevention lists Shigella as a food safety threat.
The Shigella genus comprises four different species: Shigella dysenteriae, Shigella flexneri, Shigella sonnei and Shigella boydii. These organisms are further divided into multiple serotypes, based on the structure of the O-polysaccharide portion of their outer membrane lipopolysaccharide (LPS), thereby increasing their antigenic variability. One serotype in particular, S. dysenteriae serotype 1, which produces Shiga toxin, is responsible for the most severe infections, including hemolytic uremic syndrome and it is the cause of epidemic dysentery. Additionally, serotypes can drift during outbreaks, further limiting the efficacy of vaccines that are restricted to particular serotypes. The emergence of strains resistant to antimicrobial drugs, including ciprofloxacin, currently the first-line antibiotic treatment against Shigella infections, heightens the difficulty of controlling this pathogen and makes the development of an effective vaccine even more urgent.
Despite being a longstanding priority for the World Health Organization, and the progress made in recent years, no licensed vaccine for Shigella spp. currently exists. Efforts to develop a vaccine against this pathogen have included the use of killed bacteria, live attenuated and recombinant carrier organisms, polysaccharide conjugates and LPS/protein mixtures. When tested in humans, these vaccines were either too reactogenic or poorly immunogenic. A major disadvantage of these candidates is the O antigen restriction, which limits the scope of protection they can offer and requires the development of a multi-serotype vaccine to provide adequate protective coverage in Shigella endemic areas.
The type III secretion system (TTSS) is a common virulence mechanism in many Gram-negative pathogens. Resembling a molecular needle and syringe, the TTSS apparatus provides a conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm. Residing atop the TTSS needle tip is a tip complex composed of IpaB and IpaD sequentially assembled, which is required for pathogenesis. Several studies have reported the presence of antibodies against Ipa proteins in serum from infected individuals, in subjects immunized with live attenuated organisms during clinical trials and following vaccination in animal models. The conserved nature of the IpaB and IpaD and their critical role in pathogenesis make them ideal targets for vaccine development. An Ipa-based vaccine would provide broader coverage across multiple serotypes. It would also simplify vaccine production and formulation.
The present invention provides broad-based, protective O antigen-independent vaccines against Shigella and related pathogens. The vaccines are advantageously not subject to O antigen restriction and thus may be used to confer protection against a wide range of Shigella species and related pathogens. The vaccines have been developed using the surface-exposed proteins at the tip of the type III secretion (TTS) apparatus needle from Shigella, as well as a cognate chaperone protein. The two TTS apparatus needle tip proteins from Shigella are the initial tip protein IpaD and the first ‘translocator’) protein IpaB. Within the cytoplasm of Shigella, IpaB forms a complex with its cognant chaperone IpgC. For vaccines, recombinant IpaB was prepared with its chaperone by co-expression in E. coli and co-purification via column chromatography. In addition, IpaB was prepared alone by inducing its release from the chaperone using various detergents, thereby leaving IpaB for independent purification. Thus, the IpaB/IpgC complex, IpaB alone, and IpaD were examined as protective antigens by immunizing mice intranasally and challenging the mice with S. flexneri and S. sonnei. The challenge results showed that all three of these antigen preparations (individually and/or in combination) were effective in protecting the mice against homologous S. flexneri challenge and heterologous (O-serotype independent) S. sonnei challenge. This discovery provides a significant advancement over the live-attenuated and lipopolysaccaride-based vaccines that are currently being tested. In some embodiments, the proteins are advantageously administered as chimeric proteins which comprise two or more of the proteins encompassed in single recombinant protein vaccine.
The immunogenicity of the Shigella proteins IpaB, IpaD and IpgC administered alone or combined (e.g. as an IpaB/IpgC complex), have been shown to provide protective immunity to mammals against the otherwise serious consequences of exposure to and infection by Shigella. Significantly, the administration of these proteins afforded protection in an O-independent manner, i.e. protection was not limited to a single O-type. Thus, vaccines formulated from the proteins may be used to protect recipients from disease caused by a broad range of Shigella and related organisms. In some embodiments, the proteins are administered together with a mucosal adjuvant such as the double mutant heat-labile toxin (dmLT) from enterotoxigenic E. coli.
The proteins which may be used in the practice of the invention include IpaB, IpaD and IpgC, administered alone or in various forms and combinations as described herein. The full length sequences of each of these proteins are provided in
The proteins, polypeptides or peptides which may be used in the practice of the invention include IpaB, IpaD and IpgC, and variants or derivatives thereof which have about at least about 50, 55, 60, 65, 70, 75, 80, 85, or 90% identity to the sequences presented herein, and preferably the level of identity is at least about 92, 93, 94, 95, 96, 97, 98 or 99%. Those of skill in the art are familiar with methods and software programs for sequence comparison to determine identity.
The invention also encompasses the use of nucleic acids encoding the proteins/polypeptides/peptides described herein, i.e. nucleic acid vaccines are also contemplated. Those of skill in the art are aware of the many protocols for preparing and administering nucleic acid vaccines, such as those described in issued U.S. Pat. Nos. 7,927,870 and 7,094,410, the complete contents of which is hereby incorporated by referenced in entirety.
Those of skill in the art will appreciate that the genetic code is redundant and that many sequences may encode a given amino acid sequence. All nucleic acid sequences (e.g. DNA, RNA, cDNA, DNA/RNA hybrids, etc.) which successfully encode and express the proteins/polypeptides/peptides described herein, in a manner that results in protection of a subject to whom the vaccine is administered, also as described herein, may be used in the practice of the invention. Also encompassed are vectors which contain such nucleic acid sequences (e.g. plasmids, cosmids, various expression vectors, etc.), and host cells such as bacteria, insect cells, etc., which comprise the nucleic acid sequences and/or the vectors. Such vectors typically include one or more expressible gene sequences encoding one or more or the proteins/polypeptides/peptides of interest, which may be a chimera, operably linked to at least one transcription element (e.g. a promoter) that drives expression of the gene.
In some embodiments of the invention, the proteins, polypeptides or peptides that are used in the vaccine are chimeric proteins. By a “chimeric protein” we mean a protein that is translated from a single, contiguous nucleic acid molecule, and which comprises sequences from at least two different proteins or antigenic regions thereof. For example, a chimera of the invention may include two or more of IpaB and/or IpaD and/or IpgC, or antigenic regions of two or more of these. Typically, the individual sequences are joined via a linker or spacer sequence of e.g. from about 2 to about 20 amino acids, usually from about 2 to about 10 amino acids. The amino acids in linking sequences are typically uncharged and the linker sequence usually does not exhibit secondary or tertiary structure, but does allow the fused protein segments to adopt functional secondary, tertiary, etc. conformations.
The present invention provides compositions for use in eliciting an immune response and/or vaccinating an individual against Shigella, or against disease symptoms caused by Shigella infection, and/or infection by related organisms. The compositions include one or more substantially purified proteins, polypeptides or antigenic regions thereof as described herein, or substantially purified nucleic acid sequences (e.g. DNA cDNA, RNA, etc.) encoding such proteins, polypeptides or antigenic regions thereof, and a pharmacologically suitable carrier. By “substantially purified” we mean that the molecule is largely free of other organic molecules, cellular debris, solvents, etc. when tested using standard techniques known to those of skill in the art (e.g. gel electrophoresis, column chromatography, sequencing, mass spectroscopy, etc.). For example, the molecule is generally at least about 50, 55, 60, 65, 70, or 75% pure, and preferably is at least about 80, 85, 90, 95% or more pure (e.g. 96, 97, 98, 99 or even 100% pure). The preparation of proteins, polypeptides, and peptides as described herein is well-known to those in the art, and includes, for example, recombinant preparation; isolation from a natural source; chemical synthesis; etc. The purification of proteinaceous materials is also known, and new improved methods that are advantageous for the proteins utilized in the practice of the invention are described in the Examples section below.
The preparation of compositions for use as vaccines is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared (e.g. lyophilized, freeze-dried forms, etc.). The preparation may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of protein, polypeptide or peptide (or encoding nucleic acids) in the formulations may vary. However, in general, the amount in the formulations will be from about 1 to about 99%.
In addition, the composition may contain adjuvants, many of which are known in the art. For example, adjuvants suitable for use in the invention include but are not limited to: bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof. Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 de-O-acylated monophosphoryl lipid A. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529. Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly (dG) sequences have also been shown to be immunostimulatory. The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. e.g. replacement of guanosine with 2′-deoxy-7-deazaguanosine. The CpG sequence may include, for example, the motif GTCGTT or TTCGTT. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed so that the 5′ end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3′ ends to form “immunomers”.
Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (e.g. E. coli heat labile enterotoxin “LT”), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants and as parenteral adjuvants are known. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, is known. Such adjuvants are described, for example, in issued U.S. Pat. No. 8,039,007 (the complete contents of which is hereby incorporated by reference in entirety). Various interleukins may also be used as adjuvants to increase the immune response in a subject. In preferred embodiments, the adjuvant is a mucosal adjuvant such as, for example, the double mutant heat-labile toxin (dmLT) from enterotoxigenic E. coli.
The vaccine compositions (preparations) of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, inhalation, orally, intranasally, topically, etc. In preferred embodiments, the mode of administration is intranasal. In addition, the compositions may be administered in conjunction with or in a composition which contains other antigens of interest. In other words, they may be administered as a component of a multivalent vaccine which also contains antigens against other related or non-related infectious diseases, especially childhood diseases, such as polio, whooping cough, tetanus, diphtheria, etc.
Recipients of the vaccine of the invention are generally mammals, frequently humans, but this need not always be the case. Veterinary applications are also contemplated. The vaccine are often administered as or together with so-called “child hood” vaccines. Thus, the recipients are preferably human children who may be infants (e.g. up to about 1 hear of age), toddlers (e.g. up to about 2 years of age), or older, and administration may be carried out as a series of initial inoculations followed by booster doses at suitable intervals, e.g. monthly, or every 6-months, or yearly, etc. as necessary to provide protection. Thereafter, or in the case of adults who have not previously been vaccinated, the vaccines may be administered as necessary to result in a protective immune response, and subjects may be re-boosted e.g. about every 10 years throughout adult life.
Vaccine recipients may have never been exposed to Shigella, or may have been exposed or suspected of having been exposed but be asymptomatic, or may have actual symptoms of shigellosis, and still benefit from administration of the vaccine. Vaccine administration may prevent disease symptoms entirely, or may lessen or decrease disease symptoms, the latter outcome being, while not being ideal, still better than experiencing full-blown disease symptoms.
The amount of protein that is administered per dose of vaccine is typically in the range of from about 1 to about 1000 μg/kg, and is usually in the range of from about 0.02 to about 20 μg/kg of body weight of the recipient. Those of skill in the art will recognize that the precise dosage may vary from situation to situation and from patient to patient, depending on e.g. age, gender, overall health, various genetic factors, and other variables known to those of skill in the art. Dosages are typically determined e.g. in the course of animal and/or human clinical trials as conducted by skilled medical personnel, e.g. physicians.
The vaccines and immunogenic compositions of the invention may be used to provide immune protection against various Shigella species in an O-independent manner, examples of which include but are not limited to: S. flexneri, S. sonnei, Shigella dysenteriae, and Shigella boydii.
In addition, the compositions may be used to vaccinate or elicit and immune response in subject against disease causing pathogens that are related to Shigella, examples of which include but are not limited to enteroinvasive E. coli and Salmonella enterica serovars Typhi, Paratyphi A & B, Typhimurium and Enteritidis.
Administration of the compositions of the invention results in the production of a protective immune response in subjects who receive the compositions, frequently by the elicitation of antibody production against the administered antigens. Such antibodies are also encompassed by the invention, for example, those generated using laboratory techniques in experimental animals, or using cell culture, or by chemical syntheses, etc. Such antibodies may be polyclonal or monoclonal, and may be specific for the antigens (reacting with no other antigens) or selective for the antigens (reacting more strongly or preferably with the antigens, compared to other antigens). The antibodies may be multivalent. The antibodies may be used for research and/or diagnostic purposes, or alternatively, for treatment, especially of individuals who are infected with Shigella.
The invention provides methods of vaccinating, or, alternatively, of eliciting an immune response, in a subject in need thereof. The method generally involves identifying a suitable subject, and administering the composition as described herein. The method may also encompass follow-up of administration, e.g. by assessing the production of protective antibodies by the subject, or the presence (or lack thereof) of disease symptoms, etc. The immune response that is elicited may be of any type, i.e. any type of antibody may be produced in response to administration, and cell-mediated immunity may also be elicited.
The foregoing Examples serve to illustrate exemplary aspects of the invention, but should not be interpreted as limiting in any way.
Shigella ssp. are food and water-borne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains as well as emerging antibiotic resistance require the development of a broadly protective vaccine. All Shigella ssp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that forms the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella, they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, co-administered with a double mutant heat-labile toxin (dmLT) from E. coli as a mucosal adjuvant in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody and T cell-mediated immunity were induced against both proteins, particularly IpaB. Mice immunized with IpaB alone or combined with IpaD, in the presence of dmLT, were fully protected against lethal pulmonary infection with S. flexneri and S. sonnei. We show that Shigella TTSA IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccines.
Materials and Methods
Protein purification materials. Plasmids and competent E. coli were obtained from Novagen (Madison, Wis.). Metal affinity columns, Q-Sephadex, Butyl Sepharose and HiLoad 16/60 Superdex 200 preparative grade columns were obtained from GE Healthcare (Piscataway, N.J.). n-octyl-polyoxyethylene (OPOE) was obtained from Alexis Biochemicals (Lausen, Switzerland). All other chemicals were reagent grade.
The gene encoding IpaD was copied by PCR from the virulence plasmid of Shigella flexneri 2457T (accession number J04117). The PCR fragment was digested with the restriction enzymes NdeI and BamHI and ligated into NdeI/BamHI digested pET15b from Novagen. The ligation product was used to transform NovaBlue E. coli. Transformants were screened for the proper insert and subjected to double stranded sequencing. A plasmid containing the correct sequence was transformed into BL21 (DE3) E. coli (Novagen). This strain was used to inoculate LB media containing ampicillin. The bacteria were grown to an absorbance at 600 of about 0.6 at which time they were induced to over express IpaD with Isopropyl-β-D-thio-galactoside (IPTG). The bacteria were grown an additional three hours to allow protein expression to occur. Alternatively, a starter culture of 100 ml was used to inoculate a 5- or 10-liter fermentor vessel containing TB media. After growing overnight (16 hours), the bacteria were induced and allowed to express protein for three hours. The bacteria were collected by centrifugation, resuspended in binding buffer (see below for recipe), and lysed by microfluidization. This suspension was clarified by centrifugation and was loaded onto a nickel charged immobilized metal affinity column (IMAC). The column (5 ml) was washed with 10 bed volumes each of binding and wash buffers and then subjected to a gradient of 0% to 40% elution buffer. Peak fractions were combined, buffer exchanged into 10 mM Tris-HCl pH 7.5, 10 mM NaCl and loaded onto a Q-Sephadex anion exchange column (5 ml). The IpaD was eluted via a gradient from 0-16% 1M NaCl in 10 mM Tris pH 7.5 over 15 column volumes. The peak fractions were combined and dialyzed against PBS. Pure protein was stored at −80° C.
The gene encoding IpaB was copied by PCR from the virulence plasmid of Shigella flexneri 2457T (accession number J04117). The PCR fragment was digested with the restriction enzymes NdeI and BamHI and ligated into NdeI/BamHI digested pET15b from Novagen. The ligation product was used to transform NovaBlue E. coli. Transformants were screened for the proper insert and subjected to double stranded sequencing. The gene encoding IpgC was copied by PCR from the virulence plasmid of Shigella flexneri 2457T (accession number J04117). The PCR fragment was digested with the restriction enzymes NdeI and XhoI and ligated into NdeI/XhoI digested pACYCDuet-1 from Novagen. The ligation product was used to transform NovaBlue E. coli. Transformants were screened for the proper insert and subjected to double stranded sequencing. A plasmid containing the correct sequence for IpaB and a plasmid containing the correct sequence for IpgC were transformed into Tuner (DE3) E. coli (Novagen). This strain was used to inoculate LB media containing ampicillin and chloramphenicol. The bacteria were grown to an absorbance at 600 of about 0.6 at which time they were induced to over express IpaB/IpgC with Isopropyl-β-D-thio-galactoside (IPTG). The bacteria were grown an additional three hours to allow protein expression to occur. Alternatively, a starter culture of 100 ml was used to inoculate a 5- or 10-liter fermentor vessel containing TB media. After growing overnight (16 hours), the bacteria were induced and allowed to express protein for three hours. The bacteria were collected by centrifugation, resuspended in binding buffer (see below for recipe), and lysed by microfluidization. This suspension was clarified by centrifugation and was loaded onto a nickel charged immobilized metal affinity column (IMAC). The column (5 ml) was washed with 10 bed volumes each of binding and wash buffers and then subjected to a gradient of 0% to 40% elution buffer. Peak fractions were collected, the buffer exchanged into 1M ammonium sulfate in 50 mM sodium phosphate pH 7.0 and loaded onto a Butyl Sepharose High Performance column (5 ml) with a linear gradient from 1M ammonium sulfate in 50 mM sodium phosphate pH 7.0 to 50 mM sodium phosphate. For preparation of His-tag IpaB, the IMAC-bound protein complex was incubated overnight in the presence of 1% OPOE. The chaperone was removed in the flow through and subsequent wash steps. His-tag IpaB was eluted in the presence of OPOE to maintain the protein in a soluble form. His-tag IpaB was then further purified using size exclusion chromatography using a HiLoad 16/60 Superdex 200 Prep Grade column. All proteins were concentrated by ultrafiltration and dialyzed into PBS pH 7.2. Protein concentrations were determined via absorbance at 280 nm using extinction coefficients based on the amino acid composition of each protein.
Buffers that were utilized are listed below:
4× Charge Buffer: 500 ml
8× Binding Buffer: 1Liter
8× Wash Buffer: 500 mL
4× Elution Buffer: 500 mL
4× Strip Buffer: 500 mL
Far-UV circular dichroism (CD) spectra were collected using a Jasco J815 spectropolarimeter equipped with a Peltier temperature controller (Jasco Inc., Easton, Md.). Spectra were acquired using a 0.1 cm path length cuvette at 10° C. A resolution of 1.0 nm and a scanning speed of 50 nm/min with a 2-s data integration time were employed. Spectra presented are an average of three consecutive measurements. The thermal unfolding of the proteins was followed by monitoring the ellipticity at 222 nm over a temperature range of 10 to 90° C. with a resolution of 2.5° C. and 15° C./h heating rate. Protein concentration was 0.5 mg/mL for IpaD and 0.3 mg/ml for IpaB and IpaB/IpgC. CD signals were converted to mean residue molar ellipticities [θ]R and the thermal transitions were analyzed using the Jasco Spectral Manager™ and Microcal Origin™ 6.0 software.
Challenge Organisms. S. flexneri 2a 2457T and S. sonnei 53G inocula for challenge were prepared as previously described. Briefly, the strains were grown on tryptic soy agar (TSA) with Congo Red (final concentration 0.02%); 20-25 colonies were picked and grown in a volume of 125 ml Luria-Bertani (LB) broth (Athena Environmental Sciences, Baltimore, Md.) with agitation for 3-4 hours at 37° C. or until an Abs600 of 0.8-1.3 was achieved. Cultures were centrifuged and bacterial pellets were resuspended in phosphate buffered saline (PBS). Colony-forming units (CFU) of each challenge dose were determined by plating serial dilutions of the culture on Congo Red agar plates. The mouse lethal dose 50% (MLD50) for the challenge strains were calculated by the method of Reed and Muench and determined to be ˜5.4×106 CFU for S. flexneri and 1.2×107 CFU for S. sonnei.
Mice, Immunizations and Challenge Procedures. Female Balb/c mice (8-10 week old, Charles River Laboratories, Wilmington, Mass.) were immunized intranasally (i.n.) on days 0, 14 and 28 with IpaB (2.5 μg), IpaB complexed with IpgC (3.25 μg; equivalent to 0.75 μg IpgC and 2.5 μg IpaB) or IpaD (10 μg). The proteins were administered alone or combined, admixed with 2.5 μg of the E. coli dmLT adjuvant. Control groups received 2.5 μg of dmLT or PBS. Immunization was performed under Isoflurane anesthesia (Abbott Laboratories, North Chicago, Ill.) dispensed through a precision vaporizer. The inoculum volume was 25 μl and it was administered by means of a pipette and tip, delivering half of the volume in each nare. Animals were allowed to completely recover before they were returned to their cages, and they were monitored daily after immunization. On day 56 after primary immunization, mice were challenged with virulent Shigella strains via the nasal route to induce a pulmonary infection, as previously described. Two lethal dosage levels were used for both S. flexneri (6×107 and 1.3×108 CFU), corresponding to 11 and 24 MLD50, and S. sonnei (1.1×108 and 2×108 CFU), corresponding to 5 and 9 MLD50, respectively. The challenge inoculum was given in a volume of 30 μl PBS. Mice were monitored daily for 14 days after the challenge, and their health status, daily weight and survival were recorded. Mice were euthanized if they reached the moribund state or lost more than 20% of their initial body weight and did not recover within 48 h. Animal studies and procedures were approved by the University of Maryland Institutional Animal Care and Use Committee.
Measurement of Antibodies. Serum IgG antibodies to IpaB, IpaD and dmLT were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, Immulon II ELISA plates (Thermo Scientific, Waltham, Mass.) were coated with IpaB (0.1 μg/ml), IpaD (1 μg/ml) or dmLT (1 μg/ml) diluted in PBS, for 3 h at 37° C. Plates were washed with PBS Tween (PBST) and blocked overnight with PBS containing 10% dry milk (Nestle, Solon, Ohio). Serum samples were added in serial dilutions in PBST 10% dry milk and incubated for 1 h at 37° C. Antibodies were detected with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG followed by TMB substrate (KPL, Gaithersburg, Md.). The reaction was stopped by adding 1M H3PO4. Plates were read using an Ascent microplate reader (Thermo Scientific, Waltham, Mass.). Titers were calculated by interpolation of Abs values of experimental samples in the regression curve of a calibrated positive control; they are reported in ELISA Units (EU) per ml and correspond to the inverse of the serum dilution that produces an Abs450 of 0.2 above the blank. For measurement of stool antibodies, 5-6 fecal pellets from individual animals were weighted and resuspended 10% W/V in PBS containing 0.2% sodium azide (Sigma-Aldrich, St Louis, Mo.). Samples were centrifuged and supernatants stored at −20° C. with 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich). IgA antibodies to IpaB, IpaD and dmLT were measured in stool supernatants by ELISA, as described above, using HRP-labeled goat anti-mouse IgA as the secondary antibody (SouthernBiotech, Birmingham, Ala.).
Antibody Secreting Cells (ASC). The frequency of IgG- and IgA-secreting cells was measured by ELISpot as previously described, with modifications. Spleen, bone marrow and nasal associated lymphoid tissue (NALT) were collected on day 39 and 56 after immunization and single cell suspensions were prepared. Cells were incubated overnight in Immulon II flat bottom plates (Thermo Scientific) previously coated with 5 μg/ml IpaB or IpaD; PBS-coated wells were used as negative controls. HRP-labeled goat anti-mouse IgG (KPL) and IgA (SouthernBiotech) were added in agarose overlay. True Blue (KPL) was used as substrate. For all ELISpot measurements, spots were counted using a stereomicroscope and results were expressed as mean spot-forming cells (SFC) per 106 cells from quadruplicate wells.
IFN-γ Secretion. The frequencies of IFN-γ-secreting T cells were measured as previously described. Spleens were collected on day 56 following primary immunization and single cell suspensions were prepared. Cells (in quadruplicate wells) were incubated with IpaB or IpaD (10 μg/ml) in plates previously coated with anti-IFN-γ capture antibody (IFN-γ ELISpot kit, BD Biosciences, San Diego, Calif.) for 48 h at 37° C., 5% CO2. IFN-γ production was detected using a biotinylated anti-IFN-γ antibody, followed by Streptavidin-HRP. 3-Amino-9-ethylcarbazole was used as a peroxidase substrate (Calbiochem, San Diego, Calif.). SFC were calculated and reported as described above.
Statistical Methods. All graphics and statistical analysis were generated using GraphPad Prism 5. Data distribution was first examined using the D'Agostino & Pearson normality test. One-way ANOVA and Tukey's tests were used for subsequent group comparisons. Survival curves were plotted in Kaplan-Meier curves; treatments were compared using a log rank test. A P<0.05 was considered significant in all analyses.
Results
Purification of Recombinant Shigella IpaB and IpaD. Purification of recombinant IpaD and IpaB was performed by modification to previous methods. In addition to the standard IMAC purification, contaminating proteins were removed from the His-tag IpaD via anion exchange chromatography (
Far-UV CD was employed to assess the secondary structure of the purified proteins. These spectra are routinely collected to ensure the purification process has resulted in proteins maintaining their proper conformation. Thus, CD was used to demonstrate that the proteins were unaffected by the additional purification steps. Indeed, the spectra for IpaD, IpaB and the IpaB/IpgC complex exhibit the typical α-helical minima at 208 and 222 nm (
To determine the initial stability of the proteins under standard storage conditions, IpaD, IpaB and IpaB/IpgC in PBS were stored at 4° C. or −80° C. and assessed for degradation weekly, for a three-month period, using SDS-PAGE. IpaD remained stable for up to three months regardless of temperature (data not shown). Both IpaB and IpaB/IpgC were stable for three months at −80° C. In contrast, IpaB and IpaB/IpgC were stable for only three and four weeks at 4° C., respectively. In light of the thermal stability described above, it was surprising that the IpaB/IpgC complex degraded 25% faster than IpaB alone at 4° C., albeit only one week. While these data appear contradictory to the thermal unfolding where the complex was more stable, it is likely that binding of IpgC to IpaB results in unstructured regions that are unique to the complex and are available to proteolytic cleavage.
Mucosal immunization with IpaB/D proteins elicits strong antibody responses. The kinetics of serum IgG responses against Shigella IpaB and IpaD and the E. coli dmLT adjuvant are shown in
Very strong serum IgG responses were also produced against the adjuvant E. coli dmLT. Elevated titers were detected 2 weeks after vaccination and reached a plateau 2 weeks after the second dose. No differences were seen in titers among the different treatment groups. The dmLT-specific IgG responses were consistently high in all the experiments performed.
Ipa-specific IgA- and IgG-secreting cells in systemic tissues. In addition to serum antibodies, we measured the frequency of vaccine-induced ASC in spleen and bone marrow. These cells represent pools of long-lived plasma cells and memory cells, respectively, which have the capacity to maintain circulating antibody levels. IgG and IgA ASC specific for IpaB and IpaD were detected in the spleen of vaccinated mice one month after the last immunization (
Similarly, vaccine-induced IgG- and IgA-secreting cells against IpaB were found in the bone marrow of vaccinated mice (
Stool IgA and nasal ASC responses following immunization with IpaB and IpaD. To demonstrate the induction of antibodies in the gastrointestinal mucosa, which represent the first line of adaptive immune defense against enteric pathogens, we measured IgA responses to IpaB and IpaD in stool supernatants from vaccinated and control mice (
IgG and IgA ASC specific for IpaB and IpaD were detected in the nasal tissue on day 56 after immunization (
Intranasal immunization with IpaB and IpaD elicits robust T cell-mediated immunity. In addition to antibodies, T cells are also believed to contribute to protection against Shigella infection through the secretion of pro-inflammatory mediators that will further activate innate immune cells and T helper cytokines that will support antibody production. T cells might also contribute to clearing the infection by promoting T cytotoxic- or NK-mediated killing of infected cells. The frequency of IFN-γ secreting T cells specific for IpaB and IpaD was measured in spleens of vaccinated and control mice, one month after the last immunization (
Protection against S. flexneri and S. sonnei lethal pulmonary challenge. To determine the protective efficacy of IpaB and IpaD, vaccinated and control mice were subjected to a lethal pulmonary infection with homologous S. flexneri 2a or heterologous S. sonnei (
S. flexneri
S. sonnei
S. flexneri
S. sonnei
Discussion
Despite intense efforts, the development of an effective vaccine against Shigella remains a daunting task. One of the main challenges in achieving an effective prophylactic tool against this pathogen is the need to protect against multiple serotype strains. Antibodies to LPS have been associated with serotype-specific protection against Shigellosis, yet the existence of structurally and antigenically different LPS molecules limits the applicability of an LPS-containing vaccine.
Our approach to develop a safe and effective vaccine to prevent this disease involves the use of conserved proteins that could provide broad protection against multiple serotypes. Among potential cross-protective antigens, we focused our attention on the exposed proteins of the TTSA. These proteins are encoded as part of the mxi-spa operon located on the large virulence plasmid that is present in all Shigella ssp. Thus, in addition to being nearly identical among pathogenic Shigella strains, they mediate critical steps of bacterial pathogenesis. IpaB and IpaD were selected because of their significant roles during TTSS-mediated events that lead to bacterial invasion of epithelial cells. IpaD localizes to the needle tip where it controls further assembly of the TTSA, as well as TTSS effector secretion. IpaB is subsequently recruited to the tip of the needle where it interacts with host membranes to initiate formation of the translocon/pore complex. Immunological effector mechanisms (i.e. antibodies) that could interfere with their function in vivo could potentially prevent bacterial infection, making them very attractive vaccine target antigens.
We examined the immunogenicity of IpaB and IpaD (alone or combined) in a mouse intranasal immunization model, using the E. coli dmLT as adjuvant, and demonstrated that both proteins were able to induce robust serum and mucosal antibody levels and elevated frequencies of antibody-secreting cells in mucosal and systemic tissues. They also induced strong antigen-specific cell-mediated immunity responses, evidenced by the presence of IFN-γ secreting T cells. Most important, very high levels of protection (80 to 100%) were achieved against a homologous and a heterologous strain using a lethal pulmonary challenge model. The dmLT molecule is a derivative of the heat-labile enterotoxin of E. coli with two attenuating mutations in the enzymatically active A-subunit (R192G/L211A); these mutations effectively reduce enterotoxicity without impacting the adjuvant properties of the molecule. It was chosen as an adjuvant for our studies because it has been shown to enhance the production of vaccine-specific humoral and cell-mediated immune responses in both the systemic and mucosal compartments when it is included as part of a vaccine formulation.
IpaB was the most immunogenic of the two proteins, consistently and across measurements. Both serum IgG and stool IgA responses to IpaB appeared faster and achieved a higher magnitude than the responses induced by IpaD. This superior immunogenicity was also seen at the T cell response level. The co-administration of proteins had no effect on their individual serological responses, although it appeared to have an impact in the T cell responses as discussed below. The presence of high frequencies of IpaB/D specific ASC in the spleen indicates the capacity of the vaccine to induce a large pool of functional plasma cells, which are the source of systemic antibodies. The demonstration of antigen-specific ASC in the bone marrow indicates the existence of B memory cells, which can reactivate upon a subsequent antigen encounter to mount a rapid and strong anamnestic response and thus contribute to long-term protection.
The ability to generate mucosal antigen-specific ASC and to produce intestinal sIgA are also important features of our IpaB/D-based vaccine, as these immunological effectors would be necessary to prevent mucosal enteric infection in humans. Although the mechanisms of protection against Shigella have not been elucidated, mucosal immune defenses are sought, as they block pathogens in the intestinal lumen, preventing their attachment and infection of epithelial cells. Ipa-specific sIgA antibodies are expected to neutralize TTSS interactions at the mucosal epithelial barrier thereby preventing bacterial invasion of host cells. IgG antibodies might also contribute to protection when infection has occurred, facilitating opsonophagocytic killing by polymorphonuclear neutrophils and macrophages.
In addition to antibodies, both IpaB and IpaD induced high frequencies of IFN-γ-secreting T cells demonstrating the presence of robust cell-mediated immunity. Production of IFN-γ is required for protection of mice against Shigella pulmonary infection. Likewise, T cell-mediated immunity is thought to be necessary for protection of humans against Shigellosis. IFN-γ activated macrophages abrogate bacterial growth whereas, in the absence of IFN-γ, Shigella replicates intracellularly and further disseminates, which suggests an IFN-γ mediated mechanism for controlling infection. Production of IFN-γ in response to IpaB has been demonstrated using peripheral mononuclear cells (PBMC) from infected individuals or from subjects that received live attenuated vaccine organisms.
In our studies, the production of IFN-γ appeared to be increased when IpaB/D were administered together, as opposed to the proteins given alone, suggesting a synergistic or more effective activation of antigen-presenting cells or Th1 lymphocytes. Interestingly, the increased responses appeared to be limited to T cells, as they were not seen in the antibody or ASC measurements. This effect was not due to cross reactivity or contamination, as cells from mice immunized with IpaB did not respond against IpaD and vice versa.
Although IpaB degrades when expressed in the E. coli cytoplasm, it can be stably co-expressed and purified with its cognate chaperone, IpgC. The complex can then be separated by the addition of the detergent OPOE, which results in stable, multimeric IpaB. While IpaB would be the obvious antigen, separation from IpgC requires an additional purification step and the use of a non-GRAS detergent, OPOE, which is currently the only detergent found to efficiently separate the complex. This lengthens the purification process and increases production costs. Thus, the question emerged as to whether IpaB/IpgC is as equally immunogenic as IpaB and could serve as vaccine antigen. We found no significant differences between IpaB and IpaB/IpgC in any of the responses measured. This finding could facilitate the production of a vaccine with a more simple production process and reduced costs.
Mice immunized with IpaB/D were protected against Shigella lethal infection in a pulmonary challenge. Heterologous protection was also observed using Shigella sonnei. Protection in the lower dose challenge was significant for all combinations of Ipa proteins tested. In agreement with the lower immune responses, immunization with IpaD afforded lower protection compared with IpaB. Protection conferred by IpaB/IpgC was comparable to that induced by purified IpaB.
Contrary to what we expected, the combination IpaB/D did not result in increased protection in any of the challenge conditions. We believe, however, that it would still be advantageous to maintain both antigens in a future vaccine candidate since IFN-γ responses seemed to be improved, which might be important to enhance protection in humans.
Another vaccine candidate that contains Shigella Ipas is the “Invasin-LPS complex” or Invaplex. This vaccine consists of a mixture of IpaB, IpaC and LPS (and possibly other antigens) present in water extracts from intact Shigella. Two chromatographic fractions, designated Invaplex 24 and 50, were immunogenic and protective against homologous virulent strains in mice and guinea pigs. The S. flexneri 2a Invaplex 50 vaccine was safe and immunogenic when administered intranasally to human volunteers in Phase 1 clinical studies. However, this vaccine failed to protect subjects from a subsequent oral challenge with virulent S. flexneri 2a 2457T. Unlike the Invaplex, our vaccine contains known quantities of highly purified IpaB, along with IpaD, and it does not contain LPS.
Although antibodies to LPS are known to mediate protection against shigellosis, our study provides the first evidence that protective immunity can be induced by the IpaB/D vaccine combination, and in the absence of LPS. A protein-based, LPS-free vaccine would be safer and amenable for use in infants, young children and immune-compromised individuals. The demonstration of cross-protective immunity using purified TTSA proteins is also novel and has broader implications as the TTSA tip proteins from other Gram-negative enteric pathogens could prove to be protective antigens as well. Through the use of efficient antigen delivery systems, TTSA-based vaccines could potentially be administered to humans to induce mucosal immunity.
In sum, a subunit-based IpaB/D vaccine could be an effective broadly protective vaccine for prevention of shigellosis.
Two proteins sit at the tip of the Shigella TTSA needle. IpaD resides at the top of the TTSA needle; however, upon activation of IpaD, IpaB mobilizes to a position at the needle tip distal to IpaD and is responsible for host cell contact by Shigella. These two proteins are exposed to the immune system and are thus prime vaccine candidates.
Example 1 describes purification of IpaD and IpaB via His-tag technology using pET plasmid vectors (commercially available from Novagen). While IpaD can be purified by standard methods, IpaB requires co-expression with its chaperone IpgC in a method similar to that published by Birket, et al. (Biochemistry [2007] 46:8128-8137). In order to release IpgC from IpaB, the detergent OPOE was added. OPOE is not a GRAS reagent and therefore must be removed from the purification scheme or replaced. Therefore, in addition to IpaD and IpaB alone, the IpaB/IpgC complex was used to vaccinate mice intranasally, followed by Shigella challenge (
However, the use of a protein complex in a vaccine composition may not be optimal. Thus, a third method of preparation of the proteins in the form of a chimeric protein of IpaD-IpaB was undertaken. The genes for IpaD and IpaB were genetically fused and inserted into the vector pET15b. To produce the plasmid, an NdeI/XhoI PCR fragment of IpaD without a stop codon was subcloned 5′ to an XhoI/BamHI IpaB in pET15b. The plasmid was cotransformed into Tuner (DE3) with IpgC/pACYCDuet-1. This strain was used to inoculate LB media containing ampicillin and chloramphenicol. The bacteria were grown to an absorbance at 600 of about 0.6 at which time they were induced to over express the fusion/IpgC with Isopropyl-β-D-thio-galactoside (IPTG). The bacteria were grown an additional three hours to allow protein expression to occur. Alternatively, a starter culture of 100 ml was used to inoculate a 5- or 10-liter fermentor vessel containing TB media. After growing overnight (16 hours), the bacteria were induced and allowed to express protein for three hours. The bacteria were collected by centrifugation, resuspended in binding buffer (see below for recipe), and lysed by microfluidization. This suspension was clarified by centrifugation and was loaded onto a nickel charged immobilized metal affinity column (IMAC). The column (5 ml) was washed with 10 bed volumes each of binding and wash buffers and then subjected to a gradient of 0% to 40% elution buffer. Peak fractions were collected, the buffer exchanged into 1M ammonium sulfate in 50 mM sodium phosphate pH 7.0 and loaded onto a Butyl Sepharose High Performance column (5 ml) with a linear gradient from 1M ammonium sulfate in 50 mM sodium phosphate pH 7.0 to 50 mM sodium phosphate. For preparation of His-tag fusion, the IMAC-bound protein complex was incubated overnight in the presence of 1% OPOE. The chaperone was removed in the flow through and subsequent wash steps. His-tag fusion was eluted in the presence of OPOE to maintain the protein in a soluble form. His-tag fusion was then further purified using size exclusion chromatography using a HiLoad 16/60 Superdex 200 Prep Grade column. All proteins were concentrated by ultrafiltration and dialyzed into PBS pH 7.2. Protein concentrations were determined via absorbance at 280 nm using extinction coefficients based on the amino acid composition of each protein.
This method produces a chimeric IpaD-IpaB protein that is stable and soluble. An SDS-PAGE picture of the purified protein is shown in
Additional testing and characterization of the IpaD-IpaB fusion protein (also referred to as “DB” or “DB fusion”) has also been carried out. In one experiment, mice were divided into 4 groups, A, B, C and D, and immunized as follows: Group A: 12 μg IpaB+7 μg IpaD+2.5 μg dmLT; Group B: 20 μg DB fusion+2.5 μg dmLT; Group C: 20 μg DB fusion; and Group D: PBS (control). Intranasal immunization was performed at days 0, 14 and 28. Serum IgG levels were monitored at days 0, 14, 28, 42 and 56. At Day 56, selected mice were euthanized and ASC and IFNγ secretion were analyzed, and other mice were challenged with either S. flexneri or S. sonnei.
Serum IgG levels in the mice at days 0, 14, 28, 42 and 56 are presented in
As can be seen, the DB fusion was able to generate a higher specific cytokine response in the case of IFN-γ secreting cells and secretion levels of IL-17 and IL-2, compared to the administration of IpaB and IpaD. This demonstrates that the DB fusion protein has a unique advantage in the generation of cell-mediated immunity, which can be important for control of Shigella infection.
The detergent effects on the secondary structure of the IpaD/B fusion protein were assessed using circular dichroism (CD). The experiments were carried out using 0.3 mg.ml of protein in the presence of the detergents octyl polyoxyethylene (OPOE) and lauryldimethylamine-oxide (LDAO), compared to a combination of IpaD and IpaB proteins in the presence of OPOE, and IpaD/B fusion in phosphate buffered saline (PBS) after dialysis to remove LDAO detergent. The results are presented in
The effects of temperature on the secondary structure of DB protein in the presence and absence of OPOE and LDAO were also investigated by monitoring thermal melting using CD. The results are presented in
The thermal stability of DB protein was also investigated using CD. CD spectra of IpaD plus IpaB proteins (1:1 in 0.5% OPOE, DB in 0.5% OPOE, DB in 0.05% LDAO and DB in PBS were acquired at 10° C., and the solution was then heated to 90° C. at a rate of 15° C./hour. Spectra were reacquired after the temperature was returned to 10° C. The results are shown in
DB fusion was also characterized with respect to its ability to mimic IpaB's ability to disrupt phospholipid membranes. IpaB is well characterized and is known to form tetramers in OPOE that efficiently disrupt liposomes through pore formation. A sulforhodamine B (SRB) dye release assay was used to assess the ability of DB to do so. In the assay, membrane disruption releases fluorescent dye from lipid vesicles, giving rise to an increase in fluorescence. The results showed that IpaB in OPOE, IpaD+IpaB in OPOE and the DB fusion protein in OPOE all has similar membrane disruption efficiencies. Therefore, the DB fusion likely maintains at least some of the native structural characteristic of IpaB. (Notably, in LDAO detergent and in PBS buffer, DB and IpaB are both unable to cause membrane disruption.)
Next, the quaternary oligomeric structure of DB was investigated. Chemical crosslinking studies with and without the reducing agent dithiothreitol (DTT) and analyzed by gel electrophoresis identified the formation of extremely large DB fusion protein complexes. Both 0.5% OPOE and 0.05% LDAO supported the monomeric form of the protein to some extent (OPOE more so than LDAO), although most of the protein did not migrate into the gel even in the presence of detergent. Once dialyzed into detergent free PBS, the protein exclusively formed large complexes (much larger than 500 kDa) which were unable to migrate into the gel. Dynamic light scattering was used to determine the physical size of the DP oligomers. Consistent with crosslinking results, two predominant populations were identified: an ˜5 nm diameter population (which may represent the monomer) and an ˜25 nm population (which may represent the oligomer). In the presence of OPOE, the smaller species was primarily present while the presence of LDAO allowed the formation of the larger oligomer (which was also observed in detergent free PBS. These results showed that the DB protein is stable in LDAO for long term, but when diluted into PBS in preparation for immunization forms a large multimer that would be similar to the principle associated with virus-like particles (VLP). Thus, the multimer has a greater probability for success in humans.
Additional biophysical characterization if DB and subsequent analysis via an empirical phase diagram (EPD) has demonstrated that, in general, the fusion maintains its structure at pH 8 until higher temperatures are reached (not shown). At 60° C., the fusion protein in pH 6-7 buffer enters a molten globule state. The fusion in lower pH buffers does not exhibit exceptional stability at any temperature with high light scattering and noisy CD thermal melting curves (not shown). Interestingly, the fusion appears to exhibit mixed stability characteristics. At pH 8 the fusion appears to be more stable than IpaB with a transition temperature closer to 70° C. At pH 6, the fusion is less stable than either IpaB or IpaD associated with IpaB with an increase in stability which is more like IpaD. EPD provides a measure of the effects of physical conditions on protein stability. This in turn allows development of improved formulation taking into account conditions that are best suited to a stable vaccine and those conditions not conducive to vaccine stability. In the latter case, it also provides the basis for the screening of stabilizing agents from a library of GRAS reagents.
In the studies described in Example 1, IpaD and IpaB were produced from plasmids that were maintained in E. coli by ampicillin resistance. Since ampicillin cannot be used in proteins produced for use in humans, the ipaB and ipaD genes were moved into pET28b and pET9a, respectively. Both plasmids are kanamycin resistant and therefore the resulting proteins can be used in humans The ipaD/pET9a-Tuner (DE3) strain was used to over-express IpaD using the typical shake flask method as well as in the 10 L fermentor. Both methods produced over 10 milligrams of IpaD per liter as was seen for the initial pET15b plasmid, showing that this strain and the resulting protein are ready for vaccine development.
The ipaB/pET28b plasmid was also used to co-transform Tuner (DE3) with ipgC/pACYC-Duet1. It should be noted that protein expression of this strain is sensitive to bacterial growth media when grown in shake flasks and can be problematic. Maximal protein expression typically occurs in Terrific Broth (TB) media with overnight expression at 18° C. Nevertheless, milligram quantities of protein were purified from the co-transformed Tuner (DE3) cells. When protein expression was performed in a 10 L fermentor, excellent expression was also observed. The IpaB/IpgC complex was further purified by passage over a hydrophobic interaction chromatography (HIC) column and IpaB alone (i.e. without IpgC) was purified by immobilized metal ion affinity chromatography (IMAC) in the presence of OPOE. Triton X-100 has also been shown to release IpgC from IpaB.
A small pilot study was performed to confirm the lack of toxicity of the Ipa proteins following intranasal administration with target doses in guinea pigs. Two animals each were inoculated intranasally with 1) IpaB (25 μg)+IpaD (100 μg) or 2) IpaB (25 μg)+IpaD (100 μg)+dmLT (10 μg). Animals were observed for 5 days and all animals tolerated the vaccination well with no abnormal clinical signs. The doses were thus determined to be safe for use in the immunogenicity/protection study.
Intranasal (IN) Immunization of Guinea Pigs
An experiment was conducted to assess intranasal (IN) immunization of guinea pigs using IpaB, IpaD, IpaB/IpgC and dmLT. The doses of protein given to the guinea pigs were: 25 μg of IpaB, 100 μg of IpaD, 32 μg IpaB/IpgC and 10 μg of dmLT. The volume of inoculation was 30-50 μl (see Table 2). Immunization was performed on days 0, 14 and 28. Sera, tears (for measurement of secretory IgA) and fecal samples were collected to examine immune responses. Three weeks after the last immunization (day 49) the guinea pigs were challenged intraocularly with S. flexneri 2a 2457T (1-5×108 CFU).
The challenge results are shown in Table 3. As can be seen, 100% of animals immunized with the positive control strain CVD 1208S were protected against challenge. There were 75% and 100% attack rates in the negative control groups E and F immunized with PBS or dmLT respectively. Immunization with IpaB+IpaD+dmLT provided the highest level of protection (73%). Immunization with IpaB/IpgC+dmLT provided 20% protection. Immunization with IpaB+dmLT or IpaB/IpgC+IpaD+dmLT provided no protection against challenge.
S. flexneri 2a
S. flexneri 2a
S. flexneri 2a
S. flexneri 2a
S. flexneri 2a
S. flexneri 2a
S. flexneri 2a
Immunology data was compiled to assess serum IgG and mucosal (tears and fecal) IgA responses to each antigen. All animals that were immunized with IpaB (Groups A, B, C, D) responded with strong serum IgG anti-IpaB responses following 3 doses (
Serum and mucosal responses to IpaD were robust in Groups B and D which received the IpaD antigen (
Serum and mucosal responses to dmLT were very strong following a single dose in all groups that received the antigen (Groups A, B, C, D, F) and were boosted to higher levels following each subsequent immunization (
As expected serum and mucosal responses to S. flexneri 2a LPS were measured only in Group G, immunized with CVD 1208S (
Conclusions from intranasal immunization. Serum and mucosal responses to the IpaB and dmLT antigens were similar in Groups A, B, C, and D. Responses to IpaD were similar in Groups B and D. The only striking difference in immune responses in Group B, the highly protected group, was an earlier strong response in all animals to IpaB following 2 doses instead of following 3 doses.
Immunogenicity and protective efficacy of IpaB and IpaD when administered orally using the mouse pulmonary model are remarkably lower compared with the responses induced when these proteins are given intranasally. Thus, an intradermal delivery approach was assessed.
In order to define the adequate amounts of IpaB, IpaD and dmLT to deliver through intradermal injection, we performed a dose finding experiment (
The proteins were administered in the presence of 100 ng of E. coli dmLT. The volume of inoculum was 25 μl and it was dispensed using the NanoPass MicronJet 600 microneedle device attached to a Hamilton syringe. Mice immunized with intranasally with 2.5 μg of IpaB/IpgC and 10 μg of IpaD along with 2.5 μg of dmLT were included as positive control. Negative control groups received dmLT (100 ng) and PBS via the i.d. route. A total of 3 doses were administered on days 0, 14 and 28. Serum and stool samples were collected before the first immunization and every two weeks until day 56. On day 56, mice were subjected to lethal pulmonary challenge with Shigella flexneri 2a. Five additional animals were euthanized at this time to measure systemic ASC and cell-mediated immunity.
Serum Antibody Responses
Fecal Antibody Responses
IpaB- and IpaD- specific stool IgA titers were found in the positive control group immunized with IpaB and IpaD i.n. but not in the animals immunized i.d., regardless of the amount of antigen given. Very high stool IgA responses against dmLT were observed in the positive control group, while very little (although detectable) IgA responses were found in mice immunized i.d.
Mucosal ASC
In order to assess the induction of mucosal immunity in the lungs following intradermal immunization with the Ipas, we measured the frequency of IpaB, IpaD and dmLT-specific IgG and IgA ASC in lymphocytes from the lungs on day 56 after immunization; this time corresponds to the time of challenge (
Very strong ASC responses against dmLT were observed in the control group.
Systemic ASCs
The frequency of IpaB and IpaD-specific ASC was measured in the spleen and bone marrow of i.d. vaccinated mice on day 56 after immunization (
IgA ASC responses specific for dmLT were also induced following i.d. vaccination, although at lower levels. It is interesting to notice that while i.n. immunization led to the production of both IgG and IgA ASC in the spleen, intradermal vaccination led to the production of IgG ASC only, and this was true for all 3 antigens. Only the group immunized i.n. had detectable levels of IgA ASC in the spleen.
A similar pattern of responses was found in the bone marrow (
Cell-Mediated Immunity
We measured the production of cytokines by spleens cells obtained on day 56 after immunization. To assess antigen-specific cytokine production, cells were stimulated with IpaB or IpaD for 48 hours. The supernatants were tested using a TH1/TH2 multiplex system. The levels IFN-γ, TNF-α and IL-1 produced in culture supernatants of antigen-stimulated cells are shown in
No differences were seen in the production of IL-12, most likely because this cytokine is non-specific and mainly produced by macrophages and antigen presenting cells, rather than antigen-specific T cells. Animals immunized i.d. produced IL-1β in response to IpaB in levels comparable to those of the positive control group immunized i.n. (
When these animals were challenged with virulent organisms using the pulmonary challenge model, up to 70% protection was observed in mice immunized with the medium dosage level, as described in detail below. Surprisingly, no protection was seen in mice receiving the highest dose of IpaB and IpaD. These observations prompted us to perform a second experiment where we included groups receiving both the medium and the high doses of antigen used in the previous experiment. To confirm the immune enhancement effect of the dmLT, the proteins were given in the presence and absence of dmLT (
The kinetics of serum IgG specific for IpaB, IpaD and dmLT from the previous experiment is shown in
Mucosal ASC
To assess the induction of mucosal immunity, we measured the frequency of IgG and IgA ASC in the NALT, lungs and PP (
For the most part, these responses were lower than those of mice immunized i.n. and did not follow a defined pattern. Detectable IgA and IgG ASC responses to both IpaB were observed in the Peyer's patches of i.d. immunized mice at levels comparable with those induced in the intranasal positive control. IpaD-specific IgG and IgA responses were seen in the i.d. vaccinated mice.
Systemic ASCs
IpaB and IpaD-specific IgG and IgA ASC responses were measured in the spleen and bone marrow tissue collected on day 35 (
Antibodies in BAL
In agreement with the presence of IgG ASC in the lung tissue, we also detected IgG antibodies specific for IpaB and IpaD in bronchoalveolar lavages from mice immunized i.d. (
Conclusions: The results demonstrate that intradermal immunization of mice with IpaB and IpaD in the presence of dmLT, using the NanoPass MicronJet 600 microneedles, induce a very strong serum IgG and systemic IgG ASC response as well as Th1 and Th2 cytokine production.
Survival Against Lethal Pulmonary S. Flexneri Challenge
Mice were immunized with increasing doses of IpaB and IpaD along with dmLT on days 0, 14 and 28 as described for
Survival Against Lethal Pulmonary S. Flexneri Challenge
High and medium doses of IpaB and IpaD, along with dmLT were included; 10 mice from each group were challenged with S. flexneri on day 56 after the initial immunization. The challenge doses used was 5.4×107 CFU in a 30 μl volume. The group receiving the medium dose+dmLT again showed 70% survival (
Survival Against Lethal Pulmonary S. Sonnei Challenge
Another subgroup of 10 mice from each immunization group was challenged with S. sonnei. The challenge dose used was 1.35×108 CFU of S. sonnei in a 30 μl volume. The highest survival, 50%, was seen in the group immunized with the medium dose of proteins in the presence of dmLT (
Microneedle and adapter devices were tested for use in the guinea pig. The adapter method was chosen since this device was readily available and i.d. injection was efficient using the adapter. The intradermal experiment was completed in which guinea pigs (Hartley strain), 10 per group, were immunized i.d. with the IpaB/D vaccine candidates and dmLT as described in Table 4. The doses chosen were based on results from mouse studies wherein optimal efficacy was achieved using the middle dose tested. The middle mouse doses were adjusted for body weight of guinea pigs. For intradermal inoculation 1.0 μg IpaB, 2.5 μg IpaD and 1.0 μg dmLT were used. For the repeated intranasal immunization (Group D) inoculation with 25 μg IpaB, 100 μg IpaD and 10 μg dmLT were used.
S. flex 2a
S. flex 2a
S. flex 2a
S. flex 2a
S. flex 2a
Sera, tears (for measurement of secretory IgA) and fecal samples were collected prior to the first dose and 2 weeks following each dose to examine immune responses to each antigen included. Four weeks after the last immunization the guinea pigs were challenged intraocularly with S. flexneri 2a 2457T (1-5×107CFU).
Challenge results are shown in Table 5. 100% of animals immunized with the positive control strain CVD 1208S were protected against challenge. There was 100% attack rate in the negative control Group C immunized with PBS. Intradermal immunization with IpaB+IpaD+/−dmLT did not provide protection against challenge. Intranasal immunization with IpaB+IpaD+dmLT conferred 33% protection against challenge. However, the progression of disease in this group was slower than that in Groups A, B or C (
Immunology data was compiled to assess serum IgG and mucosal (tears and fecal) IgA responses to each antigen. All animals that were immunized with IpaB (Groups A, B and D) responded with strong serum IgG anti-IpaB responses following 2 doses (
Mucosal anti-IpaD IgA was only measured in Group D, immunized by the i.n. route. Immunization with CVD 1208S did not elicit serum or mucosal anti-IpaD responses. Serum anti-dmLT IgG responses were elicited in Groups B, and D (
Conclusions from Intradermal Immunization. Intradermal immunization was able to elicit serum anti-IpaB and anti-IpaD responses equivalent to those elicited by intranasal immunization. However, i.d. immunization did not stimulate mucosal IgA responses to IpaB, IpaD or dmLT.
Summary of Guinea Pig Studies. The guinea pig serves as an alternative model for assessing the immunogenicity and protective capacity of Shigella vaccines. The most protective regimen in these studies was i.n. immunization with IpaB+IpaD+dmLT. The combined efficacy of this regimen from the 2 experiments was 46%.
Due to the similarity of their immune response and skin thickness with humans, piglets were also chosen as an animal model. Piglets were immunized three times, two weeks apart either intradermally, sublingually or a combination of the two (Table 6).
Immune responses were measured at the time of immunization and two weeks after the last immunization. Although a serum IgG response was elicited by IpaB+IpaD alone, the highest response was seen after admixing dmLT (
The protective efficacy of the fusion protein was tested using DB fusion or DB fusion plus dmLT, in comparison to vehicle (PBS) and IPAB+IpaD+dmLT. The results are shown in
While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.
Thus, the present invention is well adapted to carry out the objects and attain the ends and advantages mentioned above as well as those inherent therein. While presently preferred embodiments have been described for purposes of this disclosure, numerous changes and modifications will be apparent to those skilled in the art. Such changes and modifications are encompassed within the spirit of this invention as defined by the appended claims.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/568,918 filed on Dec. 9, 2011, and incorporates said provisional application by reference into this document as if fully set out at this point.
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| Number | Date | Country | |
|---|---|---|---|
| 20130149329 A1 | Jun 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61568918 | Dec 2011 | US |
