WO2011149955A2 - Therapeutic method and composition - Google Patents

Abstract

Live attenuated therapeutic compositions provide a route for effective immune system priming allowing a subject to more effectively combat inevitable exposure to pathogens. Molecular targets for effective attenuation of a pathogen for use in a therapeutic composition are not commonly conserved across species, however, the expression of anchorless surface proteins is conserved across many pathogens allowing for attenuation. Exemplary embodiments provide therapeutic compositions for protection against a wide array of pathogens, and methods for attenuating pathogens to generate the therapeutic compositions.

Description

THERAPEUTIC METHOD AND COMPOSITION

Inventor: Vijay Pancholi

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC §1 19(e) to US provisional application 61 /347,729 filed on 24 May 2010, which is hereby incorporated by reference.

TECHNICAL FIELD

[0002] The present invention is directed to the fields of molecular biology and medicine. More particularly, it concerns compositions and methods of treating and preventing infectious diseases in a patient with therapeutic agents.

BACKGROUND OF THE ART

[0003] Infectious disease has long been a leading cause of death both in the U.S. and abroad. One of the best strategies for combating infection is the widespread use of therapeutic compositions.

[0004] Immunity to microbial infection by means of a prompt immune system response is one of the most effective means by which a host avoids a clinical episode. Exposure to fully pathogenic miro-organisms in the absence of an appropriate immune response often leads to full-blown infection and the associated morbidity or possibly mortality that comes with it. If a patient survives this initial exposure, their immune system will oftentimes have mounted an appropriate response and will be prepared for future exposure to the pathogen. However, it is advantageous for would-be patients to have their immune systems primed for response to pathogens without the necessity of a full-blown clinical episode.

[0005] Exposure to live but attenuated pathogenic micro-organisms by way of vaccination has proved a highly effective means of priming a host's immune system to mount an effective response to a particular pathogen. Therapeutic compositions that introduce live but attenuated micro-organisms provide the advantage that, once the animal host has been vaccinated, entry of the microbial pathogen into the host induces an accelerated recall of earlier, cell-mediated or humoral immunity which is able to control further growth of the organism before it can reach clinically significant proportions. Therapeutic compositions based on killed pathogens are generally less effective at producing this type of response.

[0006] Development of therapeutic compositions for infectious pathogens involves many challenges. Often the largest hurdle takes the form of finding a way to attenuate the virulence of the pathogen of interest while not hampering its life-cycle. Obviously the first step in this process, discovering which molecular components of the pathogen are essential for the pathogen's virulence is necessary and that must then be coupled with a breakthrough in attenuating the factor such that the pathogen may be administered to a patient for inoculation without risking full-blown infection.

[0007] For live attenuated therapeutic compositions there exists a paradox; an approach for attenuating bacteria is removal of one or more virulence factors. In many cases however, virulence factors also play a role in inducing immunity. In those cases, deletion of virulence factors unavoidably impairs the immunogenic capacity of the bacterium. A live therapeutic composition should preferably retain the antigenic capacity of the wild-type strain without being virulent.

[0008] Molecular targets that would seem to be of particular interest include those that are present in many different pathogens, prove to be essential for virulence, yet not essential for pathogen growth. Thus, a need exists for a method to attenuate the virulence pathogens while leaving the pathogen in a state that it may be administered for vaccination.

SUMMARY OF THE INVENTION

[0009] Anchorless proteins (such as glyceraldehyde-3-phosphate dehydrogenase (SDH)) have been identified on the surface of Group A Streptococcus (GAS), and other gram-positive and negative bacterial pathogens, fungal pathogens and protozoans and parasites. SDH belongs to class of surface-located metabolic enzymes that are generally found in the cytoplasm and lack C-terminus hydrophobic tails and N-terminal signal sequence required for export to the cell surface, they are thus referred to as anchorless proteins. Transposon mutagenesis or knockout mutant strains have failed to produce a strain lacking surface located SDH, as SDH is apparently essential for GAS survival. A thorough discussion of the role of SDH in various functions of GAS is presented in Boel, Jin and Pancholi; Inhibition of Cell Surface Export of Group A Streptococcal Anchorless Surface Dehydrogenase Affects Bacterial Adherence and Antiphagocytic Properties (Infection and Immunity, Oct. 2005) which is hereby incorporated by reference as though recited in its entirety. Disclosed herein is a novel method for achieving an attenuated micro-organism therapeutic composition. Potential pathogens of interest for this approach include those that cause pneumonia, diarrhea, tuberculosis etc.

[0010] Accordingly, embodiments include a pharmaceutical composition for a mammalian subject, comprising: a therapeutically effective amount of a transgenic bacterium, the transgenic bacterium comprising a modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (e.g., a modified sdh gene), the modification being sufficient to substantially inhibit surface exportation of the GAPDH protein (e.g., streptococcal GAPDH-SDH (hereinafter SDH)), thereby attenuating pathogenicity of the transgenic bacterium towards the mammalian subject, relative to a wild type bacterium; and a pharmaceutically-acceptable carrier.

[0011] In some embodiments, the modified sdh gene comprises a nucleotide sequence encoding an SDH polypeptide fused to a hydrophobic anchor peptide. In some embodiments, the hydrophobic anchor peptide comprises the amino acid sequence IVLVGLVMLLLS (SEQ ID NO:4).

[0012] In various embodiments, the transgenic bacterium may be derived from almost any pathogenic bacterial strain. In some embodiments, the transgenic bacterium is derived from a group A streptococcal strain. In other embodiments, the transgenic bacterium may be derived from various other pathogenic bacteria including, but not limited to, mycobacteria, pneumococcus, diarrheagenic bacteria, and/or anthrax bacteria. The pharmaceutical composition may further comprise an adjuvant. The composition may be formulated for intraperitoneal administration. The composition may be formulated for intranasal administration.

[0013] The pharmaceutical composition may additionally comprise at least one additional antigen, such that the additional antigen triggers an immune response that protects the subject against another disease or a pathological condition.

[0014] In various embodiments, the therapeutically effective amount of a transgenic bacterium is about 100 to about 1 x10⁸ cells. [0015] Embodiments include a method of eliciting an immune response in an animal, comprising introducing into the animal a composition comprising a therapeutically effective amount of a transgenic bacterium comprising a modified sdh gene, the modification being sufficient to substantially inhibit surface exportation of SDH, thereby attenuating pathogenicity of the transgenic bacterium towards the mammalian subject, relative to a wild type bacterium. In some embodiments, the modified sdh gene comprises a nucleotide sequence encoding an SDH polypeptide fused to a hydrophobic anchor peptide.

[0016] Embodiments also include a method of generating antibodies specific for a bacterium, comprising introducing into the animal a composition comprising a therapeutically effective amount of a transgenic bacterium comprising a modified sdh gene, the modification being sufficient to substantially inhibit surface exportation of SDH, thereby attenuating pathogenicity of the transgenic bacterium towards the mammalian subject, relative to a wild type bacterium. In various embodiments, the modified sdh gene comprises a nucleotide sequence encoding an SDH polypeptide fused to a hydrophobic anchor peptide. The introducing step may comprise a systemic route of administration.

[0017] The present embodiments provide a novel therapeutic agent and method for vaccination against pathogens. Various embodiments comprise strains of pathogens wherein the expression of one or more anchorless proteins is modified to prevent full transport.

[0018] In an exemplary embodiment a mutant strain of GAS is produced. The mutant strain has been modified to express SDH identical to wild type with an additional hydrophobic tail at its C-terminal end.

[0019] In an exemplary embodiment a mutant strain of GAS is produced, wherein the enzyme activity of modified SDH and the growth patterns of the strain that expresses the modified protein, are substantially the same as those of the wild-type strain.

[0020] Exemplary embodiments relate to the production of attenuated strains of GAS. The embodiments may relate to the modification of the expression and transport of anchorless proteins including altered SDH exportation. [0021] Exemplary embodiments provide cultures of cells of a strain derived from a pathogenic parent strain of a species of GAS, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects a patient against a clinical episode when administered as a live therapeutic composition.

[0022] Exemplary embodiments provide therapeutic compositions to protect a host against a clinical episode, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain of a species of pathogen, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects a host against a clinical episode when administered as a live therapeutic composition, and an acceptable carrier. Therapeutic compositions of the invention may further comprise one or more other components including, for example, an adjuvant. Therapeutic compositions of the present invention may be administered to any subject species susceptible to infection and disease.

[0023] Exemplary embodiments provide methods for preparing cultures of attenuated cells from a pathogenic strain of a pathogen comprising an anchorless surface protein for use in a therapeutic composition that protects a subject against a clinical episode, comprising modifying cells from a pathogenic parent strain to produce modified anchorless surface proteins; selecting and clonally propagating one or more modified cells that exhibit attenuated pathogenicity compared to cells of the parent strain; and selecting and clonally propagating one or more attenuated cells which are capable of triggering an immune response that protects the subject against a clinical episode when administered in a live therapeutic composition.

[0024] Exemplary embodiments provide methods for preparing a therapeutic composition that protects a subject against a clinical episode, comprising modifying cells from a pathogenic parent strain of a pathogen comprising anchorless surface proteins; selecting and clonally propagating those modified cells that exhibit attenuated pathogenicity compared to cells of the parent strain but which are capable of triggering an immune response in the subject that protects against a clinical episode when administered in a live therapeutic composition; and combining an immunologically effective amount of the attenuated cells with an acceptable carrier in a form suitable for administration as a live therapeutic composition to the subject.

[0025] Exemplary embodiments provide methods for vaccinating a subject against a clinical episode, comprising administering to the subject an immunologically effective amount of a therapeutic composition comprising live cells of a strain derived from a pathogenic parent strain, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the subject against disease or a clinical episode when administered as a live therapeutic composition, and an acceptable carrier. The therapeutic compositions may further comprise one or more other components including, for example, an adjuvant.

[0026] Exemplary embodiments provide combination therapeutic compositions, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the subject against a clinical episode when administered as a live therapeutic composition; one or more other antigens that trigger an immune response that protects the subject against a disease or a clinical episode; and an acceptable carrier. The combination therapeutic compositions may further comprise one or more other components including, for example, an adjuvant.

[0027] In an aspect of the invention, a virulent pathogen is attenuated by modifying transport of a protein. The protein may play a significant role in pathogenicity of the organism. In a more preferred aspect, the pathogen is attenuated by preventing transport of a protein to the surface of the organism. In an even more preferred aspect, the protein is an anchorless prokaryotic GAPDH protein (e.g., streptococcal GAPDH- SDH) SEQ ID NO:1 .

[0028] The pharmaceutical preparations of the invention may, optionally, include pharmaceutically acceptable carriers, adjuvants, fillers, or other pharmaceutical compositions, and may be administered in any of the numerous forms or routes known in the art. [0029] Exemplary embodiments include an article of manufacture comprising a composition according to any one of the aforementioned compositions within a pill, a tablet, a capsule, or a syringe.

[0030] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIGURE 1 is a schematic showing how the mutant strain (M1 -SDHHBtail) was derived from the wild-type strain (M1 -SF370) using the pFW5-SDH_HBtaii suicide shuttle vector and by achieving double crossover during allelic-exchange events.

[0032] FIGURE 2 shows the amino acid sequences of the SDH protein (only residues 1 to 20 and 301 to 336 are noted for brevity) and an inserted hydrophobic tail (³³⁷IVLVGLVMLLLS³⁴⁸; in bold) at the C-terminal end {sdh/plr in the M1 genome is annotated as SPy0274).

[0033] FIGURE 3 shows the growth characteristics of the wild-type and mutant strains of GAS.

[0034] FIGURE 4 shows a comparison of the levels of sdh-specific mRNA expression with housekeeping gene by real-time PCR.

[0035] FIGURE 5 shows a Western immunoblotting of whole bacterial lysates of equal number of wild-type and mutant strains using anti-sdh monoclonal antibody.

[0036] FIGURE 6 shows a comparison of GAPDH enzymatic activities of GAS strains.

[0037] FIGURE 7 shows a comparison of GAPDH enzyme activities of cell wall extracts (CW).

[0038] FIGURE 8 shows a comparison of GAPDH activities of intact wild-type and mutant GAS strains.

[0039] FIGURE 9 shows a Western blot analysis of the presence of SDH in culture supernatant and subcellular fraction using monoclonal antibody.

[0040] FIGURE 10 shows a semiquantitative analysis of the presence of SDH in the cytoplasm and cell wall fractions of wild-type and mutant strains. [0041] FIGURE 1 1 shows the plasminogen binding activities of serially diluted purified SDH and SDH HBtaii proteins under native conditions using the slot blot device- based protein-ligand-binding method.

[0042] FIGURE 12 shows plasminogen binding activity of M1 -WT and M1 - SDH HBtaii strains as measured in 96-well microtiter plate-based ligand-binding assay using intact bacteria and Alexafluor-488 labeled human plasminogen.

[0043] FIGURE 13 shows the growth profile for the M1 -WT and M1 -SDH HBtaii in human blood expressed as a multiplication factor.

[0044] FIGURE 14 shows a Western blot analysis of cell-wall and cytoplasm associated proteins using M1 protein reacting 10B6 monoclonal antibody.

[0045] FIGURE 15 shows the results of a bacterial adherence assay using confluent human pharyngeal cells; the M l Aemmi was used as an internal control.

[0046] FIGURE 16 shows a table of differential virulence related gene expression profiles for the M1 -SDH_HBtaii mutant.

[0047] FIGURE 17 shows a mortality curve for mice infected with M1 -SF370 or

M1 -SDH HBtail-

[0048] Figure 18 shows a comparison of the amino acid sequences of wild-type SDH (SEQ ID NO: 1 ) and an exemplary mutant SDH (SEQ ID NO: 2) having a fused hydrophobic tail.

DETAILED DESCRIPTION

[0049] Sequence Listing: the sequence listing submitted with this application titled OSU1 159-286B SEQ listing_ST25 created on 24 May 201 1 and 1 1 KB in length is hereby incorporated by reference in its entirety.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the exemplary

embodiments, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. It will be appreciated that there is an implied "about" prior to metrics such as temperatures, concentrations, and times discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0052] A "gene" or a "sequence which encodes" a particular protein is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of

appropriate regulatory sequences. The boundaries of the gene are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence. Typically, polyadenylation signal is provided to terminate transcription of genes inserted into a recombinant virus.

[0053] The term "polypeptide" or "protein" means a linear polymer of amino acids joined in a specific sequence by peptide bonds. As used herein, the term "amino acid" refers to either the D or L stereoisomer form of the amino acid, unless otherwise specifically designated.

[0054] The nucleic acid molecules and or polypeptides are not limited strictly to molecules including the sequences set forth. Rather, specific embodiments encompasses nucleic acid molecules carrying modifications such as substitutions, small deletions, insertions, or inversions, which nevertheless encode proteins having substantially the biochemical activity of the polypeptide according to the specific embodiments, and/or which can serve as hybridization probes for identifying a nucleic acid with one of the disclosed sequences. Included in the invention are nucleic acid molecules, the nucleotide sequence of which is at least 70% identical (e.g., at least 75%, 85%, 95%, or 99% identical) to the nucleotide sequences shown. The amino acid sequences include sequences at least 60% identical (e.g., at least 75%, 85%, 95%, or 99% identical) to the sequences shown.

[0055] The determination of percent identity or homology between two sequences is accomplished using the algorithm of Karlin and Altschul (1990) Proc. Nat'l Acad. Sci. USA 87: 2264-2268, modified as in Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the N BLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

[0056] The term "transgene" refers to a particular nucleic acid sequence encoding a polypeptide or a portion of a polypeptide to be expressed in a cell into which the nucleic acid sequence is inserted. The term "transgene" is meant to include (1 ) a nucleic acid sequence that is not naturally found in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in the cell into which it has been inserted; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or a similar nucleic acid sequence naturally occurring in the cell into which it has been inserted; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been inserted. By "mutant form" is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and/or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that the transgene product may be secreted from the cell.

[0057] The term "attenuated" as used herein describes a cell, culture, or strain of a pathogen exhibiting a detectable reduction in infectivity or virulence in vitro or in vivo as compared to that of the parent strain of the pathogen from which the attenuated cell, culture, or strain is derived. Reduction in virulence (or pathogenicity) encompasses any detectable decrease in any attribute of virulence, including infectivity in vitro or in vivo, or any decrease in the severity or rate of progression of any clinical symptom or condition associated with infection.

[0058] The term "parent strain" refers to a strain of the pathogen which exhibits a relatively higher degree of pathogenicity when administered to a subject than an attenuated strain which is derived therefrom by one or more passages in vivo or in vitro and/or one or more attenuation steps. The term parent strain may include the wild type strain as understood by those of skill in the art.

[0059] An effective amount of an agent of the invention will generally be a therapeutically effective amount. A "therapeutically effective amount" generally refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as lysis of a target cell. A therapeutically effective amount a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also generally one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

[0060] The live attenuated cells of the invention are capable of triggering an immune response that protects a subject against a clinical episode after one or more administrations as a live therapeutic composition. A "protective immune response" is defined as any immunological response, either antibody or cell mediated immunity, or both, occurring in the subject that either prevents or detectably reduces subsequent infection, or eliminates or detectably reduces the severity, or detectably slows the rate of progression, of one or more clinical symptoms or conditions associated with the infectious agent. The term "immunologically effective amount" refers to that amount or dose of therapeutic composition or antigen that triggers a protective immune response when administered to a subject. A "clinical episode" refers to the onset of conditions or symptoms consistent with infection by the relevant micro-organism and may also include a protective immune response.

[0061] Exemplary embodiments provide therapeutic compositions against a clinical episode, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the subject against a clinical episode when administered as a live therapeutic composition, and optionally, acceptable carrier.

[0062] Exemplary embodiments further provide methods for preparing a therapeutic composition that protects a subject against a clinical episode, comprising modifying cells from a pathogenic parent strain; selecting and clonally propagating those modified cells that exhibit attenuated pathogenicity compared to cells of the parent strain but which are capable of triggering an immune response in the subject that protects against a clinical episode when administered in a live therapeutic composition; and combining an immunologically effective amount of the attenuated cells with an acceptable carrier in a form suitable for administration as a live therapeutic composition to the subject.

[0063] Exemplary embodiments further provide methods of vaccinating a subject against a clinical episode, comprising administering to the subject an immunologically effective amount of a therapeutic composition comprising live cells of a strain derived from a pathogenic parent strain, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the subject against a clinical episode when administered as a live therapeutic composition, and an acceptable carrier. [0064] Therapeutic compositions of the invention comprises live cells of an attenuated strain of a pathogen, either as the sole antigenic component or in

combination with one or more other antigens that trigger an immune response that protects a subject against a, clinical episode, disease or pathological condition which may or may not be related. Thus, the present invention further provides combination therapeutic compositions, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain, which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the subject against infection by the virulent strain when administered as a live therapeutic composition; one or more other antigens that trigger an immune response that protects the subject against a disease or a

pathological condition; and an acceptable carrier. The combination therapeutic compositions may further comprise, one or more other components including, for example, an adjuvant.

[0065] The therapeutic composition is conventionally administered parenterally, for example, either by subcutaneous or intramuscular injection. However, the therapeutic composition may also be administered by intraperitoneal or intravenous injection, or by other routes, including orally, intransally, rectally or vaginally, and where the therapeutic composition is so administered, an acceptable carrier is appropriately selected. The therapeutic composition may simply comprise attenuated cells in culture fluid, which are administered directly to the subject. Alternatively, the therapeutic composition may comprise attenuated cells combined with a veterinarily or

pharmaceutically acceptable carrier selected from those known in the art based on the route of administration and its ability to maintain cell viability. Non-limiting examples of such carriers include water, saline, buffered vehicles and the like. Suitable other therapeutic composition vehicles and additives are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.

[0066] The therapeutic composition may further comprise one or more other components such as an immunomodulatory agent including, for example, interleukin-1 , or another immuno-enhancing substance such as a veterinarily acceptable adjuvant. Non-limiting examples of adjuvants include Freund's complete and incomplete adjuvants, mineral gels including, for example, aluminum hydroxide, and oil-in-water or water-in-oil formulations. Immunomodulatory agents are selected based on their ability to maintain both viability of the attenuated cells and ability of the cells to trigger a protective immune response in the vaccinated subject.

[0067] An effective dosage may be determined by conventional means, starting with a low dose of attenuated cells and then increasing the dosage while monitoring the effects, and systematically varying the dosage as well. Numerous factors may be taken into consideration when determining an optimal dosage per host. Primary among these is the species, the size of the subject, the age of the subject, the general condition of the subject, the presence of other drugs in the subject, the virulence of a particular strain against which the subject is being vaccinated, and the like. The actual dose would preferably be chosen after consideration of the results of other subject studies.

EXAMPLES

[0068] The following examples are included to demonstrate embodiments. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

[0069] General Methods

[0070] Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used, for example, for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid, and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

2000). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of described herein are those well known and commonly used in the art.

[0071] Bacterial strains, vectors, growth conditions, and DNA techniques. S. pyogenes wild-type strain M1 -SF370 (M1 -WT; ATCC 700294; American Type Culture Collection, Manassas, VA) was grown in Todd-Hewitt broth (Difco Laboratories) supplemented with 0.5% yeast extract or on proteose peptone-3 blood agar plates supplemented with spectinomycin (up to 500 μ9/ιηΙ), when required. E. coli strain XL1 - Blue was used for the cloning experiments and grown in Luria-Bertani (LB) broth or on LB agar plates. Vector pFW5 containing the spectinomycin resistance gene (aad-9) was used to transform the M1 -SF370 strain.

[0072] Construction of insertion and allelic-exchange

mutations in GAS. Gram-positive bacteria surface proteins contain a signal sequence, an LPXTGX hexapeptide motif, and a hydrophobic tail for anchoring in the membrane lipid bilayer and subsequently for surface export. To create streptococcal mutant strains expressing SDH with a hydrophobic tail (IVLVGLVMLLLS) at the C-terminal end, but still lacking a signal sequence, the sdh gene (0.92-kb DNA fragment) and a 1 .1 18-kb DNA fragment corresponding to the downstream region of the sdh gene in the M1 - SF370

chromosome were PCR amplified using primer pairs SDH-Sall-F-/SDHBamHI-R (5'- ACGCGrCG/ACATGGTAGTTAAAGTTGGTATTAACGG-3' (SEQ ID NO: 5) and 5' CGCGG/ArCCTTATTTAGCAATTTTTGCGAAGTACTCAAGAGTACG-3' (SEQ ID NO: 6)) and SDH_DwN-Pstl-F/SDH_DwN-Ndel-R (5'-TTTTCrGC4GCTTGGTTTATGCT

GAGTTCTTTTTCG-3 (SEQ ID NO: 7) and 5'-GGGAATTCC>¾7^",47GTATTGGAAAATGC TATCGTGCG-3' (SEQ ID NO: 8)), respectively. As shown schematically in Figure 1 , the PCR-amplified products were cloned in the multiple cloning sites located upstream and downstream of the spectinomycin resistance gene, aad-9, in the suicide plasmid pFW5 (2.7 kb) to construct pFW5-sdh. To insert a DNA sequence encoding the putative hydrophobic tail at the 3' end of the sdh gene in the pFW5-sdh vector, a QuickChange II site-directed mutagenesis kit was used according to the manufacturer's instructions (Stratagene) with the plasmid pFW5-sdh as the template and a pair of complementary primers, SDH_HBtaii-F (5 '-TACTTCG C A AA AATTG CTA AAATTGTTCTTGTTGGCTTAGT TATGCTTCTTC TTTCTTAATAGGATCCTCGAGCTCTAG-3' (SEQ ID NO: 9)) and S DHHBtaii-R (5'-CTAGAGCTCGAGGATCCTATTAAGAAAGAAGAAGCATAACTAAG CCAACAAGAA CAATTTTAGCAATTTTTGCGAAGTAC-3' (SEQ ID NO: 10)). The resulting plasmid, pFW5-sdh_HBtaii, was introduced into strain M1 -WT by electroporation, using a Gene Pulser II electroporator (Bio-Rad). The mutant strain thus obtained was called M1 -SDH_HBtaii (SEQ ID NO:3). The confirmation of mutations and insertions at various stages of the experiments was obtained by one or more methods of PCR, DNA sequencing, and Southern hybridization. Using a similar strategy, a mutant strain lacking the M1 protein (M1 Demml ) was created. The primers used to create this mutant were emm1 -F (5'-CGCG7CG4CTAGGTCAAAAAGGTGGC-3' (SEQ ID NO: 1 1 )), emm1 -R (5'-CGCGG/4rCCGCATTCTCTAATCTCGCTT-3' (SEQ ID NO: 12)), emm1 _DwN-F (5'-AAC7GC4GGACTTGGACGCATCACGTGAA-3' (SEQ ID NO: 13)), and emml DWN-R (5 '-G ATTC CA TA 7GCTGTCTCTTAGTTTCCTTCATTGG TGC-3' (SEQ ID NO: 14)). The italicized sequences in these primers represent Sail, BamHI, Pstl and Ndel restriction sites, respectively. PCR, DNA sequencing, and Southern hybridization assays with the mutant strain M1 -SDH_HBtaii confirmed that the two-crossover allelic exchange event between the M1 -SF370 genome and pFW5sc//?HBtaii occurred only at the desired locus.

[0073] Cell fractionation and whole-cell extraction. M1 -WT and M1 -SDH_HBtaii (mutant) strains were grown to late log phase/early stationary phase (optical density at 600 nm [OD₆₀o], 0.8), and bacteria were harvested by centrifugation. The culture supernatants from these strains were saved, and the proteins therein were precipitated with 25% trichloroacetic acid (TCA). The resulting precipitates were separated by centrifugation (20,000 x g for 30 min at 4°C), washed twice with acetone, and

suspended in 1 /50 of the original volume of 50 mM Tris-HCI, pH 7.0. The harvested bacteria were subjected to mutanolysin treatment in a cell wall-extracting buffer (50 mM Tris-HCI, pH 7.0, 30% raffinose) containing 250 U/ml of mutanolysin (Sigma) as described previously to achieve nearly complete wall extraction. The resulting

protoplasts were lysed in a hypotonic buffer, followed by repeated freezing and thawing. Bacterial membrane and cytoplasmic fractions were separated from the lysed

protoplasts by ultracentrifugation (100,000 x g for 45 min at 4°C) as described previously.

[0074] SDH and SDH_HBtaii purification. Clear whole lysates obtained as described above from 500 ml THY broth culture with each of the M1 -WT and M1 - SDHHBtaii strains were dialyzed against 0.05 M NH4HC03 and precipitated at 40%, 60%, and 80% ammonium sulfate saturation. The protein precipitates obtained from 60% to 80% ammonium sulfate saturation, which contained the major amount of SDH and SDH_HBtaii, were purified by Hi-trap Blue-HP column (1 ml) chromatography

(Pharmacia) after equilibration with a starting buffer (50 6238 BOEL ET AL. INFECT. IMMUN. mM Tris-HCI, pH 7.4). Proteins other than SDH and SDH_HBtaii were eluted first by the five-column volume of starting buffers containing 0.5 M NaCI followed by the five- column volume of the same buffer containing 1 .OMNaCI. The remaining column-bound proteins were eluted with the five-column volume of the starting buffer containing 5 mM NAD. The NAD-eluted fractions (1 ml each) contained only SDH or SDH_HBtaii, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The pooled fractions containing SDH or SDHHBtaii were dialyzed against the starting buffer and stored at -70 °C until further use.

[0075] Preparation of monospecific anti-SDH antibody. Anti-SDH specific immunoglobulin G (IgG) was purified from the SDH-immunized rabbit serum using an SDH-linked diaminodipropylamine M-phase (Pierce) affinity column. The purified IgG was then treated with immobilized papain enzyme (Pierce) at 4°C overnight to cleave them between their Fab- and Fc regions. The latter was then removed by passing the cleaved products on a protein A column (Pierce) per the manufacturer's instructions. The unbound initial fall-through was used as Fab-specific anti-SDH IgG.

[0076] Electrophoresis on SDS-polyacrylamide gels and Western blotting. Purified SDH, SDH_HBtaii, and cell wall-associated proteins were separated by SDS- PAGE and Western blotted onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the presence of SDH was detected by reacting PVDF membranes with anti-SDH monoclonal antibody (clone 13D5) or monospecific rabbit anti-SDH IgG followed by corresponding alkaline phosphatase-labeled conjugate antibody (Fab- specific). SDH-specific protein bands on PVDF membranes were visualized using a chromogenic or a chemiluminescence-based substrate.

[0077] GAPDH activity. The ability of SDH and SDH_{H B}taii to convert D- glyceraldehyde-3-phosphate (G-3-P) to 1 ,3-diphosphoglycerate in the presence of NAD- was measured spectrometrically at 340 nm. Briefly, the reaction was initiated by adding 3 μg of purified SDH or SDHHBtail proteins or 6 μg of each cell wall extract (strains M1 - SF370 and M1 -SDHHBtail) in a reaction buffer at a final volume of 600 μΙ containing 50 mM Tris-HCI, pH 7.4, 4 mM NaH₂P0₄, 4 mM NAD, 4 mM G-3-P, and 1 mM

dithiothreitol. The conversion of NAD to NADH was monitored every 10 s

spectrometrically (Beckman Coulter model DU640). To measure the GAPDH activity of intact bacteria, instead of a kinetic measurement as described above, only an endpoint OD₃₄₀ was measured. M1 -WT and M1 _HBtaii strains were grown until late log phase (OD₆oo = 0.8) and washed twice in phosphatebuffered saline (PBS) to finally adjust the OD₆oo of the bacterial suspension to 1 .0 (~5 x 10⁸ CFU). One milliliter of washed bacterial suspension was centrifuged, and the 600 μΙ of complete enzyme reaction was resuspended as described above for 5 min. At the end of incubation, the bacteria were removed by centrifugation and the absorbance of the supernatant was measured at 340 nm as described above.

[0078] Real-time quantitative PCR. Total RNA was extracted from M1 -SF370 and M1 -SDH_HBtaii strains grown to an OD of 0.8 using a commercially available RNA isolation kit (RNAwiz; Ambion). The total RNA (2 μg) isolated from the wild-type and mutant strains were converted to first-strand cDNA using an SYBR green iScript cDNA synthesis kit (Bio-Rad) in a final volume of 40 μΙ. Real-time PCR was performed in a reaction mixture (25 μΙ) containing 2.5 mM MgCI2, 250 μΜ deoxynucleoside

triphosphate mix, 1 .25 U of AmpliTaqGold DNA polymerase, 500 pM of each primer, and 1 μg of template. Gene-specific primer pairs used in this study were as follows: sdh (sdh-F, 5'-ATTAACGGTTTCGGTCGTATCGGACG-3' (SEQ ID NO: 15); sdh-R, 5'-GGATCACGTTCAGCAGAAACTTTG-3' (SEQ ID NO: 16)),

gyrA (gyrA-F, 5'-TTCGTATGGCTCAGTGGTTTAGTT-3' (SEQ ID NO: 17);

gyrA-R,5'-CTGGTTCTCTTTCGCTTCCATCGT-3' (SEQ ID NO: 18)), and

proS (proS-F, 5'-GGGTGGTTCTTGACAAGTCTATTGCG-3' (SEQ ID NO: 19); proS-R, 5 '-TTCTG CCAAG G CATCTTCAG CA-3 ' (SEQ ID NO: 20)).

The mRNA copies specific to sdh were normalized with those specific to the

housekeeping genes gyrA and proS for the M1 -WT and M1 -SDH_HBtaii strains, and data were evaluated as relative mRNA copy number ratios. Different concentrations of genomic DNA were used to obtain a standard curve for each gene. The reaction mixtures were incubated for 10 min at 95 °C to activate DNA polymerase, followed by 40 cycles each of denaturing at 95 °C for 30 s, annealing at 58 °C for 50 s, and extension at 72 °C for 30 s. Fluorescence was measured in every well during each annealing step throughout the course of each reaction. Data were automatically analyzed by software of the real-time PCR instrument (Stratagene Mx-4000).

[0079] Immunofluorescence microscopy. The M1 -WT and M1 -SDH_HBtaii strains (106 CFU/ml of PBS) were attached to polylysine-treated eight-well glass slides and blocked with DAKO blocking buffer. Adherent bacteria were incubated with affinity- purified anti-SDH IgG (Fab' fragment, diluted 1 :10 in DAKO dilution buffer) at 4°C for 8 h. The wells were washed several times with PBS and reacted with fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (1 :50 dilution) and incubated at room temperature for 1 h. The slides were washed several times with PBS, and the bacteria were stained with DAPI (4',6'-diamidino-2-phenylindole, 1 μg/ml) in SlowFade equilibration buffer (Molecular Probes) for 5 min. The stained bacteria were kept in SlowFade buffer containing glycerol and observed under a Nikon Eclipse C600 fluorescence microscope coupled with a Diagnostic RT color camera and the Spot software (v3.5.9). Bacterial cells stained with only conjugated antibody were treated as a control.

[0080] Fluorescence labeling of plasminogen. Human Pig was labeled with AlexaFluor-488-carboxylic acid succinimidyl ester (antibodies Em 495 and Em 519; Molecular Probes) using EDC (1 -ethyl-3-[3-dimethylamino-propyl]carbodiimide) as per the manufacturer's instructions. Fluorescence of the labeled plasminogen was measured with a POLARstar Galaxy fluorimeter (BMG Labtechnologies GmbH,

Offenburg, Germany). The values of fluorescence units versus different amounts of labeled plasminogen (1 to 5 μg) were plotted to obtain a standard curve.

[0081] Plasminogen-binding assay. Pig-binding activities of purified SDH proteins (SDH and SDH_HBtaii) were determined using a solid-phase ligand-binding assay and a slot blot apparatus (Bio-Rad). The binding of Pig to serially diluted SDH proteins was detected by rabbit anti-human Pig antibody (DAKO, Denmark), followed by corresponding alkaline-phosphatase-labeled conjugate. To determine Pig-binding activities of the intact M1 -WT and M1 -SDH HBtaii strains, late-log-phase THY broth cultures (OD₆oo, 0.8) were adjusted to an OD₆oo of 1 .0 after two washes in 50 mM Tris, pH 8.0, buffer. The cultures were then centrifuged, and the resulting bacterial pellets were resuspended in TBST (Tris-buffered saline containing 0.5% Tween 20, 0.5% gelatin, and 0.5% bovine serum albumin) blocking buffer in a final volume that was 1 /10 of the original volume. GAS strains (100 μΙ, ~2 x10⁹ CFU) were then mixed with twofold serially diluted labeled Pig in a deep-well 96-well plate for 2 h under constant mixing at room temperature. At the end of incubation, unbound labeled Pig was removed and the amount of bound Pig was determined with a fluorimeter as described above. Each dilution of Pig was tested in triplicate wells. Based on the standard curve, the exact amount of Pig bound to GAS strains was calculated. Data were statistically evaluated by unpaired f test with Welch's correction.

[0082] Phagocytosis/bactericidal assays. The ability of M1 -WT and M1 -

SDH HBtaii to resist phagocytosis and survive in heparinized human blood was

determined by a bactericidal test. An overnight culture of each bacterial strain was diluted 1 :50 in THY broth and grown without agitation to an OD₆₀o of 0.15 at 37°C in a C0₂ incubator. The bacterial suspension was diluted in THY broth (1 :10,000) to obtain ~4 x 10³ CFU/ml for the initial inoculum of the assay. The exact number of bacteria added to the blood was determined by plating the bacterial culture and counting CFU on sheep blood agar plates. Fifty microliters of the suspension containing approximately 200 CFU was added to 1 ml of freshly heparinized blood in sterile glass tubes (100 by 10 mm) and was incubated with end-over-end rotation at 37°C for 3 h. The number of CFU in the inoculum used and those obtained after growth in human blood were quantified by the pour plate method using sheep blood agar plates. Blood for these experiments was drawn from healthy volunteers in 10-ml Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing 143 U of freeze-dried heparin.

Opsonophagocytosis assays were performed essentially in the same way as the bactericidal assays except that the former assays were carried out in the presence of increasing amounts of purified anti-SDH antibody (SDH-specific IgG).

[0083] Estimation of the hyaluronic acid of the bacterial capsule. The hyaluronic acid capsule produced by wild-type, mutant, and complemented mutant strains was measured. Briefly, a standard curve was generated by the addition of 2 ml of a chromogenic reagent {20 mg of 1 -ethyl-2- [3-(1 -ethylnaphtho-[1 ,2-c/]thiazolin-2- ylidene)-2-methylpropenyl]naptho-[1 ,2-d thiazolium bromide (Kodak) and 60 μΙ of glacial acetic acid in 100 ml of 50% formamide} into different concentrations of hyaluronic acid (0 to 30 μ9/ιτιΙ) in a 50-μΙ volume (ICN/MP Biomedicals LLC) and measuring absorbance at 640 nm. Bacterial cells from a 10-ml late-log-phase culture were washed with water and resuspended in 0.5 ml water. Capsule was released by shaking with 1 ml of chloroform and centrifuged. The aqueous phase (50 μΙ) was then processed as described above, and the hyaluronic acid content was determined by comparing the absorbance values with the standard curve as described above.

[0084] Streptococcal adherence assays. Streptococcal adherence to Detroit pharyngeal cells was carried out in 24-well tissue culture plates. Briefly, overnight cultures of M1 -WT and M1 -SDH_HBtaii were washed and adjusted to an OD₆oo of 1 .0 in minimal essential medium without fetal bovine serum. Cells were infected with these strains (multiplicity of infection, 50 bacteria to 1 cell, ~4 x 10⁷ to 5 x 10⁷ CFU/well) and incubated in a humidified C0₂ incubator at 37°C for 3 h. Nonadherent bacteria were removed by repeated washing, Detroit cells with adherent bacteria were lysed, and the resulting cell lysates containing cell-associated bacteria were counted as CFU on sheep blood agar plates.

[0085] Figure 2 shows the strategy to insert a hydrophobic tail at the C-terminal end of SDH. (A) Amino acid sequences of the SDH protein (only residues 1 to 20 and 301 to 336 are noted for brevity) and an inserted hydrophobic tail

(³³⁷IVLVGLVMLLLS³⁴⁸; in bold) at the C-terminal end (sdh/plr m the M1 genome is annotated as SPy0274). The sequence of the hydrophobic tail for SDH was derived from that of the hydrophobic tail portion (²⁰²⁰IVLVGLGVMSLLLGMVLY²⁰³⁷ (SEQ ID NO: 21 )) of the product of the ep gene (SPy0737) of the M1 -SF370 genome.

[0086] Figure 3 demonstrates that M1 -WT and and M1 -SDH_HBtaii strains grew similarly in THY broth throughout the lag and log phases of the growth period and until the stationary phase. It is apparent that the hydrophobic tail at the C-terminal had no effect on bacterial growth.

[0087] To determine the effect of the insertion of a hydrophobic tail at the C- terminal end of SDH on protein expression, first SDH-specific mRNA expression was measured in wild-type and mutant strains grown to the late log phase (OD600 of 0.8) by real-time quantitative PCR. Figure 4 demonstrates that the expression of SDH-specific mRNAs with respect to housekeeping gene {gyrA and proS)-specific mRNAs in both the wild-type and mutant strains was found to be essentially the same without a statistically significant difference {gyrA, P = 0.137; proS, P = 0.213).

[0088] Figure 5 shows that western immunoblot analysis of whole-cell extract using anti-SDH antibody indicated the presence of SDH and the SDH_HBtaii protein in their respective strains. Thus, the introduction of hydrophobic residues at the C terminus of SDH does not affect metabolic functions or the growth of the resultant M1 -SDH_HBtaii strain.

[0089] The wild-type SDH and mutant SDH_HBtaii proteins were purified and as described above from whole-cell extracts and their GAPDH activities were determined using the same amount of protein. Figure 6 demonstrates that both SDH and

SDHHBtaii show similar kinetics for the conversion of G-3-P to 1 ,3-diphosphoglycerate in the presence of NAD, indicating that the hydrophobic tail does not interfere with the catalytic activity of the protein.

[0090] To demonstrate the effect of insertion mutagenesis in the sdh gene on the surface export of SDH, initially GAPDH activities per unit amount of total protein of the whole-cell extract of the wildtype and mutant strains were determined. In both cases, ratios of the slopes of the kinetic activities were found to be similar. However, as Figure 7 shows, the GAPDH activity of the cell wall extract of the mutant M1 -SDH_HBtaii strain was 5.5-fold or 76% less than in the cell wall extracts of the wild-type M1 -SF370 strain, indicating that the insertion of the hydrophobic tail at the C-terminal end of SDH results in its retention in the cytoplasm, while significantly affecting its export to the surface and, hence, its association with the cell wall. Figure 8 shows that GAPDH activity of the intact mutant M1 -SDH_HBtaii strain was also found to be significantly less than that of the wildtype M1 -WT strain, confirming the above results.

[0091] The presence of SDH was detected by Western immunoblot analysis using SDH-specific monoclonal antibody (13D5). The results, shown in Figures 9 and 10, showed the minimal amount of secreted SDH in the supernatant of both the wild- type and mutant strains. The immunoblot analysis of serially diluted samples derived from equal amounts of cell wall-associated proteins from these two strains showed that the cell wall fraction of the mutant strain contained approximately fourfold less SDH than the cell wall fraction of the wild-type strain. On the other hand, the cytoplasmic fraction of the mutant strain contained more SDH than that in the cytoplasmic fraction of the wild-type strain. These results corroborated those obtained by enzyme kinetic assay and confirmed that the insertion of a hydrophobic tail at the C-terminal end of SDH results in the retention of SDH primarily in the bacterial cytoplasm and to some extent also within the bacterial cell walls. These results indicate that the hydrophobic tail may interfere with the secretion of SDH.

[0092] Role of SDH in plasminogen binding. Both SDH/PIr and SEN directly bind Pig; however, their relative contributions to the overall Pig-binding activity of GAS are not known. To determine the contribution of SDH to the Pig binding activity of GAS, serially diluted purified SDH and SDH_HBtaii proteins (starting concentration, 2 μg each) were subjected to Pig binding and then detection of Pig binding by anti-Pig antibody in a slot blot-based direct ligand-binding Western blot assay. Figure 11 shows that Pig- binding activities of serially diluted SDH and SDH_HBtaii revealed that both proteins bind to Pig equally, indicating that the insertion of hydrophobic tail at the C-terminal end of SDH does not interfere with the Pig-binding activity of the SDH molecule. To determine Pig binding activities of the wild-type and mutant strains, each strain (10⁹ CFU) was mixed with various concentrations of AlexaFluor-488-labeled Pig, and the amount of bound Pig was estimated based on the standard curve (labeled Pig versus fluorescence units) incorporated in each assay. Figure 12 shows that at the highest concentration (300 nM) of exogenously added labeled Pig, the mutant strain acquired almost 60% less Pig than the wild-type (M1 -WT) strain (-2.4 pmol in the mutant strain versus -6.0 pmol in the wild type; P < 0.001 ), indicating that surface expressed SDH plays a significant role in plasminogen-binding activity.

[0093] Contribution of SDH to the antiphagocytic activity of GAS.

Antiphagocytic properties of the wild-type and mutant strains were determined by measuring their abilities to multiply in fresh human blood during a 3-h incubation period as described above. The numbers of live bacteria present before and after incubation were determined by counting CFU on blood agar plates. As shown in Figure 13, the results expressed in the form of multiplication factors (MF) (the number of CFU at 3 h over the number of CFU at time zero) revealed that, whereas the wild-type (M1 -WT) strain survived in the blood (MF = 59.63 ± 6) as expected, the mutant strain (M1 - S DHHBtaii) were readily killed (MF = 0.58 ± 0.36), indicating that the antiphagocytic activity of the mutant strain was completely inhibited (99% inhibition ; P < 0.0001 ). The ΔΕΜΜ1 strain was used as a negative control. SDH, therefore, either directly or indirectly is involved in the antiphagocytic process of GAS.

[0094] Western immunoblot analysis of the cell wall as well as cytoplasm- associated proteins of the wild-type and mutant strains was carried out using the class I M protein-specific 1 0B6 mouse monoclonal antibody (raised against the M6 protein). Figure 14 illustrates the absence of the expression of M1 protein in both fractions of the M1 -SDH_HBtaii mutant strain. In a parallel study of capsular polysaccharide estimation, no significant change (P > 0.05) in the capsule polysaccharide associated with the mutant M1 -SDH_HBtaii strain (22.6 0.1 μ9/ιηΙ) from that in the wild-type strain (20.3 0£ §1π\\) was found. Together, these results demonstrate that the insertion of the hydrophobic tail at the C-terminal end of SDH affects M1 protein expression but not capsule formation in GAS.

[0095] Contribution of SDH in GAS adherence. M1 -WT and M1 -SDH_HBtaii strains were examined for their ability to adhere to pharyngeal cells. The GAS adherence assay revealed that the mutant strain, M1 -S DHHBtaii, adhered to pharyngeal cells threefold less (-70% less) than the wild-type strain Figure 15. The results showed that the M1 Aemm1 mutant strain adhered in a way similar to that of the wild-type strain (P = 0.268), indicating that SDH and not the M protein plays an important role in GAS adherence to pharyngeal cells.

[0096] Figure 16 shows the results of a differential virulence related gene expression for M1 -SDH_HBtaii mutant. The expression results show down-regulation of major virulence related genes along with down-regulation of carbohydrate metabolism genes and up-regulation of lipid biosynthesis genes.These results show that SDH may be a novel regulator of micro-organism virulence.

[0097] Figure 17 shows a Kaplan-Meier survival curve for mice infected with M1 SF370 (Wild-type) or M1 SDH_HBtaii- Note the loss of virulence by prevention of surface exportation of SDH. The M1 -SDH_HBtaii mutant was injected intraperitonally for this experiment.

[0098] Amino acid sequences for wild-type SDH (SEQ ID NO:1 ) and SDH_{H B}taii (SEQ ID NO:2) are set forth in Figure 18.

[0099] References

The following documents are hereby incorporated by reference (there is no admission thereby made with respect to whether any of the documents constitute prior art with respect to any of the claims):

[00100] Ferretti, J. J., W. M. McShan, D. Ajdic, D. J. Savic, G. Savic, K. Lyon, C. Primeaux, S. Sezate, A. N. Suvorov, S. Kenton, H. S. Lai, S. P. Lin, Y. Qian,

H. G. Jia, F. Z. Najar, Q. Ren, H. Zhu, L. Song, J. White, X. Yuan, S. W. Clifton, B. A. Roe, and R. McLaughlin. 2001 . Complete genome sequence of an M1 strain of

Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98:4658-4663.

OTHER EMBODIMENTS

[00101] It is to be understood that while various embodiments have been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, combinations and modifications are within the scope of the following claims.

Claims

Claim 1 . A pharmaceutical composition for a mammalian subject, comprising:

a therapeutically effective amount of a transgenic bacterium, the transgenic bacterium comprising a modified sdh gene, the modification being sufficient to substantially inhibit surface exportation of SDH, thereby attenuating pathogenicity of the transgenic bacterium towards the mammalian subject, relative to a wild type bacterium; and

a pharmaceutically-acceptable carrier.

Claim 2. The pharmaceutical composition of claim 1 , wherein:

the modified sdh gene comprises a nucleotide sequence encoding an SDH polypeptide fused to a hydrophobic anchor peptide.

Claim 3. The pharmaceutical composition of claim 2, wherein:

the hydrophobic anchor peptide comprises the amino acid sequence IVLVGLVMLLLS.

Claim 4. The pharmaceutical composition of claim 1 , wherein the transgenic

bacterium is a group A streptococcal strain.

Claim 5. The pharmaceutical composition of claim 1 , further comprising an

adjuvant.

Claim 6. The pharmaceutical composition of claim 1 , wherein the composition is formulated for intraperitoneal administration.

Claim 7. The pharmaceutical composition of claim 1 , wherein the composition is formulated for intranasal administration.

Claim 8. The pharmaceutical composition of claim 1 , further comprising an antigen, such that the antigen triggers an immune response that protects the subject against another disease or a pathological condition, and an acceptable carrier.

Claim 9. The pharmaceutical composition of claim 1 , wherein:

the therapeutically effective amount of a transgenic bacterium is from about 100 cells to about 1 x10⁸ cells.

Claim 10. A pharmaceutical composition for a mammalian subject, comprising:

a therapeutically effective amount of a transgenic bacterium, the transgenic bacterium comprising a modified prokaryotic GAPDH gene, the modification being sufficient to substantially inhibit surface exportation of a prokaryotic GAPDH protein, thereby attenuating pathogenicity of the transgenic bacterium towards the mammalian subject, relative to a wild type bacterium; and

a pharmaceutically-acceptable carrier.

Claim 1 1 . A method of eliciting an immune response in an animal, comprising

introducing into the animal a composition comprising a therapeutically effective amount of a transgenic bacterium comprising a modified sdh gene, the modification being sufficient to substantially inhibit surface exportation of SDH, thereby attenuating pathogenicity of the transgenic bacterium towards the mammalian subject, relative to a wild type bacterium.

Claim 12. The method of claim 1 1 , wherein:

the modified sdh gene comprises a nucleotide sequence encoding an SDH polypeptide fused to a hydrophobic anchor peptide.

Claim 13. The method of claim 1 1 , wherein the introducing step comprises a

systemic route of administration.

Claim 14. The method of claim 13, wherein the systemic route of administration is by parenteral administration.

Claim 15. The method of claim 1 1 , wherein the transgenic bacterium is a group A streptococcal strain.

Claim 16. A method of generating antibodies specific for a bacterium, comprising introducing into the animal a composition comprising a therapeutically effective amount of a transgenic bacterium comprising a modified sdh gene, the modification being sufficient to substantially inhibit surface exportation of SDH, thereby attenuating pathogenicity of the transgenic bacterium towards the mammalian subject, relative to a wild type bacterium.

Claim 17. The method of claim 16, wherein:

the modified sdh gene comprises a nucleotide sequence encoding an SDH polypeptide fused to a hydrophobic anchor peptide.

Claim 18. The method of claim 16, wherein the introducing step comprises a systemic route of administration.

Claim 19. The method of claim 18, wherein the systemic route of administration is by parenteral administration.

Claim 20. The method of claim 16, wherein the transgenic bacterium is a group A streptococcal strain.