Patent Application
20230190906
The present disclosure relates to non-toxic listeriolysin O polypeptides and vaccine compositions, and uses thereof.
Listeria
monocytogenes is a foodborne pathogen and the causative agent of the life-threatening disease listeriosis. The risk and severity of listeriosis are significantly increased among pregnant women, the elderly, infants, and individuals with a compromised immune system. Listeriosis clinical manifestations include septicemia, meningitis, encephalitis, miscarriage, stillbirth and severe infection of neonates with an associated mortality rate ranging from 16-25% despite treatment. Although the food industry has rigorous standards for prevention and surveillance of Listeria contamination, the reported number of listeriosis cases in the US more than doubled from 2007-2014. With increasing incidence of listeriosis and its associated high fatality rate, a vaccine targeting L.monocytogenes can offer an effective preventative measure to reduce the risk of this deadly disease in susceptible populations such as pregnant women and the elderly. In particular, the aging population representing approximately 80% of listeriosis patients is constantly increasing. Therefore, what is needed is a vaccine for preventing or treating L.monocytogenes infection.
Disclosed herein are non-toxic listeriolysin O polypeptides and vaccine compositions, and methods of use thereof. Disclosed herein is the generation of a full-length LLO toxoid (LLOT) in which the Thr-Leu (T515G/L516G) cholesterol recognition motif in domain 4 was substituted with two glycine residues. Using LLOT and adjuvant, a novel vaccine was created that protects against infection by L.monocytogenes. This vaccine elicits CD4+ Th1 and CD8+ cells producing IFN-y and B cells producing LLO-neutralizing antibodies. The advantages of developing a LLOT-based subunit vaccine are safety, the fact that LLOT binds antigen-presenting cells and contains all native antigens for efficient activation of T and B cell responses, while LLO toxicity is abrogated. Finally, this vaccine elicited a response that neutralizes LLO, which is the most critical virulence factor of the bacterium.
In some aspects, disclosed herein is a polypeptide comprising: a non-toxic listeriolysin O comprising an amino acid substitution at one or more amino acid positions when compared to SEQ ID NO: 1, wherein the one or more amino acid positions are selected from the group consisting of 515 and 516.
In some embodiments, the amino acid substitution is at amino acid position 515. In some embodiments, the amino acid substitution is at amino acid position 516. In some embodiments, the non-toxic listeriolysin O comprises amino acid substitutions at amino acid positions 515 and 516.
In some embodiments, the amino acid substitution at amino acid position 515 is selected from the group consisting of T515G and T515A. In some embodiments, the amino acid substitution at amino acid position 516 is selected from the group consisting of L516G and L516A. In some embodiments, the non-toxic listeriolysin O binds to a cell membrane. In some embodiments, the non-toxic listeriolysin O binds to an antigen-presenting cell.
In some aspects, disclosed herein is a nucleic acid comprising a genetically modified listeriolysin O gene comprising one or more point mutations, wherein the genetically modified listeriolysin O gene encodes a polypeptide of any preceding aspect.
In some aspects, disclosed herein is a recombinant DNA vector comprising the nucleic acid of any preceding aspect.
In some aspects, disclosed herein is a vaccine comprising: a non-toxic listeriolysin O comprising an amino acid substitution at one or more amino acid positions when compared to SEQ ID NO: 1, wherein the one or more amino acid positions are selected from the group consisting of 515 and 516.
In some embodiments, the vaccine further comprises one or more adjuvants. In some embodiments, the one or more adjuvants are selected from the group consisting of cholera toxin including the β subunit of cholera toxin (CTB), and other detoxified derivatives of cholera toxin. Additional adjuvants can include Freund’s incomplete adjuvant, Freund’s Complete adjuvant, monophosphoryl lipid A, QS-21, salts, i.e., AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4)2, silica, kaolin, carbon polynucleotides, i.e., poly IC and poly AU. Still other adjuvants can include QuilA, Alhydrogel, and the like. Optionally, the vaccine contemplated herein can be combined with immunomodulators that stimulate Toll-like receptors (such as poly(I:C) and CpG motifs) and cytosolic immune sensor (such as cyclic di-nucleotides such as c-di-AMP, c-di-GMP and the like; bacterial mRNA) and immunostimulants (such as interleukins, interferons and the like).
In some aspects, disclosed herein is a method of preventing a Listeria infection, comprising administering to a subject an effective amount of a vaccine comprising: a polypeptide comprising: a non-toxic listeriolysin O comprising an amino acid substitution at one or more amino acid positions when compared to SEQ ID NO: 1, wherein the one or more amino acid positions are selected from the group consisting of 515 and 516.
In some embodiments, administering the vaccine activates CD4+ Th1s, CD8 T cells, and B cells.
In some embodiments, the Listeria is Listeriamonocytogenes. In some embodiments, the subject is a human.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. In the text below, the listeriolysin O toxoid is referred to as LLOT.
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The L.monocytogenes pore-forming exotoxin listeriolysin O (LLO) is an essential virulence factor required for host cell invasion and pathogenesis, with LLO-deficient L.monocytogenes strains being avirulent. Indeed, LLO plays essential roles in the intracellular lifecycle of L.monocytogenes including mediating the disruption of the phagosome to release the bacterium into the host cell cytosol, and mediating the spreading of the bacterium from cell to cell, among other functions.
In addition to its role as a virulence factor, LLO is a major source of CD4+ and CD8+ T cell antigens during the adaptive immune response to L.monocytogenes in mice. CD4+ and CD8+ T cell responses are critical for sterilizing immunity against Listeriamonocytogenes. In addition, the passive transfer of LLO neutralizing antibodies can protect naive mice against lethal doses of Listeriamonocytogenes. Therefore, disclosed herein is a LLO toxoid-based vaccine that elicits both i) T cell (CD4+ and CD8+) immunity involving LLO antigenic peptides and ii) LLO-neutralizing antibodies, which can efficiently protect humans against Listeriamonocytogenes infection. Beyond the interest of developing a listeriosis vaccine, L.monocytogenes and its virulence factor LLO display immune stimulatory functions that have raised considerable interest in the field of cancer immunotherapy. Hence, live-attenuated L.monocytogenes strains have shown promise in providing protection against L.monocytogenes infection and cancer in experimental animal models and several cancer vaccines are currently being tested in clinical trials.
However, the potential dangers of L.monocytogenes live-attenuated strains in immunocompromised individuals have been reported. Given that populations at higher risk for listeriosis and cancer patients are characterized by a weak or altered immunity, a subunit vaccine can prevent the risk of vaccine-related infections. As such, subunit vaccines that utilize important L.monocytogenes virulence factors have been developed. Most of these vaccines induce potent T cell responses (CD4+ and CD8+), which are essential for the acquisition of sterilizing immunity against L.monocytogenes and play critical roles in anti-cancer immunity.
The pore-forming exotoxin listeriolysin O (LLO) secreted by L.monocytogenes is required for host cell invasion and pathogenesis, with LLO-deficient L.monocytogenes strains being avirulent. Indeed, LLO plays an essential role in the intracellular lifecycle of L.monocytogenes by promoting phagosomal escape of the bacterium into the host cell cytosol. In addition to its role as a virulence factor, LLO has been shown to constitute a major source of CD4+ and CD8+ T cell antigens during the adaptive immune response to L.monocytogenes in mice. Finally, native as well as non-hemolytic LLO and truncated LLO variants have been shown to stimulate cancer antigen-specific T cell responses. In the present disclosure, a novel LLO toxoid (LLOT) is generated by substituting with Glycine residues a Threonine-Leucine pair located in domain 4, which is critically involved in LLO pore formation. The potency of LLOT as a vaccine antigen alone or in combination with various adjuvants in inducing specific B and T cell responses to protect against L.monocytogenes infection is tested using the murine model.
Described herein is a polypeptide comprising a non-toxic listeriolysin O toxoid that comprises one or more amino acid substitutions at positions 515 and/or 516 relative to SEQ ID NO: 1, and the methods for preventing and treating a Listeria infection.
Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.
Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicant desires that the following terms be given the particular definition as defined below.
As used herein, the article “a,” “an,” and “the” means “at least one,” unless the context in which the article is used clearly indicates otherwise.
The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.
As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
The terms “about” and “approximately” are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment, the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1 %.
A “composition” is intended to include a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
“Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington’s Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.
As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, and improvement or remediation of damage.
“Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the treatment of a Listeria infection. In some embodiments, a therapeutic result is the prevention of a Listeria infection. In some embodiments, a desired therapeutic result is the treatment of an inflammatory disorder. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as coughing relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
The term “cell membrane”, “plasma membrane”, or “cytoplasmic membrane” as used herein refers to a biological membrane that separates the interior of all cells from the extracellular environment which protects the cell from its environment. Cell membrane is consisted of a lipid bilayer, including cholesterol that sit between phospholipids to maintain their fluidity under various temperature, in combination with proteins. Cholesterol in a plasma membrane may be accumulated in microdomains with specific phospholipids such as sphingomyelin. These domains, often called lipid rafts, ubiquitously distribute from yeast to mammals, playing important roles in cellular functions, such as signal transduction and membrane traffic. In some embodiments, the listeriolysin O toxoid (LLOT) disclosed herein still binds to the host cell membrane despite the destruction of the cholesterol-recognition domain.
The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides.
The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.
The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed 20 of deoxyribonucleotides.
The term “oligonucleotide” denotes single- or double-stranded nucleotide multimers. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22: 1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPSTM technology. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded,” as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes.
The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers.
The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.
The term “recombinant” refers to a human manipulated nucleic acid (e.g. polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g. polynucleotide), or if in reference to a protein (i.e, a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g. polynucleotide). In some embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise nucleic acids (e.g. polynucleotides) combined in such a way that the nucleic acids (e.g. polynucleotides) are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g. polynucleotide). One of skill will recognize that nucleic acids (e.g. polynucleotides) can be manipulated in many ways and are not limited to the examples above.
The term “expression cassette” or “vector” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. In some embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)).
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) nucleotide sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g. enhancers and coding sequences) do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In some embodiments, a promoter is operably linked with a coding sequence when it is capable of affecting (e.g. modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
The term “gene” or “gene sequence” refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a “gene” as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term “gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof. The term “gene” or “gene sequence” includes, for example, control sequences upstream of the coding sequence.
The term “point mutation” means a change in the nucleotide sequence of a gene that results in a single amino acid change in a protein encoded by the gene. For example, a point mutation in a gene can result in the deletion of a single amino acid in a protein encoded by the gene or can result in the substitution of an amino acid in a wildtype version of the encoded protein with a different amino acid. Non-limiting examples of point mutations in listeriolysin O toxoid genes are described herein.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
In some aspects, disclosed herein is a polypeptide comprising: a non-toxic listeriolysin O comprising an amino acid substitution at one or more amino acid positions when compared to SEQ ID NO: 1, wherein the one or more amino acid positions are selected from the group consisting of 515 and 516.
It is understood herein that “listeriolysin O” is a member of the largest family of bacterial pore-forming toxins, the cholesterol-dependent cytolysins (CDCs), a hallmark of which is the formation of large oligomeric pores in cholesterol-rich membranes of nucleated cells and erythrocytes. CDC binding to cholesterol is indispensable for the prepore-to-pore transition of the toxin and the cholesterol-binding domain is identified as a conserved Threonine-Leucine pair located in their C-terminal domain 4 (D4). Accordingly, “listeriolysin O toxoid” or the abbreviation “LLOT”, or “non-toxic listeriolysin O” refers to the listeriolysin O toxoid that lacks the conserved Threonine-Leucine motif and displays drastically reduced toxicity. In some embodiments, the listeriolysin O toxoid polypeptide comprises the sequence set forth in SEQ ID NO: 1, or sequence having at or greater than about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1.
In some embodiments, the amino acid substitution is at amino acid position 515. In some embodiments, the amino acid substitution is at amino acid position 516. In some embodiments, the non-toxic listeriolysin O comprises amino acid substitutions at amino acid positions 515 and 516.
In some embodiments, the amino acid substitutions at amino acid positions 515 and 516 can be, for example, T515G, T515A, L516A, L516G, or any other amino acid substitution(s) that causes the destruction of the cholesterol recognition motif, but does not abolish LLO binding to host membranes. In some embodiments, the amino acid substitution at amino acid position 515 is selected from the group consisting of T515G and T515A. In some embodiments, the amino acid substitution at amino acid position 515 is preferably T515G. In some embodiments, the amino acid substitution at amino acid position 516 can be, for example,T515G, T515A, L516A, L516G, or any other amino acid substitutions that cause the destruction of the cholesterol recognition motif, but do not abolish LLO binding to host membranes. In some embodiments, the amino acid substitution at amino acid position 516 is selected from the group consisting of L516G and L516A. In some embodiments, the amino acid substitution at amino acid position 516 is preferably T516G. In some embodiments, additional amino acid substitutions in listeriolysin O can be used that affect its ability to form pores and/or to bind host cells.
In some embodiments, the polypeptide is isolated. In some embodiments, the polypeptide is recombinant. In some embodiments, the polypeptide is a non-naturally occurring polypeptide.
In some embodiments, the non-toxic listeriolysin O binds to a cell membrane.
In some embodiments, the non-toxic listeriolysin O binds to an antigen-presenting cell.
In some aspects, disclosed herein is a nucleic acid comprising a genetically modified listeriolysin O toxoid gene comprising one or more point mutations, wherein the genetically modified listeriolysin O toxoid gene encodes a polypeptide of any preceding aspect.
In some embodiments, the nucleic acid is isolated. In some embodiments, the nucleic acid is recombinant. In some embodiments, the nucleic acid is a non-naturally occurring nucleic acid.
In some aspects, disclosed herein is a recombinant DNA vector comprising the nucleic acid of any preceding aspect.
In some aspects, disclosed herein is a vaccine comprising: a non-toxic listeriolysin O comprising an amino acid substitution at one or more amino acid positions when compared to SEQ ID NO: 1, wherein the one or more amino acid positions are selected from the group consisting of 515 and 516.
It should be understood that the one or more adjuvants described herein can be any of the adjuvants that can stimulate the production of LLO neutralizing antibodies and T cell immunity. In some embodiments, the vaccine further comprises one or more adjuvants. In some embodiments, the one or more adjuvants are selected from the group consisting of cholera toxin including the β subunit of cholera toxin (CTB), and other detoxified derivatives of cholera toxin. Additional adjuvants can include Freund’s incomplete adjuvant, Freund’s Complete adjuvant, monophosphoryl lipid A, QS-21, salts, i.e., AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4)2, silica, kaolin, carbon polynucleotides, i.e., poly IC and poly AU. Still other adjuvants can include QuilA, Alhydrogel, and the like. Optionally, the vaccine contemplated herein can be combined with immunomodulators that stimulate Toll-like receptors (such as poly(I:C) and CpG motifs) and cytosolic immune sensor (such as cyclic di-nucleotides such as c-di-AMP, c-di-GMP and the like; bacterial mRNA) and immunostimulants (such as interleukins, interferons and the like). Still other adjuvants can include bacterial toxin derivatives. Many vaccine formulations are also known to those of skill in the art.
In some embodiments, the vaccine further comprises a pharmaceutically acceptable carrier.
In some aspects, disclosed herein is a method of preventing, inhibiting, reducing, and/or treating a Listeria infection, comprising administering to a subject an effective amount of a vaccine comprising: a polypeptide comprising: a non-toxic listeriolysin O comprising an amino acid substitution at one or more amino acid positions when compared to SEQ ID NO: 1, wherein the one or more amino acid positions are selected from the group consisting of 515 and 516.
In some aspects, disclosed herein is a method of inducing immune response specific to a Listeria, comprising administering to a subject an effective amount of a vaccine comprising: a polypeptide comprising: a non-toxic listeriolysin O comprising an amino acid substitution at one or more amino acid positions when compared to SEQ ID NO: 1 selected from the group consisting of 515 and 516. It is understood herein that the induced immune response prevents, inhibits, reduces, or treat the Listeria infection.
As the timing of an infection can often not be predicted, it should be understood the disclosed methods of treating, preventing, reducing, and/or inhibiting a Listeria infection, can be used prior to or following the infection of the Listeria infection, to treat, prevent, inhibit, and/or reduce the infection or an infection-associated disease. Where, the disclosed methods can be performed any time prior to the infection. In one aspect, the disclosed methods can be employed 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 years, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 months, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 days, 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 hours, 60, 45, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute prior to infection; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 or more days, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 years after infection.
The vaccines of the present invention can be administered to the appropriate subject in any manner known in the art, e.g., orally intramuscularly, intravenously, sublingual mucosal, intraarterially, intrathecally, intradermally, intraperitoneally, intranasally, intrapulmonarily, intraocularly, intravaginally, intrarectally or subcutaneously. They can be introduced into the gastrointestinal tract or the respiratory tract, e.g., by inhalation of a solution or powder containing the conjugates. In some embodiments, the compositions can be administered via absorption via a skin patch. Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained.
A pharmaceutical composition (e.g., a vaccine) is administered in an amount sufficient to elicit production of antibodies and activation of CD4+ T cells and CD8+ T cells as part of an immunogenic response. It is understood herein that CD4+ T cell is a group of heterologous lymphocytes having different subsets, including, for example, Th1, Th2, Th17, Tfh, and Treg. In some embodiments, the CD4+ T cell is a Th1 cell. It should also be understood herein that the term “activation” or “activates” refer to a response of a CD4+ T cell, a CD8+ T cell, or a B cell. Such response includes, for example, enhanced proliferation and increased IFN-y+ production of the CD4+ T cell (e.g. Th1), enhanced proliferation and increased IFN-y+ production of the CD8+ T cell, and/or antibody production of the B cell. In some embodiments, administering the vaccine of any preceding aspects activates a CD4+ Th1, a CD8 T cell, and/or a B cell. Dosage for any given patient depends upon many factors, including the patient’s size, general health, sex, body surface area, age, the particular compound to be administered, time and route of administration, and other drugs being administered concurrently. Determination of optimal dosage is well within the abilities of a pharmacologist of ordinary skill.
In some embodiments, the subject is a human. In some embodiments, the human has or is suspected of having Listeria infection. It should be understood herein that the “ Listeria” refers to a genus of bacteria that comprises, for example, L.aquatica, L.booriae, L.cornellensis, L.costaricensis, L.goaensis, L.fleischmannii, L.floridensis, L.grandensis, L.grayi, L.innocua, L.ivanovii, L.marthii, L.monocytogenes, L.newyorkensis, L.riparia, L.rocourtiae, L.seeligeri, L.thailandensis, L.weihenstephanensis, and L.welshimeri. In some embodiments, the Listeria is L.monocytogenes. In some embodiments, disclosed herein is a method of preventing, inhibiting, reducing, and/or treating L.monocytogenes infection.
The vaccine compositions are administered to subjects which may become infected by a Listeria described herein, including but not limited to dogs, cats, rabbits, rodents, horses, livestock (e.g., cattle, sheep, goats, and pigs), zoo animals, ungulates, primates, and humans. In some embodiments, the preferred subject is a human.
The following examples are set forth below to illustrate the compounds, systems, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
LLO is a member of the largest family of bacterial pore-forming toxins, the cholesterol-dependent cytolysins (CDCs), a hallmark of which is the formation of large oligomeric pores in cholesterol-rich membranes of nucleated cells and erythrocytes. CDC binding to cholesterol is indispensable for the prepore-to-pore transition of the toxin and the cholesterol-recognition domain was identified as a conserved Threonine-Leucine pair located in their C-terminal domain 4 (D4). A full length LLO toxoid (LLOT) was generated by substitution of the cholesterol-recognition threonine-leucine pair with glycines (T515G/L516G). The properties of LLOT was compared relative to native LLO, a truncated LLO D1-3 variant devoid of the host cell binding domain D4, and a full-length LLO variant with the amino acid substitution W492A in domain 4 that was previously reported as non-hemolytic (LLO W492A). Recombinant 6-histidine-LLO, -LLOT, -LLO D1-3, and -LLO W492A were purified and characterized by SDS-PAGE (
Sterilizing immunity against L.monocytogenes is well known to involve CD4+ and CD8+ T cells. In addition, the passive transfer of monoclonal LLO-neutralizing antibodies was shown to efficiently protect naive mice against sub-lethal and lethal doses of L.monocytogenes. To determine if LLOT can promote immunization of mice against L.monocytogenes, LLOT was administered in the presence or absence of the experimental adjuvant cholera toxin (CT). Cholera toxin was selected for its broad effects on stimulating both T and B cell responses. Mice were treated with PBS, LLOT, CT, or LLOT + CT via intraperitoneal injections at weekly intervals for 3 weeks. At day 28 after initial immunization, mice were challenged with 2 x 104L.monocytogenes by tail vein injection and bacterial burden was determined by CFU enumeration in the spleen and liver three days post-infection. As shown in
To test whether production of anti-LLO antibodies alone can play a role in the protection of mice, the effectiveness of Alum was examined. Alum is a widely used vaccine adjuvant that predominantly induces strong Th2 responses and antibody responses to antigens. After a similar immunization procedure as described previously with CT, mice that received LLOT+ Alum were not protected against L.monocytogenes as previously observed with CT + LLOT (
To address the difference in the ability of CT and Alum as adjuvants to protect mice immunized with LLOT against L.monocytogenes challenge, the production of anti-LLOT IgG titers in the various groups of animals was measured. Data summarized in
To establish the nature of the T helper responses elicited by the protective (LLOT + CT) and non-protective (LLOT + Alum) vaccine formulations, in comparison to LLOT alone and control PBS, splenocytes were collected from the different groups of mice and in vitro stimulated with LLOT. After 5 days of culture, cells were extracellularly stained with fluorescent anti-CD3 and anti-CD4 antibodies to denote CD4+ T helper cells, intracellular stained with fluorescent antibodies to identify Th1 (IFN-γ, and TNF-α)-, Th2 (IL-5, IL-4, and IL-10)-, Th17 (IL-17A)-, and Tfh (IL-21)-type cytokines. This labeling strategy can also characterize the production of cytokines by CD8+ T cells, identified as CD3+CD4- cells that were positive for any of the tested cytokines.
Th1 cells and their characteristic cytokines (IFN-γ and TNF-α) promote cell-mediated immunity, including cytotoxic CD8+ T cells and the activation of macrophages, both of which are important for protection against intracellular pathogens, including L.monocytogenes. Flow cytometry analysis of CD4+ CD3+cells (CD4+ T cells) showed that immunization with LLOT + CT led to a significant increase in IFN-y producing T helper cells when compared to all other treatments (
Th2 cells are known to produce cytokines (IL-5, IL-4, and IL-10) that support the production of antibodies. The main products of Tfh cells (IL-21) and Th17 cells (IL-17A) also facilitate antibody production and their affinity maturation. Immunization with LLOT + Alum or CT led to significant increases in cytokine producing T helper cells when compared to the PBS control group and the groups given the adjuvants alone. Additionally, immunization with LLOT alone led to increases in IL-5 producing T helper cells (
The protective immunization regimen (LLOT + CT) was characterized by both increased LLO-specific neutralizing antibody production (
Generation of LLO variants and LLO toxoid. The gene coding for six-His-tagged LLOT with the substitutions T515G and L516G was generated by PCR-based site-directed mutagenesis using the pET29b plasmid harboring wild type hly (the gene coding LLO) as a template and mutagenic primers (Forward - 5-gaa ata tct cca tct ggg gca ccg ggg gtt atc cga aat ata gta ata aag-3 (SEQ ID NO:2) and Reverse - 5-ctt tat tac tat att tcg gat aac ccc cgg tgc ccc aga tgg aga tat ttc-3 (SEQ ID NO:3)) as described previously. The gene coding for six-His-tagged LLOW492A was also generated using the same strategy and the mutagenic primers (Forward - 5-ggt tta gct tgg gaa tgg gcg aga acg gta att gat gac cgg-3 (SEQ ID NO:4) and Reverse - 5-ccg gtc atc aat tac cgt tct cgc cca ttc cca agc taa acc-3 (SEQ ID NO:5)). The gene coding for the six-His-tagged truncated listeriolysin O LLO (LLO D1-3) was amplified by PCR from the wild type sequence of hly using the Forward - 5′-aac gtg cat atg gat gca tct gca ttc aat aaa G-3′ (SEQ ID NO:6) and Reverse - 5′-att ctc gag tgt ata agc ttt tga agt tgt-3′ (SEQ ID NO:7) and cloned into pET29b using NdeI and XhoI restriction sites. LLO variants were purified. LLO variants were aliquoted in 50 mM phosphate, pH=6, 1 M NaCl and stored at -80° C. until used. Endotoxin measurements were performed as directed by the manufacturer using the Chromogenic Endotoxin Quant Kit (Pierce), and LLOT was inoculated at 200 µg/ml with endotoxin levels strictly below the recommended limit of 36 EU/ml. For detection of the toxin derivatives by SDS-PAGE, 1 µg of recombinant LLO, LLOW492A, LLOT, or LLOD1-3 were diluted in Laemmli sample buffer with β-mercaptoethanol and denatured by heating at 95° C. for 5 min. Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 10% polyacrylamide gels. Gels were stained with Coomassie blue and imaged using a ChemiDoc XRS imaging system (Bio-Rad).
Circular Dichroism (CD) spectroscopy. CD spectra for LLO and LLOT were acquired on a Jasco J-815 spectrometer at 10° C. with a 1 mm cuvette at a protein concentration of 0.5 mg/ml in 20 mM sodium phosphate buffer at pH=6. Spectra were recorded at wavelength intervals of 1 nm (190 to 255 nm). The spectra are the average of 3 scans
Cholesterol Binding Assay. Spots (2 µl) of a serially diluted ethanol-cholesterol solution were deposited onto a PVDF membrane and air-dried. The membranes were saturated by incubation in a 20 mM Tris buffer (TBS) containing 4% nonfat milk and 0.2% Tween 20 at pH 7.4. LLO and LLOT (20 µg/ml) were incubated at 4° for 3 h in TBS with 0.2% Tween 20. After washes, rabbit anti-LLO Abs (Abcam) were incubated for 1 h in TBS with 0.1% Tween 20, followed by washes and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies in TBS with 0.1% Tween 20. LLO was detected with ECL Western Blotting Detection Kit (Amersham). Western blotting was performed to verify that the rabbit anti-LLO antibodies recognize LLO and LLOT with similar efficiency.
LLO binding assays HepG2 invasion assays. HeLa cells (1.5 x 105 /well) were grown for 24 h in 6-well plates in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HIFBS), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen). Cells were incubated for 30 min in FBS-free medium +/- 5 mM methyl-β-cyclodextrin (mβCD) at 37° C. to deplete cholesterol. Cells were incubated for 10 min in FBS-free medium with LLO, LLOT, or LLO D1-3 at 1, 2, or 5 nM at 4° C. Cells were then washed with PBS and lysed with lysis buffer (150 mM NaCl, 20 mM Tris/HCl, 2 mM EDTA, 1% NP-40, and protease inhibitor cocktail (Roche)). Cell lysates were subjected to western blot analysis using an anti-LLO (Rabbit polyclonal from Abcam) or anti-actin antibodies (Cell Signaling) and secondary antibodies conjugated to HRP (Cell Signaling). Detection was performed using the Amersham ECL Select Reagent Kit (GE Healthcare) and a ChemiDoc XRS Imaging System (Bio-Rad). THP-1 cells were cultured in RPMI-1640 supplemented with 10% HIFBS, 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen). 2 x 106 cells were washed with FBS-free medium and incubated with 1 nM and 5 nM LLO or LLOT for 10 min at 4° C. THP-1 cells were washed with PBS, lysed and subjected to western blot analysis as described above. For invasion assay, HepG2 cells were cultured in 24-well plates and incubated with bacteria at multiplicity of infection 5 for 30 min in the presence or absence of LLO-neutralizing antibodies. Cells were washed, fixed and labeled to measure the percentage of bacterial internalization as described in 31. All human cell lines used in this study were authenticated by ATCC.
Hemolysis Assays. Human blood was drawn in heparinized Vacutainer tubes, from healthy adult volunteers with approval of the Ohio State University Institutional Review Board. After centrifugation of blood on Polymorphprep (Axis-Shield, Oslo, Norway), erythrocytes were collected from the lower cell layer and were washed with Alsever’s solution. The concentrations of LLO and its derivatives leading to 50% hemolysis (EC50) were determined by performing a hemolysis assay as follows. Erythrocytes were washed three times with phosphate buffered saline (PBS) and diluted to a concentration of 4 x 107 cells/ml. Duplicate serial dilutions of native LLO, LLOW492A, LLOD1-3, and LLOT were made at 4° C. in a round bottom 96-well plate, and 160 µl of cold erythrocytes suspension were added in each well. Concentration ranges tested were: native LLO (100 nM - 0.1 nM), LLOW492A (3,000 nM - 1.5 nM), LLOT (10,000 nM - 5 nM), LLOD1-3 (6,000 nM - 3 nM). Plates were then incubated for 30 min at 37° C., centrifuged, and the supernatants were transferred to a flat bottom 96-well plate for reading their absorbance (540 nm) in a spectrophotometer. Erythrocytes were treated with 0.1% Triton X-100 (100% hemolysis) and with PBS (no hemolysis) as positive and negative controls, respectively. The concentration of toxin leading to 50% hemolysis (EC50) was determined by polynomial regression using Graph Pad Prism 7 software (GraphPad Software Inc, La Jolla, CA).
Immunization. All animal protocols were approved by The Ohio State University’s Institutional Laboratory Animal Care and Use Committee. Seven to eight week-old C57BL/6 or C57BL/6-Igh-6tm1Cgn (B cell-deficient, also known as µMT-/-)30 mice, purchased from The Jackson Laboratory (Bar Harbor, ME), were housed in the university vivarium for one week before starting immunization. Mice were immunized on days 0, 7, and 14 by intraperitoneal injection of 100 µl of injectable grade PBS containing one of the following: 20 µg LLOT alone, 20 µg LLOT plus 1 µg cholera toxin (List Biological Laboratories, Inc, Campbell, CA), 20 µg LLOT adsorbed on 40 µg alum (ThermoFisher Scientific, Waltham, MA). Control groups received 100 µl of PBS alone, or 1 µg cholera toxin, or 40 µg of alum. For the preparation of alum plus LLOT, LLOT was adsorbed to alum via gentle mixing for 45 min at 4° C. Blood was collected from mice via submandibular cheek bleed during the immunization procedure on days 14, 21, and 28. Serum was obtained by centrifugation of the clotted blood (1,500 x g for 15 min at 4° C.). For IgG isolation, larger volumes of blood were obtained via cardiac puncture immediately after sacrifice of the animals.
Bacterial Cell Culture and Mouse Infection Wild type L.monocytogenes (strain DP10403S) were grown overnight at 37° C. in brain heart infusion (BHI). For infections, overnight cultures were diluted 1/20 in BHI and grown at 37° C. until OD600 = 0.7-0.8. Bacteria were washed three times and diluted in injectable grade phosphate-buffered saline (PBS). Mice were inoculated by tail vein injection with L.monocytogenes (2 x 104 bacteria in 100 µl injectable grade PBS) on day 28 after immunization. After 72 h, mice were euthanized and livers, spleens, and blood were collected. Organs were homogenized in PBS and homogenates were serially diluted, plated on BHI agar plates and incubated at 37° C. for 48 hours. Bacterial colonies were enumerated to determine the colony forming units (CFUs).
Evaluation of LLO-specific antibody titers. To determine the LLO-specific antibody titers, ELISA was performed with LLO-coated plates. Briefly, 100 µl of LLOT (5 µg/ml in PBS) were added to microtiter plates and incubated at 4° C. overnight. Plates were washed three times with cold PBS and blocked for 2 h with 1% BSA in PBS. Plates were washed three times and 100 µl of PBS 1% BSA containing serial dilution of sera were added. After overnight incubation at 4° C., the LLOT-specific antibodies were detected with HRP-conjugated anti-mouse IgG sera (1:3000 dilution) (Southern Biotech Associates Inc., Birmingham, AL). Alternatively, to measure IgG subclasses, biotin-conjugated rat anti-mouse IgG1, IgG2a/c, IgG2b, or IgG3 monoclonal Abs and HRP-conjugated streptavidin (BD Biosciences, San Jose, CA) were used (0.5 µg/ml). The HRP substrate ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt, Sigma-Aldrich) was added and the antibody titers were determined as the last dilution of samples with an absorbance of >0.1 above that of samples from control mice mock immunized with PBS.
Evaluation of the production of LLO-neutralizing antibodies. To test for LLO neutralization by LLOT-induced antibodies, a kinetic hemolytic assay was performed. IgG were purified from serum collected from immunized mice using protein G-agarose (Pierce) according to the manufacturer’s instructions. LLO and LLOT (5 nM in PBS) and various dilutions of purified serum IgG were pre-incubated on ice in a 96-well plate for 15 min before the addition of erythrocytes at 4 x 107 cells/ml, to test LLO activity using a kinetic assay. Triton X-100 (0.1%) and PBS served as positive and negative controls for hemolysis, respectively. Samples were transferred to a spectrophotometer at 37° C. and the absorbance (700 nm) was measured every minute for 30 min.
Analysis of LLOT-specific T helper cell cytokines responses. Spleens were aseptically removed from mice 38 days after initial immunization and minced by pressing through a cell strainer. Red blood cells were removed by incubation in 0.84 % ammonium chloride and, following a series of washes in RPMI 1640, spleen cells were suspended in RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum. The cell concentration was adjusted to 5 × 106 cells/ml, and 100 µl cells were added to each well (3 wells per spleen) of a 96-well micro-titer plate and cultured either alone or in the presence of 5 µg/ml LLOT for 5 days at 37° C. in a 5% CO2 atmosphere. Flow cytometry and intracellular cytokine staining were then used to determine the profile of T helper cell cytokine responses. For this purpose, cells were stimulated with PMA and Ionomycine (BD-Pharmangen, NJ, US) and incubated for 1 h at 37° C. in a 5% CO2 atmosphere. The Golgi function was blocked by Golgistop, (BD-Pharmangen, NJ, US), and cells were incubated at 37° C. in a 5% CO2 atmosphere for 5 h. Cells were then collected and washed twice with FACS buffer (PBS, 2% BSA, 0.01% NaN3). For labeling extracellular T-cell lineage markers, cells were incubated with Alexa Fluor 700 anti-CD3 and Alexa Fluor 750 anti-CD4 antibodies (Biolegend, San Diego, CA) for 30 min at 4° C., then washed twice with FACS buffer. For intracellular cytokine staining, cells were incubated with Fixation-Permeabilization Buffer (BD-Pharmagen, NJ, US) for 20 min at 4° C. and washed twice with the permeabilization buffer (BD-Pharmangen, NJ, US). Cells were then labeled with Th1, Th2, Th17, and Tfh cytokine-specific antibodies (Alexa Fluor 488-IFNy, PerCP Cy5.5-TNFα, PE-IL-5, Alexa Fluor 647-IL-21, PECy7 IL-10, Brilliant Violet 650 IL-17, Brilliant Violet 605 IL-4 (Biolegend, San Diego, CA)) for 30 min at 4° C. Cells were washed twice with the permeabilization buffer and then washed twice with the FACS buffer. Cells were suspended in FACS buffer and analyzed with an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA). The data were analyzed by triple gating as (CD3+CD4+Cytokines+). Statistical analyses were performed by one-way ANOVA using Graph Pad Prism7 (GraphPad Software Inc, La Jolla, CA) and significant differences were considered at p ≤ 0.05 (*) .
Disclosed here in is the generation of a full-length LLO toxoid (LLOT) in which the Thr-Leu (T515G/L516G) cholesterol recognition motif in domain 4 was substituted with two glycine residues. Using LLOT and the cholera toxin experimental adjuvant, a novel vaccine was created that protects against infection by L.monocytogenes. This vaccine elicits CD4+ Th1 and CD8+ cells producing IFN-y and B cells producing LLO-neutralizing antibodies. The advantages of developing a LLOT-based subunit vaccine are safety, the fact that LLOT binds antigen-presenting cells and contains all native antigens for efficient activation of T and B cell responses, while LLO toxicity is abrogated. Finally, this vaccine elicited a response that neutralizes LLO, which is the most critical virulence factor of the bacterium.
The cholesterol recognition motif is conserved among the cholesterol-dependent cytolysin (CDC) family members and was shown to be essential for perfringolysin O (PFO), streptolysin O (SLO), pneumolysin (PLY), and intermedilysin (ILY) binding to cholesterol. The data herein show that this motif is also required for LLO binding to cholesterol (
Adjuvants were introduced in the present vaccine design. Key players that mediate sterilizing adaptive immune response to L.monocytogenes include CD4+ Th1 cells producing IFN-y, which are known to activate the bactericidal activity of macrophages and CD8+ cytotoxic T cell responses. Studies by Edelson et al. using a murine infection model suggested that, unlike the robust T cell responses, B cell responses and the production of antibodies were limited in response to L.monocytogenes infection. However, the adoptive transfer of monoclonal LLO-neutralizing antibodies, but not of anti-LLO non-neutralizing antibodies, protected naïve mice against sub-lethal and lethal doses of L.monocytogenes. The protective effect was attributed to the neutralization of LLO within the phagosomes of infected cells. LLO-neutralizing antibodies can in addition abrogate the extracellular activities of LLO, as evidenced by their ability to inhibit LLO-mediated bacterial internalization into hepatocytes (
Cholera toxin, an experimental adjuvant, was used herein for eliciting balanced and robust T and B cells immune responses. Inoculation of LLOT plus cholera toxin significantly protected mice against L.monocytogenes (
The ability of the two adjuvants to elicit LLO-specific T helper responses was compared. The major distinction of the LLOT+ CT treatment is the significant increase in IFN-y+ CD4+ T cells, indicating a Th1 dominated response. This shows the critical role of IFN-y in the activation of CD8+ T cells and macrophages for the clearance of L.monocytogenes. IFN-y is critical for Ig class switching to the IgG2a isotype, with the associated Th1 response important for IgG2b and IgG3 production. To determine if the Th1 response, which is implicated in protection, is sufficient in the absence of LLO-specific antibodies, µMT-/- mice that lack mature B cells were immunized. This experiment led to two major conclusions. First, MT-/- mice are more resistant to L. monocytogenes infection as shown by the substantial decrease in CFU recovered from spleen and liver in comparison to WT mice. Indeed, L.monocytogenes was shown to stimulate IL-10 producing B cells leading to decreased macrophage anti-listeria responses. Similar observations were reported with SCID mice, which are deficient in both T and B cells, infected with L. monocytogenes despite the acknowledged protective role of T cells. Second, there was a significant reduction in bacteria CFUs in the livers and spleens of µMT-/- mice immunized with LLOT+CT compared to all other groups of animals (
In order to confirm the role of T cells in the anti-L. monocytogenes protection of mice immunized with LLOT + CT, T cells were depleted after immunization by administering a cocktail of CD8- and CD4-cell-depleting antibodies, or control isotypes, 48 h before and 24 h after infection. Analysis of circulating leukocytes confirmed the efficacy of T cell depletion, whereas B cells, natural killer cells, and dendritic cells remained unaffected. When isotype control antibodies were administered to mice immunized with LLOT + CT, significant decreases were observed in bacterial burden 72 h post-infection (
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SEQ ID NO: 1, AMINO ACID SEQUENCE WILD TYPE LLO:
SEQ ID NO: 2, SEQUENCE OF LLO-ENCODING GENE:
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.
This application claims the benefit of priority to U.S. Provisional Application No. 62/908,877 filed Oct. 1, 2019, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with government support under grant number R01AI107250 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/053707 | 10/1/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62908877 | Oct 2019 | US |
