Patent Application
20150017198
This application makes reference to the following U.S. patents and U.S. patent applications: U.S. Provisional Patent Application No. 61/774,895, filed Mar. 8, 2013, entitled “In Vitro and In Vivo Delivery of Genes and Proteins Using the Bacteriophage T4 DNA Packaging Machine”; U.S. Provisional Patent Application No. 60/904,168, filed Mar. 1, 2007, entitled “Liposome-Bacteriophage Complex as Vaccine Adjuvant”; U.S. patent application Ser. No. 12/039,803, filed Feb. 29, 2008, entitled “Liposome-Bacteriophage Complex as Vaccine Adjuvant”, now U.S. Pat. No. 8,148,130, issued Apr. 3, 2012; U.S. patent application Ser. No. 11/015,294, filed Dec. 17, 2004, entitled “Methods and Compositions Comprising Bacteriophage Nanoparticles”; U.S. Provisional Patent Application No. 60/530,527, filed Dec. 17, 2003, entitled “Methods and Compositions Comprising Bacteriophage Nanoparticles”; U.S. Provisional Patent Application No. 61/322,334, filed Apr. 9, 2010, entitled “Promiscuous DNA Packaging Machine From Bacteriophage T4”; U.S. patent application Ser. No. 13/082,466, filed Apr. 8, 2011, entitled “Protein and Nucleic Acid Delivery Vehicles, Components and Mechanisms Thereof”; U.S. Provisional Patent Application No. 61,731,147, filed Nov. 29, 2012, entitled “Designing a Soluble Full-Length HIV-1 GP41 Trimer”; and U.S. Provisional Patent Application No. 61/845,487 to Rao and Tao, entitled “Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia Pestis as Next Generation Plague Vaccines,” filed Jul. 12, 2013. The entire disclosure and contents of these patent applications are incorporated herein by reference.
1. Field of the Invention
The present invention relates generally to mutated immunogens from Yersinia pestis and antigen carriers.
2. Related Art
Pneumonic plague is a highly virulent infectious disease with approximately one hundred percent mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Stockpiling of an efficacious plague vaccine that could protect people against a potential bioterror attack has been a national priority. Currently, there is no FDA approved vaccine against plague. There exists a growing need to develop efficacious and easily manufacturable plague vaccines.
According to a first broad aspect, the present invention provides a recombinant protein comprising a mutated F1 antigen of Yersinia pestis, wherein an NH2-terminal β-strand of a native F1 antigen of Yersinia pestis is deleted from an NH2-terminus of the F1 antigen of Yersinia pestis to thereby form an NH2-terminal β-strand deleted F1, wherein the NH2-terminal β-strand of the F1 antigen of Yersinia pestis is fused through a first peptide linker to a COOH-terminus of the NH2-terminal β-strand deleted F1 to thereby form an NH2-terminal β-strand transplanted F1, and wherein an NH2-terminal amino acid sequence of F1 antigen of Yersinia pestis that flanks the NH2-terminal β-strand of a native F1 antigen of Yersinia pestis is duplicated at a COOH-terminus of the NH2-terminal β-strand transplanted F1 to thereby form the mutated F1 antigen of Yersinia pestis.
According to a second broad aspect, the present invention provides a fusion protein comprising a small outer capsid protein from a T4 phage and/or a T4-related bacteriophage RB69 fused through a peptide linker to a heterologous polypepeide derived from one or more antigens of Yersinia pestis to thereby form a phage capsid protein fusion protein.
According to a third broad aspect, the present invention provides a method for developing a monomeric immunogen comprising the steps that include: deleting an NH2-terminal β-strand of a F1 antigen of Yersinia pestis from an NH2-terminus of an native F1 antigen of Yersinia pestis to thereby form an NH2-terminal β-strand deleted F1, fusing the NH2-terminal β-strand of F1 antigen of Yersinia pestis to a COOH-terminus of the NH2-terminal β-strand deleted F1 via a first peptide linker to thereby form an NH2-terminal β-strand transplanted F1, and duplicating an NH2-terminal amino acid sequence of F1 antigen of Yersinia pestis flanking the NH2-terminal β-strand of F1 antigen of Yersinia pestis at a COOH-terminus of the NH2-terminal β-strand transplanted F1 to thereby form a mutated F1 antigen of Yersinia pestis.
According to a fourth broad aspect, the present invention provides a vaccine comprising one or more phage T4 nanoparticles and one or more immunogens, wherein each of the one or more immunogens comprises a fusion protein comprising a small outer capsid protein from a T4 phage and/or a T4-related bacteriophage RB69, wherein the small outer capsid protein from a T4 phage and/or a T4-related bacteriophage RB69 is fused through a peptide linker to a heterologous polypepeide derived from one or more antigens of Yersinia pestis, and wherein each of the one or more immunogens is capable of binding to each of the one or more phage T4 nanoparticles via the small outer capsid protein of a phage T4 and/or a T4-related bacteriophage RB69.
According to a fifth broad aspect, the present invention provides a method for producing a vaccine comprising the following steps: incubating one or more phage T4 nanoparticles in a buffered solution with one or more immunogens to thereby form immunogen-bound T4 nanoparticles, sedimenting the immunogen-bound T4 nanoparticles to thereby form a phage pellet and a supernatant, separating the phage pellet from the supernatant to thereby form a separated phage pellet, washing the separated phage pellet one or more times to thereby form a washed phage pellet, and suspending the washed phage pellet in a buffered solution to thereby form a vaccine solution. In the vaccine, each of one or more immunogens comprises one or more phage capsid protein fusion proteins and each of one or more phage capsid protein fusion proteins is capable of binding to each of the one or more T4 nanoparticles.
According to a sixth broad aspect, the present invention provides a method of immunization comprising a step of administering to a subject an immunogenic amount of a vaccine comprising a purified mutated F1 antigen of Yersinia pestis.
According to a seventh broad aspect, the present invention provides a method of immunization comprising administering to a subject an immunogenic amount of a vaccine that comprises one or more phage T4 nanoparticles and one or more immunogens, wherein each of the one or more immunogens comprises a fusion protein comprising a small outer capsid protein from a T4 phage and/or a T4-related bacteriophage RB69, wherein the small outer capsid protein from a T4 phage and/or a T4-related bacteriophage RB69 is fused through a peptide linker to a heterologous polypepeide derived from one or more antigens of Yersinia pestis, and wherein each of the one or more immunogens is capable of binding to each of the one or more phage T4 nanoparticles via the small outer capsid protein of a phage T4 and/or a T4-related bacteriophage RB69.
According to an eighth broad aspect, the present invention provides a method comprising following steps: culturing compatible host cells having expression vectors therein to thereby form a cell culture comprising expressed products, and purifying the expressed products from the cell culture to thereby form one or more immunogens, wherein the one or more immunogens encompassing one or more recombinant proteins as in any one of claims 1-10.
The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate exemplary embodiments of the invention, and, together with the general description given above and the detailed description given below, serve to explain the features of the invention.
Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated.
For purposes of the present invention, it should be noted that the singular forms, “a,” “an” and “the” include reference to the plural unless the context as herein presented clearly indicates otherwise.
For purpose of the present invention, the term “adjacent” refers to “next to” or “adjoining something else.”
For purpose of the present invention, the term “adjuvant” refers the components in a vaccine or therapeutic composition that increase the specific immune response to the antigen. Adjuvants are well known to those of skill in the art and may include cytokines (e.g., IFN-γ, IL-2, and IL-12) which contribute to the induction of cell-mediated immune response to an administered antigen, as well as induction of humoral immune responses. Traditional vaccine usually needs an adjuvant.
For purpose of the present invention, the phase “administration of a vaccine” refers to introduce a vaccine into a body of an animal or a human being. As is understood by an ordinary skilled person, it can be done in a variety of manners. For example, administration of a vaccine may be done intramuscularly, subcutaneously, intravenously, intranasally, intradermaly, intrabursally, in ovo, ocularly, orally, intra-tracheally or intra-bronchially, as well as combinations of such modalities. The dose of the vaccine may vary with the size of the intended vaccination subject.
For purpose of the present invention, the term “array” refers to in vitro binding of a protein on T4 phage. For example, a Soc fusion protein, a protein fused with a small outer capsid protein Soc of a T4 phage, may be arrayed by incubating Hoc−Soc− T4 phage particles with the Soc fusion protein to allow the Soc fusion protein to bind on Hoc−Soc− T4 phage particles.
For purposes of the present invention, the term “bind,” the term “binding” or the term “bound” refers to any type of chemical or physical binding, which includes but is not limited to covalent binding, hydrogen binding, electrostatic binding, biological tethers, transmembrane attachment, cell surface attachment and expression.
For purpose of the present invention, the term “bivalent” refers to a composition that has two combining sites, for example, a bivalent immunogen capable of binding to two molecules of antibodies.
For purpose of the present invention, the term “β-sheet” (also “beta sheet”) refers to a secondary form of regular secondary structure in proteins. It consists of “β-strands” (also “beta strand”) connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet.
For purpose of the present invention, the term “β-strand” (also “beta strand”) refers to a stretch of polypeptide chain. It is typically 3 to 10 amino acids long with backbone in an almost fully extended conformation.
For purposes of the present invention, the term “capsid” and the term “capsid shell” refers to a protein shell of a virus comprising several structural subunits of proteins. The capsid encloses the nucleic acids of the virus. Capsids are broadly classified according to their structures. The majority of viruses have capsids with either helical or icosahedral structures.
For purpose of the present invention, the term “capsomere” refers to a basic substructure of a capsid, an outer covering of proteins that protects the genetic materials of a virus. Capsomeres self-assemble to form the capsid.
For purpose of the present invention, the term “cleft” refers to a groove or a V-shaped indentation that runs across two protein domains.
The term “comprising”, the term “having”, and the term “including” are intended to be open-ended and mean that there may be additional elements other than the listed elements.
For purpose of the present invention, the term “correspond” and the term “corresponding” refer to that a protein sequence refer interchangeably to an amino acid position(s) of a protein. An amino acid at a position of a protein may be found to be equivalent or corresponding to an amino acid at a position of one or more other protein(s) based on any relevant evidence, such as the primary sequence context of the each amino acid, its position in relation to the N-terminal and C-terminal ends of its respective protein, the structural and functional roles of each amino acid in its respective protein, etc.
For purpose of the present invention, the term “duplicate” refers to repeat or generate another identical copy of a polynucleotide sequence or an amino acid sequence.
For purpose of the present invention, the term “epitope” refers to a molecular region on the surface of an antigen capable of eliciting an immune response and combining with the specific antibody produced by such a response. It is also called “antigenic determinant.” T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules.
For purpose of the present invention, the term “flank” refers to be situated on a side of a polynucleotide sequence or an amino acid sequence.
For purpose of the present invention, the term “fragment” of a molecule such as a protein or nucleic acid refers to a portion of the amino acid or nucleotide sequence.
For purpose of the present invention, the term “fuse” refers to join together physically, or to make things join together and become a single thing.
For purpose of the present invention, the term “fusion polypeptide” or the term “fusion protein” refers to a polypeptide or a protein created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. Usually, a fusion protein has at least two heterologous polypeptides covalently linked, either directly or via an amino acid linker. The heterologous polypeptides forming a fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order and may include more than one of either or both of the constituent polypeptides. These terms encompass conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, interspecies homologs, and immunogenic fragments of the antigens that make up the fusion protein. Fusion proteins of the disclosure may also comprise additional copies of a component antigen or immunogenic fragment thereof. Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics.
For purpose of the present invention, the term “identical” or the term “identity” refers to the percentage of amino acid residues of two or more polypeptide sequences having the same amino acid at corresponding positions.
For purposes of the present invention, the term “immune response” refers to an action by the immune system. The immune system is a system of biological structure and processes within an organism that protects against an invasion of a foreign object. The immune system can be classified into subsystems, such as the innate immune system versus the adaptive immune system, or the humoral immunity versus the cellular immunity. In humoral immunity, responses to foreign objects which include bacteria or viruses involve producing antibodies. In cellular immunity, also called “cell-mediated immunity,” responses to foreign objects including bacteria or viruses involve the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines. Cellular immunity is effective in removing virus-infected cells, but also participates in defending against fungi, protozoans, cancers, and intracellular bacteria. Cellular immunity also plays a major role in transplant rejection.
For purposes of the present invention, the term “immunization dose” refers to the amount of antigen or immunogen needed to precipitate an immune response. This amount will vary with the presence and effectiveness of various adjuvants. This amount will vary with the animal and the antigen, immunogen and/or adjuvant. The immunization dose is easily determined by methods well known to those skilled in the art, such as by conducting statistically valid host animal immunization and challenge studies.
For purposes of the present invention, the term “immunogen” and the term “immunogenic” refer to a substance or material (including antigens) that is able to induce an immune response alone or in conjunction with an adjuvant. Both natural and synthetic substances may be immunogens. An immunogen is generally a protein, peptide, polysaccharide, nucleoprotein, lipoprotein, synthetic polypeptide, or hapten linked to a protein, peptide, polysaccharide, nucleoprotein, lipoprotein or synthetic polypeptide or other bacterial, viral or protozoal fractions.
For purpose of the present invention, the term “linked” refers to a covalent linkage between two polypeptides in a fusion protein. The polypeptides are typically joined via a peptide bond, either directly to each other or via one or more additional amino acids.
For purpose of the present invention, the term “linker” refers to short peptide sequences that occur between functional protein domains and link the functional domains together. Linkers designed by researchers are generally classified into three categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. A flexible linker is often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. A linker also may play a role in releasing the free functional domain in vivo (as in in vivo cleavable linkers). Linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. The composition and length of a linker may be determined in accordance with methods well known in the art and may be tested for efficacy. A linker is generally from about 3 to about 15 amino acids long, in some embodiments about 5 to about 10 amino acids long, however, longer or shorter linkers may be used or the linker may be dispensed with entirely. A glycine linker is one that contains one or more glycines but no other amino acid residues, e.g., GlyGlyGlyGly. A glycine-rich linker is one that contains one or more glycines and may contain one or more other amino acid residues as long as glycine is the predominant species in the linker, e.g., GlyGlyGlyAsnGlyGly. A GlySer linker is one which contains both glycine and serine in any proportion, e.g. GlyGlyGlySer.
For purpose of the present invention, the term “monomer” refers to a molecule that may bind chemically to other molecules to form a polymer. The term “monomeric protein” may also be used to describe one of the proteins making up a multiprotein complex.
For purpose of the present invention, the term “mutant protein” refers to a protein product encoded by a gene with mutation.
For purpose of the present invention, the term “oligomer” refers to a molecular complex that consists of a few monomer units. Dimers, trimers, and tetramers are, for instance, oligomers respectively composed of two, three and four monomers. An oligomer can be a macromolecular complex formed by non-covalent bonding of few macromolecules like proteins or nucleic acids. In this sense, a homo-oligomer would be formed by few identical molecules and by contrast, a hetero-oligomer would be made of three different macromolecules.
For purpose of the present invention, the term “oligomerization” refers to a chemical process that converts monomers to macromolecular complexes through a finite degree of polymerization.
For purpose of the present invention, the term “polymer” refers to a compound or a mixture of compounds comprising many repeating subunits, known as monomers.
For purpose of the present invention, the term “polypeptide” and the term “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms encompass amino acid polymers in which one or more amino acid residues are artificial chemical mimetic of a corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
For purpose of the present invention, the term “protein domain” refers to a distinct functional or structural unit in a protein. Usually, a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions.
For purposes of the present invention, the term “purified” refers to the component in a relatively pure state, e.g. at least about 90% pure, or at least about 95% pure, or at least about 98% pure.
For purpose of the present invention, the term “recombinant protein” refers to a protein derived from a recombinant DNA, that is, it's code was carried by a “recombinant DNA” molecule. Recombinant DNA molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.
For purpose of the present invention, the term “recombinant vaccine” refers to a vaccine made by genetic engineering, the process and method of manipulating the genetic material of an organism. Usually, a recombinant vaccine encompasses one or more protein antigens that have either been produced and purified in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism. The vaccinated person produces antibodies to the one or more protein antigens, thus protecting him/her from disease.
For purpose of the present invention, the term “subunit” refers to a separate polypeptide chain that makes a certain protein which is made up of two or more polypeptide chains joined together. In a protein molecule composed of more than one subunit, each subunit can form a stable folded structure by itself. The amino acid sequences of subunits of a protein can be identical, similar, or completely different.
For purpose of the present invention, the term “subject” or the term “individual” refers interchangeably to a mammalian organism, such as a human, mouse, etc., that is administered a mutant protein of the present invention for a therapeutic or experimental purpose.
For purpose of the present invention, the term “suitable vector” refers to any vector (for example, a plasmid or virus) which may incorporate a nucleic acid sequence encoding an antigenic polypeptide and any desired control sequences. It may bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with a host cell into which the vector is to be introduced.
For purpose of the present invention, the term “type three secretion system (T3SS)” refers to a protein appendage found in Yersinia, a genus of Gram-negative rod shaped bacteria that cause the plague. T3SS is also called “injectisome” or “injectosome,” with a needle-like structure used as a sensory probe to detect the presence of eukaryotic organisms and secrete proteins that help the bacteria infect them. T3SS are essential for the pathogenicity of many pathogenic bacteria.
For purpose of the present invention, the term “vaccine” refers to a biological compound that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism (microbe), such as virus, bacteria, fungus, etc. Traditionally, it is often made from weakened or killed forms of the microbe, its toxins, or one of its surface proteins. The agent injected into a human or animal body stimulates the body's immune system to recognize the agent as foreign, destroy it, and keep a record of it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters.
For purpose of the present invention, the term “transplant” refers to move or transfer a fragment of a DNA or a protein to another place or situation. For example, the NH2-terminal amino acid residues of a protein may be “transplanted” to the COOH-terminus of the protein by deleting the NH2-terminal amino acid residues and fusing them to the COOH-terminus of the protein via a short linker wherein the short linker joins the deleted NH2-terminal amino acid residues to the COOH-terminus of the protein.
Plague, also known as Black Death, is one of the deadliest infectious diseases known to mankind. Yersinia pestis (Y pestis), the etiologic agent of plague, is a Gram-negative bacterium. It injects effector proteins into mammalian host cells to interfere with the host immune response, thereby enabling the pathogens to thrive. Y. pestis is transmitted from rodents to humans via fleas.[1] The bite of an infected flea results in bubonic plague which can then develop into secondary pneumonic plague, resulting in person-to-person transmission of the pathogen through infectious respiratory droplets.[2] Pneumonic plague can also be caused by direct inhalation of the aerosolized Y. pestis, leading to near approximately 100% death of infected individuals within 3-6 days.[2,3] Due to its exceptional virulence and relative ease of cultivation, aerosolized Y. pestis poses one of the greatest threats for deliberate use as a biological weapon.[4] Since the disease spreads rapidly, the window of time available for post-exposure therapeutics is very limited, usually 20-24 hours after the appearance of symptoms.[3] Although levofloxacin, a broad spectrum antibiotic, has recently been approved by the Food and Drug Administration (FDA) for all forms of plague (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm302220.htm), prophylactic vaccination is one of the most effective means to reduce the risk of plague.
Since the deadly anthrax attacks in 2001, stockpiling of recombinant anthrax and plague vaccines to protect masses against a potential bioterror attack became a national priority. However, no plague vaccine has yet been licensed. The reasons include poor stability, insufficient immunogenicity, or manufacturing difficulties associated with the current formulations of plague vaccines. A killed whole cell (KWC) vaccine was once in use in the United States and a live attenuated plague vaccine (EV76) is still in use in the states of former Soviet Union.[5] However, the need for multiple immunizations, high reactogenicity, and insufficient protection made the KWC vaccine undesirable for mass vaccination, and, consequently, it was discontinued in the United States.[6] In fact, because the highly infectious nature of the plague bacterium and the virulence mechanisms of vaccine strains have not been fully understood, the live-attenuated vaccine may not meet the requirement for the approval of FDA.[6,7] A cautionary tale related to this is a recent fatality of a researcher as a result of exposure to the attenuated pigmentation-minus Y. pestis strain, KIM/D2 (http://en.wikipedia.org/wiki/Malcolm_Casadaban).
The focus in the past two decades, thus, has shifted to the development of recombinant subunit vaccines[3,6,8,9] containing two surface-exposed virulence factors of Y. pestis: a capsular protein (Caf1 or F1; 15.6 kDa) and a low calcium response V antigen (LcrV or V; 37.2 kDa) which is a component of a type 3 secretion system (T3SS). Factors F1 and V are known as Y. pestis antigens and have been found to be capable of evoking protective immune responses in animals. The effector proteins of Y. pestis are translocated through an extracellular, hollow needle structure that forms part of the T3SS. The needle is made up of many copies of a single protein called YscF and is anchored by interactions with a T3SS base, which is embedded in the inner and outer bacterial membranes. F1 antigen assembles into flexible linear fibers via a chaperone/usher mechanism,[10] forming a capsular layer that allows Y. pestis to adhere to a host cell and escape phagocytosis.[11] The V antigen forms a “pore” at a tip of an “injectisome” structure of the T3SS needle, creating a channel that delivers a range of virulence factors, also known as the Yersinia outer membrane proteins (Yops), into the host cytosol.12] The V antigen is also critical for impairment of host's phagocytic responses.[13] Abrogation of these functions by F1 and V antibodies appears to be one of the mechanisms leading to protection of the host against lethal Y. pestis infection.
The surface-exposed Y. pestis F1 and V antigens have been the leading candidates for formulating a recombinant subunit plague vaccine for nearly two decades.[14,15,16,17] Two types of F1/V recombinant vaccines, one containing a mixture of F1 and V antigens[14], and another containing a single F1-V fusion protein, have been under investigation.[15,16] Although poorly immunogenic by themselves, their immunogenicity could be enhanced by adjuvantation with Alum[15] or by fusion with a molecular adjuvant such as flagellin.[19] Although both types of F1/V recombinant vaccines induce protective immunity against Y. pestis challenge in rodent and cynomolgus macaque models, the protection of African Green monkeys was insufficient and highly variable.[6,17] A phase I clinical trial in humans showed that a vaccine consisting of a mixture of F1 and V proteins was immunogenic, however, the antibody titers varied over a wide range leading to concerns about the consistency of vaccine efficacy.[18]
One of the problems associated with the current plague vaccines is that the naturally fibrous F1 polymerizes into heterodisperse aggregates, compromising the quality and overall efficacy of the vaccines.[15,19,20,21,22] Second, the subunit vaccines do not induce adequate cell-mediated immune responses, which appear to be essential for optimal protection against plague.[23] Third, it is unclear if inclusion of other Y. pestis antigens such as the YscF, the structural unit of the injectisome needle, can boost the potency of the F1/V vaccines. This is particularly important as F1-minus strains of Y. pestis exist in nature which are as virulent as the wild-type strains[24,25] and the significant diversity in the LcrV sequence of these F1-minus strains might render the current F1/V vaccines ineffective.[26,27] Finally, the reported immunosuppressive property of V antigen[13,28] and whether it could compromise the innate immunity of humans, are significant concerns. These questions must be addressed to generate a next generation plague vaccine that could pass licensing requirements, as well as be manufactured relatively easily for stockpiling.
New immunogen designs and vaccine platforms that could overcome some of these problems would be of great interest not only to stockpile efficacious biodefense vaccines but also to develop vaccines against a series of infectious diseases of public health importance.
The present inventors have developed a novel vaccine delivery system using the bacteriophage T4 nanoparticles.[29,30,31,32] The capsid (head) of a bacteriophage T4 is an elongated icosahedron, 120 long and 86 nm wide, composed of three essential capsid proteins: a major capsid protein, gp23*; a vertex protein, gp24*; and a portal protein, gp20. It is decorated with two non-essential proteins: Soc, a small outer capsid protein; and Hoc, a highly antigenic outer capsid protein. Binding sites for these capsid proteins appear following head “expansion,” a major conformational change that increases the outer dimensions of the capsid by approximately 15% and inner volume by approximately 50%.[33]
Approximately 870 molecules of the tadpole-shaped Soc protein (9 kDa) assemble into trimers at quasi three-fold axes, clamping to adjacent capsomers and forming a reinforced cage around the shell.[34] This stabilizes an already stable head and enables the head to withstand harsh extracellular environment (e.g., pH 10[34]. Hoc, on the other hand, is a linear “fiber” containing a string of four domains, three of which are immunoglobulin (Ig)-like.[35] One hundred and fifty five copies of Hoc fibers, with their NH2-termini projected at approximately 160 Å distance from the capsid, assemble at the center of each capsomer. Hoc binds to bacterial surfaces, apparently enriching the phage near its host for infection.[36] Although Soc and Hoc provide survival advantages to T4 phage, they are completely dispensable under laboratory conditions showing no significant effect on phage productivity or infectivity.[37] Purified Soc (or Hoc) protein binds to Hoc− Soc− capsid of a T4 phage nanoparticle with high specificity and nanomolar affinity, properties that are not compromised by attachment of a pathogen antigen at the NH2— and COOH-termini.[29,30,31,32] Individual domains, full-length proteins as large as approximately 90 kDa, or multilayered oligomeric complexes that are larger than approximately 500 kDa fused to Soc can be arrayed on T4 capsid, making it a robust antigen delivery platform.[29,30]
Disclosed embodiments provide structure-based immunogen design and T4 nanoparticle delivery approaches to engineer new and efficacious plague vaccines that could be manufactured relatively easily and could provide complete protection against pneumonic plague in at least two rodent models.
In some embodiments of the present invention, Y. pestis surface components are targeted for vaccine design.
The X-ray structure and biochemical studies have established that F1 polymerizes into a linear fiber by head to tail interlocking of F1 subunits through a donor strand complementation mechanism.[10] Each F1 subunit has an Ig-like domain consisting of a four-stranded anti-parallel β-sheet. Of the four β-strands, three belong to one subunit forming a cleft into which the NH2-terminal β-strand of the “n+1” subunit locks in, resulting in a bridge connecting adjacent subunits (inter-molecular complementation) (see Panel B of
Panel B of
In one embodiment of the present invention, the mutated F1 antigen is fused to V antigen to produce a bivalent F1mut-V immunogen that is also expressed as a soluble monomer. In one embodiment of the present invention, the mutated F1 is fused to V antigen via a flexible two amino acid peptide linker Ser-Ala.
Some embodiments of the present invention provide approaches to construct an oligomerization deficient YscF mutant antigen and a V mutant antigen lacking a putative immunomodulatory sequence. Panel C of
In some embodiments of the present invention, thirty COOH-terminal amino acid residues from 270 to 300 of V antigen of Yersinia pestis are deleted to thereby form a mutant V10 antigen. The mutant V10 may be fused to the T antigen of Yersinia pestis to thereby form a fusion protein F1mut-V10.
In some embodiments, a phage capsid protein fusion protein is constructed by fusing one or more small outer capsid proteins of a phage T4 or a T4-related phage to one or more antigens via one or more linkers. In some embodiments, the one or more small outer capsid proteins of a phage T4 or a T4-related phage encompassing Soc from phage T4 or T4-related bacteriophage RB69.
In some embodiments, a mutant antigen is fused to a Soc protein from a phage T4 or a T4-related phage RB69 via a linker to thereby form a Soc fusion protein. The linker may comprise a two amino acid linker Gly-Ser. In some embodiments, the Soc fusion protein encompasses mutated F1 antigen and V antigen of Yersinia pestis. Some embodiments of the present invention disclose that the Soc fusion protein is further fused to a mutant YscF35/67 antigen of Yersinia pestis via a linker to thereby form a fusion protein F1-V-Soc-YscF. In some embodiments, the fusion protein F1-V-Soc-YscF may be formed by fusing the mutant YscF35/67 antigen of Yersinia pestis to the Soc protein of the Soc fusion protein, and wherein the linker may be a two amino acid linker Gly-Ser.
In other embodiments of the present invention, a native V antigen of Yersinia pestis is fused to a Soc protein from a phage T4 and/or a T4-related bactgeriophage RB69 via a linker, which linker may be a two amino acid linker Gly-Ser, wherein a COOH-terminus of the linker is directly linked to an NH2-terminus of the Soc protein from a phage T4 and/or a T4-related bacteriophage RB69 and an NH2-terminus of the linker is directly linked to a COOH-terminus of the V antigen of Yersinia pestis.
Disclosed embodiments of the present invention provide that a native F1 antigen of Yersinia pestis may be fused to a Soc protein from a phage T4 and/or a T4-related bactgeriophage RB69 via a linker, which linker may be a two amino acid linker Gly-Ser, wherein a COOH-terminus of the linker is directly linked to an NH2-terminus of the Soc protein from phage T4 and/or T4-related bacteriophage RB69 and an NH2-terminus of the linker is directly linked to a COOH-terminus of the F1 antigen of Yersinia pestis.
In some embodiments of the present invention, a fusion protein F1mut-V-Soc is fused to a YscF antigen of Yersinia pestis via a linker.
In one embodiment of the present invention, the mutated antigens fused to Soc are bound on phage T4 nanoparticles.
Panel E of
Panel F of
In one embodiment of the present invention, fusion proteins encompassing mutated F1 and V of Y. pestis are expressed in bacteria E. Coli cells and purified from cell-free lysates of E. Coli cell cultures.
In some embodiments of the present invention, an immunogenic amount of a vaccine comprising a purified mutated F1 antigen of Yersinia pestis and an adjuvant is administered to a subject. This vaccine may be administered to a subject via an intramuscular route. In another embodiment of the present invention, an immunogenic amount of a vaccine comprising a purified recombinant protein F1mut-V and an adjuvant is administered to a subject. In one embodiment of the present invention, an immunogenic amount of a vaccine comprising a purified recombinant protein of F1mut-V10 and an adjuvant is administered to a subject.
In some embodiments of the present invention, vaccines encompassing T4-decorated purified recombinant proteins such as F1mut-V-Soc and F1mut-V-Soc-YscF are administered to a subject without any adjuvant. The approaches to administer the vaccines may vary. The vaccines may be administered via an intramuscular route, oral route, or any other appropriate routes.
As shown in some examples of the present application, purified bivalent F1mut-V monomers induces robust immunogenicity. In addition, the T4-decorated fusion protein F1mut-V (fusion protein F1mut-V bound on T4 nanoparticles), without any adjuvant, induced balanced TH1 and TH2 responses. Both the soluble and T4 decorated F1mut-V provide approximately 100% protection to mice and rats against intranasal challenge with high doses of Y. pestis CO92. Inclusion of YscF showed a slight enhancement in the potency of F1-V plague vaccine, whereas a replacement of V with V10 mutant, which lacks the putative immunosuppressive sequence, did not significantly alter vaccine efficacy. These results provided new insights into plague vaccine design and produced next generation plague vaccine candidates by overcoming some of the concerns associated with the current subunit vaccines.
The description of the present invention is enhanced by the various examples that follow.
This study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Texas Medical Branch, Galveston, Tex., (Office of Laboratory Animal Welfare assurance number: A3314-01) and The Catholic University of America (Office of Laboratory Animal Welfare assurance number: A4431-01).
The T7 promoter containing E. coli expression vector pET28b is used for recombinant plasmid construction. The template DNAs containing Y. pestis F1, V, or YscF are kindly provided by Dr. Richard Borschel from the Walter Reed Army Institute of Research (Silver Spring, Md.). E. coli XL-10 Gold cells are used for the initial transformation of clones. The plasmid DNAs are then re-transformed into E. coli BL21 (DE3) RIPL for expression of recombinant proteins. The Hoc−Soc− phage T4 is propagated on E. coli P301 and purified by CsCl gradient centrifugation.
The DNAs encoding F1, V, or YscF are amplified by PCR using primers containing appropriate restriction site(s) (NheI/XhoI for F1 and YscF, and NheI/HindIII for V). The PCR products are purified, digested with appropriate restriction enzymes, and ligated with pET-28b vector DNA digested with the same restriction enzymes. The resulting plasmids had F1, V, or YscF coding sequences fused in-frame with the 23 aa vector sequence containing a hexa-histidine tag at the NH2-terminus. The YscF mutant, YscF35/67, which contained point mutations at aa 35 (Asn to Ser) and 67 (Ile to Thr) is amplified by overlap PCR[61] followed by digestion with NheI and XhoI enzymes. YscF35/67 DNA is then ligated into the linearized pET28b vector. The F1mut1, in which the first 14 aa residues are deleted and fused to the COOH-terminus with a two aa (Ser-Ala) linker, is constructed by two rounds of PCR. The first round of PCR is performed to amplify F1 fragment in which the NH2-terminal 14 aa residues are deleted. This PCR product is used as a template for the second round of PCR using a forward primer containing NheI restriction site and a reverse primer containing the NH2-terminal 14 aa residues and XhoI restriction site. The PCR fragment is then inserted into NheI and XhoI linearized pET28b vector.
To construct F1mut2 in which aa residues 15 to 21 are duplicated at the COOH-terminus, a reverse primer with a 5′-tag corresponding to the 15 to 21 aa sequence and XhoI restriction site is used for PCR amplification. The F1mut2 fragment is then inserted into NheI and XhoI linearized pET28b vector. To construct F1-V recombinants, V is first amplified and inserted into BamHI and HindIII linearized pET28b vector to generate the pET-V clone. F1 and F1mut2 are amplified with primers containing NheI and BamHI restriction sites, digested with NheI and BamHI, and ligated with the pET-V vector DNA digested with the same restriction enzymes. The resulting F1-V and F1mut-V plasmids contain F1 or F1mut in-frame fusion with V and a 23-aa vector sequence containing the hexa-histidine sequence at the NH2-terminus of F1. The F1mut-V10 is amplified by overlap PCR using F1mut-V as the template and the mutated DNA is inserted into the NheI and HindIII linearized pET28b vector.
T4 Soc gene or phage RB69 Soc gene is fused with V, F1, or YscF with a two amino acid linker Gly-Ser by overlap PCR and the amplified DNA is inserted into the pET28b vector. The fused products V-T4 Soc, F1-T4 Soc, V-RB69Soc, and F1-RB69 Soc are further fused to YscF by overlap PCR to generate V-Soc (T4 or RB69)-YscF and F1-Soc (T4 or RB69)-YscF. A two amino acid linker Gly-Ser is used as a linker between Soc and YscF. To construct F1-V-Soc clones, RB69 Soc gene is first amplified with end primers containing HindIII and XhoI restriction sites and inserted into the HindIII and XhoI linearized pET28b vector. This clone is then linearized by digestion with NheI and HindIII restriction enzymes. F1mut-V and F1mut-V10 DNAs are amplified by using the end primers containing NheI and HindIII restriction sites and inserted into the above plasmid. The resulting clones contain F1mut-V or F1mut-V10 fused in-frame to the NH2-terminus of RB69 Soc and also contain the flanking vector sequences containing two hexa-histidine tags at both NH2— and COOH-termini. The F1mut-V-Soc is then fused with YscF by overlap PCR with a two amino acid linker Gly-Ser between Soc and YscF. All of the clones are sequenced (Retrogen, CA) and only the clones containing approximately 100% sequence accuracy are used for protein purification.
The structural models of F1, V, YscF, and a T4 phage nanoparticle are constructed using Chimera version1.4.1.[62] The T cell epitopes are predicted using MetaMHC, a new web server which integrates the outputs of leading predictors by several popular ensemble strategies.[63] This is shown to generate statistically significant results that are more reliable than the individual predictors.[63] For the CD4+ T cell epitope prediction, F1 protein sequence is screened against 14 human MHC-II alleles. Peptides identified as positive ones by at least one predictor method are considered as potential CD4+ T cell epitopes. For the CD8+ T cell epitope prediction, F1 is screened against 57 human MHC-I alleles. Peptides identified as positive by at least one ensemble predictor approaches are considered to be potential CD8+ T cell epitopes. Default values are used for both the T cell epitope predictions.
The E. coli BL21 (DE3) RIPL cells harboring various plague recombinant plasmids constructed as above are induced with 1 mM IPTG for 1 to 2 h at 30° C. The cells are harvested by centrifugation at 4,000 g for 15 min at 4° C. and the pellets are resuspended in 50 mM Tris-HCl (pH 8.0). Solubility analysis is carried out using bacterial protein extraction reagent (B-PER). The cells are lysed with B-PER and centrifuged at 12,000 g for approximately 10 min. The soluble supernatant and insoluble pellet fractions are analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) as follows. The samples are boiled in a buffer containing SDS and β-mercaptoethanol, and are electrophoresed on a 12% or 15% (w/v) polyacrylamide gel. Since the protein aggregates will be dissociated into monomers under these conditions. The molecular weight differences reflect sizes of the polypeptide chains of F1, F1mut1, and F1mut2. For example, F1mut1 and Fmut2 are approximately 1.6 kDa and 2.2 kDa larger than F1 because F1mut1 has a two amino acid linker Ser-Ala and an eight amino acid His-tag comprising SEQ ID NO 2 at the C-terminus. F1mut2, in addition, has the duplicated T cell epitope comprising SEQ ID NO 1.
For protein purification, the cells are resuspended in a binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl and 20 mM imidazole) containing proteinase inhibitor cocktail. The cells are lysed by a French press at 12,000 psi and the soluble fractions containing the His-tagged fusion proteins are isolated by centrifugation at 34,000 g for 20 min. The supernatants are filtered through 0.22 μm filters and loaded onto 1 ml HisTrap column pre-equilibrated with 20 ml of binding buffer. After washing with the binding buffer containing 50 mM imidazole, the proteins are eluted with 20-500 mM linear imidazole gradient. The peak fractions containing the desired protein are concentrated by Amicon Ultra-4 centrifugal filtration (approximately 10 kDa cut-off; Millipore). The proteins are further purified by gel filtration on Hi-load 16/60 Superdex 200 column (AKTA-FPLC) in a buffer containing 20 mM Tris-HCl, pH 8.0 and 100 mM NaCl. The peak fractions containing the purified proteins are concentrated and stored at −80° C. The native F1 recombinant proteins are purified from the pellet containing the insoluble inclusion bodies. The pellet is dissolved in the binding buffer containing 8 M urea and loaded onto 1 ml HisTrap column pre-equilibrated with the same buffer. The proteins are renatured by washing the column with a decreasing urea gradient (8 to 0 M) in the binding buffer. The bound proteins are then eluted with 20-500 mM linear imidazole gradient. If necessary, the peak fractions from the HisTrap column are concentrated by Amicon Ultra-4 centrifugal filtration (approximately 10 kDa cut-off). The proteins are further purified by gel filtration on Hi-load 16/60 Superdex 200 column as described above.
The levels of lipopolysaccharide (LPS) contamination in the purified recombinant Y. pestis antigens from E. coli; F1, LcrV, YscF, and F1mut-V, are determined using Endosafe PTS system. This system consists of a handheld spectrophotometer and utilizes FDA approved disposable cartridges. At least three batches of each antigen are tested. The endotoxin levels ranged from 0.05 to 0.8 EU/ml, substantially lower than the maximum recommended in gene vectors and subunit vaccines, 10 and 20 EU/ml respectively, for preclinical research.[64]
In vitro binding of plague-Soc fusion protein on Hoc−Soc− T4 phage is carried out as previously described.[29,30,32] About 3×1010 phage particles are sedimented for 45 min at 34,000 g in LoBind Eppendorf tubes and resuspended in phosphate-buffered saline (PBS) buffer (pH 7.4). Various Soc fusion proteins are incubated with the resuspended Hoc−Soc− phage at 4° C. for 45 min. The phage particles are sedimented at 34,000 g for 45 min and the supernatant containing the unbound protein is discarded. The phage pellet containing the bound plague antigen(s) is washed twice with excess buffer containing 20 mM Tris-HCl pH 8 and 100 mM NaCl. The final pellets are resuspended in PBS buffer (pH 7.4) and analyzed by SDS-PAGE. The gels are stained with Coomassie Blue R250 and the protein bands are quantified by laser densitometry. The density of Soc fusion protein, gp23*, and gp18 (major tail sheath protein; approximatley 70 kDa) bands are determined for each lane separately and the copy number of bound plague antigen molecules per capsid is calculated using the known copy numbers of gp23* (930 molecules per capsid) or gp18 (138 molecules per capsid). A saturation binding curve relating the number of bound plague protein-Soc molecules per capsid (Y) and the concentration of unbound protein in the binding reaction (X) is generated by SigmaPlot software. The apparent Kd (association constant) and Bmax (maximum copies of Soc fusion protein bound per capsid) are determined using the following equation as programmed in the SigmaPlot software:
Six to eight weeks female Balb/c mice (17-20 g) are purchased from Jackson Laboratories (Bar Harbor, Me.) and randomly grouped and acclimated for 7 days. Equivalent amounts of plague immunogen molecules, either soluble or phage-bound, are used for each immunization. For immunization of soluble antigens, the purified proteins (10 μg/mouse/immunization) are adsorbed on alhydrogel containing 0.19 mg of aluminum per dose. For the T4 displayed antigens, the phage particles are directly used without any adjuvant (10 μg of plague antigen/mouse/immunization). On days 0, 21 and 42, mice are vaccinated via the intramuscular route. Alternate legs are used for each immunization. Blood is drawn from each animal on days 0 (pre-bleeds), 35 and 49 and the sera obtained are stored frozen at −70° C. On day 56, mice are intranasally challenged with Y. pestis CO92 BEI strain [65] using the indicated LD50. Animals are monitored and recorded twice daily for mortality or other symptoms for 48 to 88 days. The animals that survived are re-challenged intranasally at 48 or 88 days post-first challenge with the indicated LD50 and monitored twice daily for a further 48 days.
Female Brown Norway rats (approximately 50-75 g) are purchased from Charles River (Houston, Tex.). Upon arrival, animals are weighed and randomized into the treatment groups and are acclimated for several days before manipulation. The plague immunogens are prepared as described above. On days 0, 21 and 42, rats are vaccinated via the intramuscular route with 15 μg antigen in 50 μl PBS buffer. Alternate legs are used for each immunization. On day 56, animals are intranasally challenged with 5,000 LD50 of Y. pestis CO92 BEI strain and are monitored twice daily for 30 days and clinical symptoms of disease and survival recorded.
The IgG titers are determined by ELISA. Briefly, 96-well microtiter plates are coated with approximately 10 ng/well of purified F1, V, YscF, F1-V or F1mutV antigen at 4° C. overnight. Following blocking and washing, sera from naïve and immunized mice are serially diluted and incubated with the affixed antigens for 1 h at room temperature. Following several washes, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody is added to the wells at a dilution of 1:10,000. After incubation for 1 h at room temperature, the unbound antibody is removed and the wells are washed several times and the TMB (3,3′,5,5′-tetramethylbenzidine) substrate is added. Following a 20 min incubation to develop the color, the reaction is quenched by the addition of 2 NH2SO4 and the absorbance is read at 450 nm using an ELISA reader. For IgG subtypes, horseradish peroxidase-conjugated goat anti-mouse IgG1 or IgG2a secondary antibodies are used.
Seven days after the second boost (day 49), mice are sacrificed and spleens are harvested to prepare splenocytes using the lymphocyte separation medium. The isolated lymphocytes are adjusted to approximately 5×106 cells/ml and 1 ml of lymphocytes seeded into each well. Triplicate cultures from each group are stimulated with purified F1-V (10 μg/ml). Additional control stimulators included medium only and concanavalin A (5 μg/ml). After approximately 48 h incubation at 37° C. in a humidified (5% CO2 in air) incubator, culture supernatants are collected. Cytokines are measured using a multiplex assay. The results are analyzed in Prism and the statistical significance is determined by one way ANOVA with Bonferroni correction.
F1 capsule antigen (caf1) [GeneID: 1172839, Sequence: NC—003134.1 (85950.86462)], lcrV [GeneID: 1172676; Sequence: NC—003131.1 (21935.22915, complement)], yscF [GeneID: 1172700, Sequence: NC—003131.1 (41026.41289)], and soc (RB69Soc) [GeneID:1494143, Sequence: NC—004928.1 (14980.15216, complement)].
Panel A of
Panel B of
Data in Panel B of
According to disclosed embodiments, shifting of the NH2-terminal β-strand of F1 to the COOH-terminus of F1 reorients the β-strand such that it fills its own cleft (intra-molecular complementation) (Panel B of
The recombinant protein F1mut1 is purified from the cell-free lysates by HisTrap affinity chromatography followed by Hi-load 16/60 Superdex 200 gel filtration. The molecular weight of F1mut1 peak fraction is calculated from the calibration curve constructed by gel filtration on the same column of standard proteins of known molecular weight [Thyroglobulin (669 kDa), Ferritin (440 kDa), Catalase (232 kDa), aldolase (158 kDa), Ovalbumin (43 kDa), RNase A (14 kDa), and Albumin (67 kDa)]. A gel filtration profile showes that the F1mut1 is eluted as a symmetrical peak corresponding to a molecular mass of approximately 19 kDa (Panel C of
In this example, a bioinformatics approach is used to determine if shifting of the NH2-terminal β-strand of F1 to the COOH-terminus of F1 disrupts the NH2-terminal epitopes of F1. The aa residues 7 to 20 are reported to contain a mouse H-2-IAd restricted CD4+ T cell epitope.
CD8+ and CD4+ T cell epitopes are predicted using MetaMHC (http://www.biokdd.fudan.edu.cn/Service/MetaMHC.html) with default values. Peptides identified as positive by at least one ensemble predictor approach are considered to be potential CD8+ T cell epitopes or potential CD4+ T cell epitopes.
Predicted CD8+ T cell epitopes are shown in Table 1 below. Highlighted cells indicate high ranking scores that predict a potential CD8+ T cell epitope. Predicted CD4+ T cell epitopes are shown in Table 2 below.
Of the fifty-three predicted 9-mer CD8+ T cell epitopes that encompassed 46 human MHC-I alleles (Table 1), four peptides (aa residues: 9-17, 10-18, 11-19 and 13-21) fall in this region; of the 9 peptides predicted to contain CD4+ T cell epitopes (Table 2), only one (aa residues 1-18) belongs to this region. The integrity of these potential linear epitopes may be restored by extending the sequence of the switched strand by up to the aa residue 21, which can be done by duplicating the NH2-terminal residues from 15 to 21 of F1 at the COOH-terminus. A recombinant protein F1mut2 is accordingly constructed (Panel A of
Panel A of
Fusion of F1mut2 to V can generate a bivalent plague vaccine. Consequently, in this example, a mutated F1-V fusion protein (F1mut-V) is produced by fusing F1mut2 to the NH2-terminus of V with a two amino acid linker (Ser-Ala) in between and the solubility of the fusion protein F1mut-V is compared to that of the native polymeric F1-V.
The Y. pestis V antigen is reported to induce interleukin (IL)-10 and suppress the production of pro-inflammatory cytokines such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which may lead to lowering of innate immunity in vaccinated individuals.[41] A truncated V in which the COOH-terminal 30 aa residues (271-300) are deleted (referred to as “V10” mutation) is reported to lack this immunomodulatory function.[41] A mutated F1mut-V10 recombinant is therefore constructed by deleting these residues (Panel A of
Expression and solubility analysis of F1-V constructs are performed using the B-PER reagent. The samples of lysates are analyzed by SDS-PAGE and Coomassie Blue staining (Panel B of
F1-V, F1mut-V and F1mut-V10 are expressed in E. coli and purified by HisTrap column chromatography followed by Hi-load 16/60 Superdex 200 gel filtration. A calibration graph in Panel C of
The native F1-V, as reported previously,[20,22] is insoluble and partitioned into inclusion bodies (lanes 5 and 6 of Panel B of
Stability of F1-V and F1mut-V proteins is tested by treatment with increasing amounts of trypsin at room temperature overnight. In Panel D of
Inclusion of YscF might expand the breadth of efficacy of F1-V plague vaccine formulation to Y. pestis strains containing variant V antigens,[26] or of those strains devoid of capsule but highly virulent in nature.[24,25] Since YscF is a structural component of the injectisome of Y. pestis, over-production of this protein caused aggregation.[42] In this example, a mutant YscF is constructed by mutating the aa residues Asn35 and Ile67, that are involved in oligomerization (Asn35 changed to Ser, and Ile67 changed to Thr) (see Panel A of FIG. 4).[43]
YscF and YscF35/67 mutant proteins are purified (Panel B of
In this example, a large number of F1, V, F1-V, and YscF recombinant proteins, both in native and mutated forms, are fused to the NH2- and/or the COOH-termini of either a phage T4 Soc or a T4-related phage RB69 Soc and screened for their solubility as well as ability to bind to T4 phage nanoparticles.
Panel A of
As shown in Panel A of
The Soc fusion proteins in panel A are over-expressed and purified as described above in Materials and Methods. The purity of the proteins is evaluated by SDS-PAGE and Coomassie Blue staining (see Panel B of
Panel C of
Saturation binding curve of F1mut-V-Soc is shown in Panel D of
Image in Panel E of
In this example, a series of nanoparticle decorated plague immunogens are prepared, including all three plague immunogens displayed on the same capsid using the F1mut-V-Soc-YscF35/67 fusion protein (Panel G of
Disclosed embodiments show that the RB69 Soc binds to T4 capsid at nearly the same affinity as T4 Soc.[34] The RB69 Soc-fused plague antigens, with the exception of the native F1-Soc, produce soluble proteins whereas the T4 Soc-fused antigens are insoluble. Several of phage RB69 immunogens are purified (see Panel B of
The 66 kDa F1mut-V-Soc bound to T4 even at a relatively low 1:1 ratio of F1mut-V-Soc molecules to Soc binding sites (Panel C of
In this example, the immunogenicity and protective efficacy of F1mut-V and other plague immunogens are evaluated in a mouse model. Balb/c mice, twelve per group, are vaccinated with various plague antigens adjuvanted with alhydrogel as shown in Panel A of
The data shows that all the three plague antigens adjuvanted with alhydrogel induce antigen-specific antibodies (Panel C of
Intranasal challenge of animals with 90 LD50 of Y. pestis CO92 [1 LD50=100 colony forming units (CFU) in Balb/c mice], one of the most lethal strains, shows that all the control mice died by day 3. However, the mice immunized with native V immunogen shows approximately 83% survival (two of twelve mice died), whereas the mice immunized with F1-V, F1mut-V, or F1-V plus YscF are approximately 100% protected (see Panel D of
The survived mice are re-challenged with a higher dose, 9,800 LD50, of Y. pestis CO92 on day-48 post-first challenge. The purpose of re-challenge is to determine if a strong adaptive immunity is generated after first infection with Y. pestis, which should in turn confer a much higher level of protection against subsequent challenges. Indeed, disclosed data shows that all of the mice survived the re-challenge except two mice in the native F1-V group that succumbed to infection (approximately 83% protection) (Panel D of
The immunogenicity of nanoparticle decorated plague antigens is tested by vaccinating mice with phage T4 particles. The immunogenicity and protective efficacy of T4 displayed plague immunogens are evaluated in a mouse model using the same immunization scheme as described and shown in Panel B of
The data In Panel C of
The challenge data shows that all the T4 decorated plague antigens, including the V alone group, provided approximately 100% protection to mice against intranasal challenge with 90 LD50 of Y. pestis CO92; all the control animals died by day 4. Upon re-challenge on day 48 post-first challenge with 9,800 LD50, all of the mice are completely protected (see Panel C of
This example demonstrates that the T4 nanoparticle displayed plague immunogens induced robust immunogenicity and protective efficacy against pneumonic plague.
Stimulation of both arms of the immune system, humoral (TH2) and cellular (TH1), is probably essential for protection against Y. pestis infection.[6,23,44,45] In mice, the TH1 profile involves induction of antibodies belonging to IgG2a subclass whereas the TH2 profile primarily involves the induction of IgG1 subclass. To determine the specificity of antibodies induced by soluble vs T4 displayed antigens, the subclass IgG titers are determined by ELISA (see
Note that the sera of the control T4 phage-immunized mice show higher background. This is because T4 phage nanoparticles, as demonstrated in previous studies, induces a strong antibody response to its components. Consequently, the sera from T4 phage-immunized mice will have increased amounts of IgGs compared to the pre-immune sera, giving more non-specific background at low dilutions of the sera. Data shown are the antibody titers of 12 mice in each group with S.D. (error bars). “*”: p<0.05; “***”: p<0.001 (ANOVA).
The immunogenicity and protective efficacy of F1mut-V vs F1mut-V10 is evaluated by three criteria: F1- and V-specific antibody titers, cytokine responses, and protection against Y. pestis CO92 challenge. The immunogenicity and protective efficacy of F1mut-V and F1mut-V10 are compared both as adjuvanted soluble antigens or adjuvant-free T4 nanoparticle decorated antigens.
The vaccine formulations used in the study, eight mice per group, are shown in Panel A of
Panel C of
Seven days after the second boost (day-49), mice (5 per group) are sacrificed and spleens are harvested. The splenocytes are cultured and stimulated by purified F1-V protein. Cytokines levels are determined as described in Materials and Methods.
Both the F1- and V-specific IgG antibodies (see Panel B of
With respect to animal survival, both the F1mut-V and F1mut-V10 immunogens, either soluble or T4 displayed, provided approximately 100% protection to mice upon intranasal challenge with 5,350 LD50 of Y. pestis CO92 (see Panel C of
To further test the efficacy of the mutated immunogens, a study on Brown Norway rat model of pneumonic plague is conducted. Rats,[47] the natural host of Y. pestis, are vaccinated with alhydrogel adjuvanted F1mut-V, and F1mut-V10 as well as the T4 nanoparticle displayed F1mut-V and F1mut-V10 (Panel A of
This present invention proposes three hypotheses to design a soluble monomeric plague vaccine, yet retaining its structural and epitope integrity. First, disclosed embodiments hypothesize that the β-strand that connects the adjacent F1 subunits requires repositioning. This is achieved by transplanting the NH2-terminal β-strand to the COOH-terminus in such a way that the reoriented β-strand fits into its own β-sheet cleft rather than that of the adjacent F1 subunit. It also eliminated the need for chaperone and usher mediated oligomerization as there would no longer be an unfilled β-sheet pocket exposed in the F1 subunit. Second, by using epitope predictions, the NH2-terminal aa residues 15-21 of F1 flanking the β-strand are duplicated at the COOH-terminal end of F1 to restore any potential T-cell epitopes that might have been lost during the switch. This is important, because, in a previous study, a simple β-strand switch produced a less stable monomer with diminished immunogenicity.[48] Third, the mutated F1 is fused to the NH2-terminus of V with a flexible linker in between to minimize interference between the F1 and V domains. The bivalent F1mut-V immunogen thus produced shows a remarkable shift in solubility, from an insoluble F1-V polymer to a completely soluble monomer (
Several lines of evidence demonstrate that the F1mut-V monomer is as efficacious as, if not better than, the native F1-V polymer. In four separate immunization studies and two animal models (
The possibility of increasing the breadth and potency of F1-V vaccine by inclusion of YscF is tested by constructing an oligomerization deficient YscF35/67 mutant.[43] Such a vaccine might be effective even against those Y. pestis strains that contain variant V antigens or lack the capsule, but are highly virulent.[26] The mutated protein, purified as a soluble dimer, elicited YscF-specific antibodies on its own, and, when it is mixed with F1-V, it enhanced the induction of V-specific antibody titers as well as survival rate in mice (
Y. pestis infection stimulates IL-10 production which in turn suppresses the production of proinflammatory cytokines IFN-γ and TNF-α. Both IFN-γ and TNF-α are important for innate immunity, as well as to elicit TH1 immune responses that might be essential for protection against pneumonic plague.[49,50,51] These immunomodulatory functions, in part, are attributed to the V antigen, specifically to the NH2-terminal aa residues 31-49.[49] Deletion of these residues, or of the COOH-terminal aa residues 271-300 (V10 mutation), have been reported to abrogate the suppression of IFN-γ and TNF-α[41], presumably by preventing the interaction of V with toll like receptor 2 (TLR2) and CD14, the receptors of the innate immune system.[49,52] The disclosed studies showed that both the F1mut-V and F1mut-V10 immunogens produced similar levels of IFN-γ and other proinflammatory cytokines, such as IL-1α and upon stimulation ex-vivo of splenocytes from immunized mice with F1mut-V. However, TNF-α is induced to significantly higher levels in the F1mut-V10 group (
Although humoral immune responses are critical for protection against plague, several studies have shown that cell-mediated immunity also plays important roles.[23,53,54] Wang et al.[53] established the role of CD8+ T cells in protection of mice against pneumonic plague evoked by Y. pestis KIM 1001 strain. This study corroborated the earlier report of Parent et al.,[23] which concluded that plague vaccines that generate both humoral- and cell-mediated immune responses will be most effective. Likewise, Philipovskiy and Smiley (3) reported that mice vaccinated with a live Y. pestis vaccine primed both CD4+ and CD8+ T cells, which when passively transferred to naïve mice, provided protection against pulmonary Y. pestis infection.[54] The adjuvant-free T4 nanoparticle decorated F1mut-V induced robust F1- and V-specific antibody responses, as well as provided approximately 100% protection to mice and rats against very high doses of Y. pestis challenge (
There has been a considerable urgency to develop a recombinant plague vaccine, but several concerns precluded licensing of current formulations. Disclosed studies have established that the F1mut-V recombinant vaccine is efficacious and easily manufacturable and should be seriously considered as a next generation plague vaccine. Future studies would include preclinical evaluation of protection against Y. pestis infection in cynomolgus macaques as well as African Green monkeys, potentially leading to human clinical trials. Although the soluble F1mut-V vaccine adjuvanted with alum would be relatively easy to manufacture, the phage T4 nanoparticle-decorated F1mut-V vaccine offers certain advantages. First, the T4 formulation provided enhanced vaccine potency in small animal models. Second, the T4 vaccine would not require an extraneous adjuvant, and third, additional antigens from other biodefense pathogens, such as the protective antigen (PA) from Bacillus anthracis could be incorporated into the same formulation generating a dual vaccine against both inhalation anthrax and pneumonic plague. The recent disclosed study demonstrats that the T4 displayed PA provided complete protection to Rhesus macaques against aerosol challenge with Ames spores of B. anthracis.[51] Fourth, the large interior of T4 head which has the capacity to package approximately 171 kb DNA can also be used to deliver DNA vaccines.[60] By combining protein display outside and DNA packaging inside the T4 nanoparticles can simultaneously deliver vaccine antigen(s) as well as vaccine DNAs, similar to that of the prime-boost strategy, potentially inducing robust and long-lasting immune responses. Finally, such prime-boost vaccines could be targeted to antigen-presenting dendritic cells (DCs) by displaying a DC-specific ligand on the capsid using Hoc, further stimulating the cell-mediated immunity. One or two doses of such potent nanoparticle vaccines might be sufficient to afford protection against multiple biothreat agents. With the recent data demonstrating the proof of the concept,[60 disclosed embodiments are currently developing these novel vaccine platforms, not only to defend against biowarfare pathogens but also to generate efficacious vaccines against complex infectious agents such as HIV-1 and malaria.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of microbiology, recombinant DNA technology and molecular biology and immunology, which are within the skills of the art.
Furthermore, in the present invention, one of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
All documents, patents, journal articles and other materials cited in the present application are incorporated herein by reference.
Although an example of the phage T4 nanoparticle arrayed immunogens comprising fusion proteins of F and V from Yersinia pestis are shown as used for generating plague vaccines, it will be appreciated that immunogens of other pathogens can be arrayed on the phage T4 nanoparticles for developing vaccines for other diseases.
The many features and advantages of the invention are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the invention which fall within the true spirit and scope of the invention. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.
The following references are referred to above and are incorporated herein by reference:
This application claims benefit of priority to U.S. Provisional Patent Application No. 61/845,487 to Rao and Tao, entitled “Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia Pestis as Next Generation Plague Vaccines,” filed Jul. 12, 2013. The entire contents and disclosures of the patent application are incorporated herein by reference in its entirety.
United States Government has rights in this invention pursuant to Contract No. NIAID U01-AI082086, NIAID AI064389 (in part), and NO1-A1-30065 awarded by National Institutes of Health; and T32 predoctoral training grant on Biodefense AI060549.
| Number | Date | Country | |
|---|---|---|---|
| 61845487 | Jul 2013 | US |
