Patent Application
20110311587
1. Field of the Invention
The present invention relates to the field of paramyxovirus biology. More specifically, the present invention relates to nipah virus-like particles and uses thereof.
2. Description of the Related Art
Since it was first recognized in 1998, Nipah virus (NiV) has caused several outbreaks in humans of encephalitic disease associated with high lethality. In the first outbreak, which was in Malaysia and Singapore, 265 humans became sick and some ˜40% of them died. Epidemiological links pointed to human contact with sick pigs in commercial piggeries, and the outbreak was brought under control through culling of approximately ˜1.1 million pigs [1,2,3,4]. Since then, the virus has re-emerged in Bangladesh and India, starting in 2001, and has caused several smaller but even deadlier outbreaks with fatality rates between 60 and 90% [5,6,7,8]. Unlike the Malaysian outbreak, the route of transmission in these outbreaks was considered to be bat-to-human via food contaminated with bat saliva [9]. In some cases, nosocomial transmissibility and person-to-person spread was also noted [5,10,11,12]. An additional concern is that Nipah virus is a potential agro-terror agent since the rate of transmission of this virus in the pig population is close to 100% [13]. Effective vaccine and therapies are needed to combat the threats posed by Nipah virus.
Nipah virus is a member of the genus Henipavirus in the subfamily Paramyxovirinae, family Paramyxoviridae. It has several distinctive genetic and biologic features [14,15,16,17,18] although its morphology and genome organization is similar to that of other members of the subfamily. Nipah virus has six genes arranged in tandem, 3′-N, P, M, F, G and L-5′ [15,16]. The N, P and L are required for reconstituting viral RNA polymerase activity, the matrix protein M is required for particle formation and budding, and the two surface glycoproteins G and F are required for attachment and entry into the host cell [19,20]. EphrinB2 and B3 have been identified as the Nipah virus entry receptors [21,22,23]. After fusion of the virus and the cell membrane, the viral ribonucleoprotein is released into the cell cytoplasm. Following transcription and replication, the viral components migrate to the plasma membrane for assembly and budding of progeny particles [24,25].
Two vaccination strategies for Nipah virus disease prevention have been explored. A canarypox virus-based vaccine vector approach was effective as veterinary vaccine [26]. The same approach for human use vaccines is undergoing extensive evaluation, largely for HIV and AIDS [27]. A soluble Nipah virus G protein approach has also shown promise [28,29]. However, subunit approaches are generally less effective than particulate immunogens, and can suffer from suboptimal presentation to the immune system [30,31,32]. Immunogenicity in mice to Nipah virus glycoproteins has been reported using two vectored approaches for gene delivery; one using Venezuelan equine encephalitis virus replicons [33] and the other involving inoculation of a mix of two complementing defective vesicular stomatitis virus (VSVΔG) vectors, one for expressing each of the two Nipah virus glycoproteins [34]. The latter approach is new and seems promising but its regulatory approval as human vaccine might be problematic [34,35].
Thus, there is a recognized need in the art for a vaccine for Nipah virus. The present invention fulfills this long-standing need and desire in the art.
The present invention is directed to a Paramyxovirus virus-like particles, wherein said virus-like particles are composed of surface glycoprotein G; surface glycoprotein F; and matrix protein M. The present invention is directed further to an expression vector comprising a nucleotide sequence that encodes the virus-like particles of the present invention. The present invention is directed further to a vaccine comprising the virus-like particles of the present invention and a pharmaceutically acceptable carrier or an adjuvant which is well known in the art. The present invention is directed further to a method of vaccinating a subject against paramyxovirus infection comprising administering the vaccine of the present invention.
The present invention demonstrated the potential of Nipah virus virus-like particles (VLPs) as a vaccine. Plasmid-mediated expression of selected viral proteins results in the spontaneous assembly and release of VLPs. These particles make highly effective immunogens because they possess several features of the authentic virus such as their surface structure and dimensions [31,36]. They are also safe because they do not contain any viral genetic material. VLPs, where one or more of the constituent proteins serve as immunogens (native VLPs), can be effective as vaccines for infectious disease. Using this approach for vaccine development, vaccines for human papillomavirus and Hepatitis B virus have been approved for human use, and many, for non-enveloped and enveloped viruses [31,32,37,38,39,40,41,42] are at various stages of development.
The budding capacity of virus proteins as VLPs, the protein-protein interactions that facilitate this process, and the central role of M protein in VLP assembly and release has been described for several paramyxoviruses such as Sendai virus (SeV), Newcastle disease virus (NDV), respiratory syncytial virus (RSV), paramyxovirus simian virus 5 (PIV-5) and human parainfluenza virus type 1 (hPIV1) [20,43,44,45,46,47]. The efficiency of VLP formation in virtually all these studies was based on M protein release in the supernatant. NiV virus-like particles have also been described [48]; the results of this study showed 1) that NiV G and F proteins individually retained some budding capacity although it was far less efficient than that of the M protein and 2) NiV N, M, F and G-containing VLPs resembled the virus in some respects but differed significantly from it with respect to ratio of VLP-incorporated F protein; most of it was present in precursor F0 form. Recently, the vaccine potential of native VLPs of NDV [49] has been described: these particles, composed of HN, F, M and NP proteins, had several virus-like properties. However, since the F protein in this formulation was modified by design to ablate the cleavage site, it remained in its precursor form; consequently, the NDV VLPs were non-fusogenic, and therefore incapable of inducing syncytia formation.
Other and further objects, features, and advantages will be apparent from the following description of the presently preferred embodiments of the invention, which are given for the purpose of disclosure.
So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. The appended drawings illustrate preferred embodiments of the invention and therefore are not to be limiting in their scope.
The present invention describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. These particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking protective efficacy studies in animal models of NiV disease.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed., CRC Press, 1997); Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course (Ream et al., eds., 1998, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag); Peters and Dalrymple, Fields Virology, 2nd ed., Fields et al. (eds.) (B.N. Raven Press, New York, N.Y.).
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” or “other” may mean at least a second or more of the same or different claim element or components thereof. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. “Comprise” means “include.” It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalents to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
The term “antigen” as used herein is defined as a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term “antigen” includes all related antigenic epitopes. “Epitope” or “antigenic determinant” refers to a site on an antigen to which B and/or T cells respond. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
An “antigen” can be a tissue-specific antigen, or a disease-specific antigen. These terms are not exclusive, as a tissue-specific antigen can also be a disease specific antigen. A tissue-specific antigen is expressed in a limited number of tissues, such as a single tissue. Specific, non-limiting examples of a tissue specific antigen are a prostate specific antigen. A disease-specific antigen is expressed coincidentally with a disease process. Specific non-limiting examples of a disease-specific antigen are an antigen whose expression correlates with, or is predictive of, tumor formation, such as prostate cancer. A disease specific antigen may be an antigen recognized by T cells or B cells.
The term “antibody” as used herein includes Immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. A naturally occurring antibody (e.g., IgG, IgM, IgD) includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term “antibody.” Specific, non-limiting examples of binding fragments encompassed within the term antibody include (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH domain; (v) an isolated complimentarily determining region (CDR); and (vi) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (e.g., see U.S. Pat. No. 4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023; Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).
The term “subject” or “animal” as used herein refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
The term conservative variation includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Non-conservative substitutions are those that reduce an activity or antigenicity.
The term “cDNA” (complementary DNA) refers to a piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
The term “diagnostic” refers to identifying the presence or nature of a pathologic condition, such as, but not limited to, prostate cancer. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives). The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. “Prognostic” is the probability of development (e.g., severity) of a pathologic condition, such as prostate cancer, or metastasis.
An “epitope” as used herein, is an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
The term “expression control sequence” refers to Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e. ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
The term “promoter” refers to a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5′ or 3′ regions of the gene. Both constitutive and inducible promoters are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.
As defined herein, the term “host cell” refers to cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.
The term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.
An “isolated” biological component (such as a nucleic acid or protein or organelle) as defined herein, has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
A “label” as defined herein, is a detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
The term “oligonucleotide” refers to a linear polynucleotide sequence of up to about 100 nucleotide bases in length.
Open reading frame (ORF) is defined as a series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.
The term “operably linked” refers to a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
As used herein, the term “vector” refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.
As used herein, the term “transduction” encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transduced cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
As used herein, the term “T Cell” refers to a white blood cell critical to the immune response. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. A CD4+ T lymphocyte is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+ T cells carry the “cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 T cell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is a suppressor T cell.
A “recombinant nucleic acid” is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
The term “polynucleotide” or “nucleic acid sequence” refers to a polymeric form of nucleotide at least 10 bases in length. A recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single- and double-stranded forms of DNA.
The term peptide, as used herein refers to any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
The term “probe” comprises an isolated nucleic acid attached to a detectable label or reporter molecule. The term “primer” includes short nucleic acids, preferably DNA oligonucleotides, 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
The term “promoter” as described herein, is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). Specific, non-limiting examples of promoters include promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used. A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the vaccines herein disclosed.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
As used herein, the term “subject” refers to any target of the treatment. Preferably, the subject is a mammal, more preferably, the subject is a canine or a human.
In one embodiment of the present invention, there are provided paramyxovirus virus-like particles, wherein said virus-like particles are composed of surface glycoprotein G; surface glycoprotein F; and matrix protein M. The paramyxovirus virus-like particles may be prepared from any paramyxovirus included but not limited to Avulavirus, Henipavirus, Morbillivirus, Respirovirus, Rubulavirus, TPMV-like viruses, Pneumovirinae, Pneumovirus and Metapneumovirus. In one preferred embodiment, the paramyxovirus virus-like particles are from Henipavirus and are from Nipah Virus. Generally, for these paramyxovirus virus-like particles, the G and F proteins mediate attachment and entry into the host cell. These paramyxovirus virus-like particles activate innate immune signaling in cells infected with paramyxovirus Virus. These virus-like particles induce robust neutralizing antibody response. Importantly, the paramyxovirus virus-like particles of the present invention are fusogenic and induce syncytia formation. The paramyxovirus virus-like particles may have biologically active G and F proteins on the particle surface and can be purified.
In another embodiment of the present invention, there is provided an expression vector comprising a nucleotide sequence that encodes the virus-like particles described above. In another embodiment of the present invention, there is provided a vaccine comprising the virus-like particles described above and a pharmaceutically acceptable carrier.
In another embodiment of the present invention, there is provided a method of vaccinating a subject against paramyxovirus infection comprising administering the vaccine of described above. The method may be applied to any subject including but not limited to a human or animals. A person having ordinary would readily recognize that the virus-like particles of the present invention may be administered by a variety of routes, including but not limited to intramuscular, subcutaneous or any other as known in the art. Further, the administering may be in a single dose or multiple doses or as needed to achieve the desired effect. Preferably, the particles are administered comprises about 5 micrograms to about 200 micrograms of said virus-like particles.
In another embodiment of the present invention, there are provided different VLP compositions and fusogenicity. The present invention demonstrated the vaccine potential of NiV virus-like particles composed of the two viral surface glycoproteins G and F, and the matrix protein M (NiVgfmn VLPs as well as those made out of N, P and L proteins in addition to G, F and N proteins, i.e., NiVmfgnpl VLPs. Both these VLP compositions produce fusogenic particles (see
The present invention also envisions that the ratios of G:F:M proteins and F0:F1 proteins may be manipulated for optimum results. For example, in the paramyxovirus virus-like particles of the present invention, G may be in the range of 0.7 to 1.3 and F may be in the range of 0.7 to 1.3 and M in the range of 2.6 to 3.4. In another aspect, G may be in the range of 0.8 to 1.2 and F may be in the range of 0.8 to 1.2 and M in the range of 2.7 to 3.3. In another aspect, G may be in the range of 0.9 to 1.1 and F may be in the range of 0.9 to 1.1 and M may be in the range of 2.8 to 3.2. In yet another aspect, G may be in the range of 0.9 to 1.1 and F may be in the range of 0.9 to 1.1 and M may be in the range of 2.9 to 3.1. Similarly, the range of F0, F1 and F1 may be varied, including but not limited to (1) F1 is in the range 40-60% and F0 in the range 60-40% with F2, F1 and F0 adding up to 100%; (2) F1 in the range 30-70% and F0 in the range 70-30% with F2, F1 and F2 adding up to 100%; (3) F1 in the range 20-80% and F0 in the range 80-20% with F2, F1 and F2 adding up to 100%; and (4) F1 is in the range 10-90% and F0 in the range 10-90% with F2, F1 and F0 adding up to 100%.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
The present invention describes NiV VLPs composed of the two surface glycoproteins G, and F, and the matrix protein M. The G and F proteins were included because they mediate attachment and entry into the host cell [50,51,52], both are major targets of neutralizing antibodies, and both are major players in vaccine induced protection [52,53,54]. NiV G and F together are also the most effective as immunogens; this was elucidated in a canary pox virus vector-based experimental protective efficacy study [26]. The M protein was included in the formulation because it is required for particle formation and release [20,25,45]. Under optimized conditions, substantial, quantifiable amounts of NiV VLPs composed of these three NiV proteins were made. This has allowed characterization of their properties in detail to show that they possessed many virus-like/vaccine desirable properties in vitro. It has also allowed testing for immunogenicity in vivo in Balb/c mice; note that although NiV does not cause disease in these animals, NiV proteins injected in them are known to induce robust neutralizing antibody response [29,33,34]. Importantly, NiV-specific mouse monoclonal antibodies are protective in the hamster model of NiV disease [55].
In this study, careful assessment of immunogenicity has shown for the first time, that these NiV VLPs are able to induce neutralizing antibody response. A detailed methodology is also provided to optimize production of the VLPs for research purposes. Beyond this, also provided is the first CryoEM study of NiV VLPs and a careful assessment of their morphology. NiV VLPs can trigger “fusion from without” upon addition to cells, a first for an enveloped VLP. Finally, it is shown that NiV VLPs activate innate immune signaling in “infected” cells and provide a transcriptional profile of this response. Based on all these attributes, NiV M, F and G-protein-containing VLPs show promise as vaccine and will be the basis for undertaking protective efficacy studies in animal models of NiV disease.
NiV expression plasmids pCAGGS-G, F, and M were all under the control of chicken beta actin promoter [56], and were constructed as described [20]. Human embryonic kidney 293 cells (ATCC, CRL-1573) and 293T cells (ATCC, CRL-11268) were grown in Dulbecco's minimum essential medium supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin, and maintained in the same medium containing 2% FBS. The minigenome was used for optimizing VLP formation as described [57]. All the initial minigenome-based optimization steps were done in BHK-T7 cells (obtained from Dr. N. Ito). These conditions were applicable to produce VLPs in 293T cells and were used throughout to generate the VLPs described herein.
293T cells were grown in Dulbecco's complete medium to achieve semi-confluent (80-90% density) cell monolayers. The cells were transiently transfected with the plasmids constructs using the lipid reagent Lipofectamine 2000 according to the guidelines provided by the manufacturers' instructions (Invitrogen Inc). At 48 hrs post-transfection, the VLP-containing cell supernatants (SUP) were harvested for concentration and purification of the VLPs. Because of the fusogenic property of these VLPs, there was widespread syncytia formation at this time point although the cells were still adherent.
VLPs released in the transfected-cell SUP were harvested and clarified by centrifugation at 3,500 rpm for 30 minutes at 4° C. and concentrated by sucrose density gradient centrifugation based on previous descriptions [44,45,58]. Briefly, the clarified SUPs were concentrated by ultracentrifugation through 20% sucrose cushion in TN buffer (0.1M NaCl; 0.05M Tris-HCL, pH 7.4) at 200,000×g for 8 hours at 4° C. The resulting VLP pellet in ˜0.5 ml volume was purified on a discontinuous sucrose gradient formed by layering 80%, 65%, 50% and 10% sucrose in TN buffer. After centrifugation at 186,000×g for 8 hours, the top ˜1.5 ml of the gradient (which included the VLP-containing band at the interface between the 10% and 50% sucrose layers) was resuspended in 20% sucrose buffer and centrifuged once more at 160,000×g for one hour. The resulting pellet was resuspended in 20% sucrose solution in endotoxin-free TN bufffer and stored at 4° C. for subsequent analysis. Supernatant of 293T cells transfected with empty pCAGGS plasmid and processed similarly (referred to as “mock” particles) served as negative control when needed.
Since the ratio of the protein expression plasmids used at transfection and the time of harvest may have a bearing on the level of VLP formation, a minigenome-based VLP infectivity assay, similar to those described previously [59,60] was used to determine the relative concentrations of the constituent plasmids, and to determine the kinetics of VLP formation for optimal production. This assay provides only a comparative assessment of VLP formation since it only accounts for VLPs that are able to incorporate and passage minigenomes. However, based on the assumption that the ratio of empty and minigenome-containing VLPs will be equivalent in each reaction, the method provides an indirect means to determine the optimal set of conditions for VLP production as determined by VLP-incorporated minigenome-encoded CAT enzyme activity. Briefly, the steps involved in the VLP infectivity assay were 1) transfection of NiV minigenome construct and co-transfection with full complement of the NiV protein expression plasmids, N, P, L, M, F and G, using Lipofectamine 2000. 2) following replication (48 hours post-transfection), passage of equal volume of VLP-containing transfected cell SUP on to fresh cells previously transfected with N, P and L plasmids and 3), determination of CAT activity in the VLP infected cells 48 hours later. Replication of the VLP-incorporated incoming minigenomes based on reporter gene activity indicates the level of particle formation and release, VLP infectivity, and successful minigenome packaging.
FAST CAT Assay kit (Molecular Probes) was used according the manufacturer's instructions and allowed accurate quantification of CAT enzyme levels over a wide linear range.
VLPs were purified as described. The particles were adsorbed on Formvar carbon coated copper grid by floating it on a drop of VLP suspension for 15 minutes, the grids were blotted, and then negatively stained with 2% aqueous uranyl acetate for viewing by transmission electron microscopy.
The VLPs were vitrified as reported [61] on holey carbon film grids (C-flat™, Protochips, Raleigh, N.C.). VLPs were imaged at 40,000× indicated magnification using a 4 k×4 k slow-scan CCD camera (UltraScan 895, GATAN, Inc., Pleasanton, Calif.) using a low-dose imaging procedure.
Unfixed VLPs were used for immunogold labeling to limit antibody reactivity to the cell surface proteins. The particles were adsorbed on formvar coated nickel grids, stained with NiV specific primary antibody (hyper immune mouse ascites fluid, HMAF, obtained from Dr. P. Rollin, CDC) diluted in buffer (1% BSA in 0.05M tris buffer) rinsed in wash buffer (0.1% BSA in 0.05M tris buffer), stained with colloidal gold labeled goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories), washed, and then negatively stained with 2% uranyl acetate for viewing by EM.
The total protein concentration of the purified VLP preparations was measured by the BCA (Bicinchoninic Acid) method (Thermo Scientific Laboratories).
VLP composition was determined by western blot analysis. Briefly, purified VLPs resuspended in endotoxin free PBS were lysed by resuspending them in equal amount of 2×SDS protein-loading buffer and loaded into a 12% SDS-polyacrylamide gel with a 4% stacking gel. 293T cell lysates processed similarly were run in parallel as negative cell control. Following electrophoresis to resolve the protein bands, and transfer to membrane, the blot was incubated with NiV-specific HMAF primary antibody at a dilution of 1:1000 dilution, overnight at 4° C., and HRP-conjugated anti-mouse secondary antibody (from GE Healthcare) at a 1:20,000 dilution for one hour at room temperature. The proteins were revealed using western blot detection reagents according to instructions provided by the manufacturer (GE Healthcare).
These studies were undertaken with the approval of the Institutional Biosafety Committee (Protocol# #01/08-2010-1) and the Institutional Animal Care and Use (IACUC) Committee (Protocol #0904028). Five to six week old female Balb/c mice (Harlan Laboratories) were housed in microisolater cage for 4 days before beginning the immunization protocol. Mice in groups of five were immunized by subcutaneous inoculation of four different concentrations of VLPs (1.75, 3.5, 7 or 14 μg/mouse, referred to subsequently as treatment groups A through D respectively) prepared just prior to use in sterile endotoxin free PBS. No adjuvant was used. A group of five mice inoculated with sterile endotoxin free PBS served as negative control group. Mice in the four treatment groups (A through D) were boosted (6pg/mouse) on days 15 and 29; the negative control group received PBS. Blood was collected from the submandibular vein of the animals on days −1, 14, 21, 28 and 35; they were euthanized on day 35.
Two-fold dilutions of test sera were made in 50 μl cell culture medium. Under biohazard level 4 conditions, each of the diluted sera were mixed with 50 μl of NiV diluted to generate ˜30 plaque forming units and incubated for 30 min at 37° C. The pre-incubated virus-antibody mix was added to Vero cell monolayers grown in 96 well plates and incubated for 30 min at 37° C. when the inoculum was removed and replaced 150 μl of cell media. After incubation at 37° C. for 24 h, the cells fixed in 100% ice-cold methanol and staining by indirect immunofluorescence assay as follows: The wells in the plate were blocked with BSA/PBS and stained with rabbit sera raised against the G protein of HeV, and goat anti-rabbit Alexa Fluor 488 conjugate (Invitrogen) diluted 1:1000 in blocking buffer. Viral plaques were visualized and counted, and neutralizing antibody titers were reported based on reduction in plaque count by 50% relative to the untreated control (PRNT50).
For IFA, NiV-specific total antibody levels were measured by using NiV G, F and M expressing 293T cells as target antigen. Thirty six hours post-transfection, the cells were harvested, fixed in paraformaldehyde, cytospun (Cytocentrifuge, Thermoscientific) on glass slides to obtain monolayered preparations and then stored at 4° C., and used as antigen within three weeks of preparation. On the day of use, the slides were washed in PBS, permeabilized with Triton-X-100 and blocked with BSA/PBS. After incubation with two fold dilutions of the test sera, the cell monolayers were washed and stained with Alexa fluor 488-conjugated goat anti-mouse antibody according the manufacturer's (Molecular Probes) instructions. Negative and positive controls were run in parallel with each batch.
VLP-mediated transcriptional activation was tested for eighty four genes involved in Toll-like receptor (TLR)-mediated signal transduction using RT2 Profiler PCR array (SABiosciences). The 96 well array format included mediators of TLR signaling including adaptors and proteins that interact with the TLRs, and members of NFKB, JNKp38, NF/IL6 and IRF signaling pathways downstream of TLR signaling. Briefly, 293 cells grown overnight in 60 mm dishes were exposed to 10 μg of purified VLPs suspended in 1 ml of OPTI-MEM (Invitrogen). “Mock particles” (see, VLP harvest and purification) resuspended similarly and exposed to 293 cells served as negative control. The inoculums were adsorbed on the cell monolayers for 3 hours at 37° C. when additional 1.5 ml of OPTI-MEM was added and the dishes further incubated. Twenty fours post VLP exposure, total cell RNA was extracted according to the manufacturer's (SABiosciences) instructions. The integrity of the RNA was verified by agarose gel electrophoresis and the same concentration of total cell RNA from the VLP-stimulated and “mock” stimulated cells were used for gene expression profiling by Real-time PCR using Eppendorf Mastercycler unit. The array plate included positive and negative controls for quality assurance, and three sets of housekeeping genes for normalization for data analysis. The fold-change in gene expression in the VLP stimulated 293 cells relative to the “mock” stimulated 293 cells was calculated by the _Ct method according to the manufacturer's instructions.
Co-expression of NiV G, F and M proteins in 293T cells resulted in the formation of VLPs that bud out into the transfected cell SUP and can be harvested, concentrated and purified as described above. However, the VLP yield was low. To improve the efficiency of VLP formation, the ratio of the three expression plasmids used at transfection was optimized. It was speculated that this would be important based on the fact that a), during replication, paramyxoviruses form a transcription gradient where the 3′ proximal genes are transcribed more abundantly than the successive downstream genes [52] and b), the stoichiometry of interaction of the viral proteins has proved to be critical in plasmid-driven minigenome and full-length rescue systems [62].
The importance of protein ratios for VLP formation was alluded to in a previous NDV study where the expression plasmids were co-transfected at “pre-determined concentrations” to produce VLP-incorporated protein ratios analogous to those in virus infected cells [49]. In a study by Patch et al [48], equivalent amounts of NiV N, M, F and G were initially used to produce the VLPs. In that study, VLPs were subsequently also made by adjusting NiV expression plasmid concentrations by experimental variations similarly to that in the NDV study [49]. The efficiency of particle formation and budding in both these, and many other paramyxovirus VLP formation systems was based on M protein release [43,44,45,46,48].
The present invention used a minigenome-based functional assay, the VLP infectivity assay (described above), to determine optimal expression plasmid ratios for efficient VLP formation based on reporter gene readouts. Briefly, 293T cells were transfected with plasmids as shown in
The reproducibility of the results was verified in an independent repeat experiment.
The optimized conditions were applied to transfect G, F and M expression plasmids in 293T cells grown in 10 cm dishes. The VLP-containing culture SUPs were harvested 48 hours later, and concentrated and purified as described. Briefly, the clarified SUPs were concentrated by ultracentrifugation through 20% sucrose cushion, and then purified on a discontinuous sucrose gradient. The VLP pellet was resuspended in TN buffer and viewed by EM after negative staining.
To verify whether the NiV proteins were incorporated into the VLPs as designed, purified particles were analyzed by western blotting using NiV-specific mouse antibody, and HRP-conjugated anti-mouse secondary antibody as described above. The right hand panel in the
The immunoreactivity of the VLP surface glycoproteins was verified by staining purified unfixed VLPs by the immunogold labeling technique using NiV-specific mouse antiserum and 6 nm colloidal gold particle-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories Inc). The particles were viewed by EM after negative staining. The use of unfixed particles assured that only the surface-exposed antigens would be reactive. Numerous VLPs with the gold particles decorating their surface were seen;
Syncytium formation is a classical feature of NiV and other paramyxovirus-induced cytopathology that can be blocked by virus-specific neutralizing antibody. A similar observation was made when 293 cells were “infected” with the NiV VLPs. Briefly, the VLPs were pre-incubated with NiV-specific antibody, Junin virus (JV)-specific antibody, and with OPTI-MEM I (Invitrogen Inc) medium only for one hour at 37° C. before inoculating onto near confluent 293 cell monolayers grown overnight in 60 mm dishes. The inoculum was removed after incubation for 3 hours at 37° C., replaced with OPTI-MEM I, and the plates were further incubated overnight at 37° C. overnight. The monolayers were then viewed for the formation of syncytia after staining with crystal violet. The results in
Mice in groups of five were inoculated subcutaneously with four different concentrations of purified VLPs and boosted as described above. The negative control group of five mice were inoculated with sterile endotoxin free PBS for each inoculation. The mice were bled from the submandibular vein on the day before primary inoculation, and then on days 14, 21, 28 and 35. For initial evaluation, sera from each treatment group were pooled, and the IFA method used to determine levels of NiV-specific antibodies. The results in
All sera were tested individually by plaque reduction neutralization method by doubling dilution of each sample (1:5 to 1:80) as described above. The results (
A PCR array format (SABiosciences) was used to investigate modulation in transcription profile of 84 genes involved in innate immune responses to include TLR signaling family and members of the downstream signaling pathways, NFKB, NF/IL6, IRF and JNKp38. These genes represent key sensors of non-self that signal, and ultimately shape the nature of innate immune response that modulates the type and duration of adaptive immune responses [66,67]. The differential expression of genes in VLP-exposed 293 cells relative to the “mock” infected 293 cells was measured by real-time PCR. Same concentration of total cell RNA from the VLP-stimulated and the “mock” stimulated control cells were used for first strand synthesis and Sybr green PCR amplification of the relevant genes as described above. The integrity of RNA in each sample was confirmed by gel electrophoresis (
Using a minigenome-based functional assay, the present invention established conditions (described above and shown in
No safe and protective vaccine for NiV disease has been developed for humans. The two vaccination strategies that have been explored are the canary pox-based vector approach [26] and soluble subunit approach [28,29]. NiV vaccine by the former method is undergoing development as a veterinary vaccine [26]. The same approach is being evaluated for human use vaccines, mainly for the prevention of HIV and AIDS [27]. The subunit approach has limitations as mentioned above [28,29,30,31,32]. One particular challenge revealed by studies that tested a soluble NiV G protein-based subunit vaccine formulated with adjuvant is the potential difficulty of eradicating infection in the central nervous system. In that study [28], live virus was present in the brain of one cat, and viral RNA was present throughout the 21 day post-challenge period in the brains of the remaining challenged animals. A recently reported vaccination strategy [34] requires simultaneous inoculation of two VSVΔG vectors, one expressing NiV G, and the other expressing NiV F proteins. It was of interest to note that supernatants of cells co-infected with these two defective viruses were infectious and could be passaged indefinitely in the absence of VSV G trans-complementation. This vaccination approach seems promising since self-propagated stock of these two viruses induced robust neutralizing antibody response in mice. However, potential pathogenicity of VSV-based vaccine vectors remains a concern [34,35]. The potential of a recombination event resulting in a single VSV vector virus expressing both these NiV proteins is unlikely, but it may still be problematic for a human use vaccine.
Native VLPs like the ones of the present invention allow the viral proteins to be presented to the immune system in the same conformation as in the virion for effective B and T cell response [31]. VLPs are particularly effective in producing a protective antibody response because of their virus-like size range, their particulate nature, and their virus-like dense, repetitive and ordered surface structure [31,36]. The spacing of the antigenic epitopes on the VLP is also optimal for B cell activation [31]: EM analysis showed that the particles of the present invention resembled the real virus in terms of size and surface structure [63,64]. The image in
NiV M, F, G and N protein-containing VLPs consistent in size and morphology to the parental virus have also been reported in a study which evaluated protein-protein interaction that facilitate VLP formation [48]. However, in that study, most of the particle-incorporated NiV F protein was predominantly in the uncleaved precursor form. This finding is clearly distinct since the VLPs of the present invention contained substantial amounts of cleaved F protein, and this may have been related to ratios of the interacting proteins expressed in 293T transfected cells. In a recent study of NDV VLPs [49], the particle-incorporated proteins were reported to have virus-like protein ratios, but the F protein remained in its precursor form because the cleavage site required to produce the fusion competent form was mutated by design. What effect a VLP-incorporated non-fusogenic F protein may have, relative to the fusogenic form, on the level and quality of VLP-induced immune response is not clear at present since, difference in immunogenicity between fusion-competent and fusion-defective VLPs has not been experimentally evaluated so far. However, a recent report suggests that viral fusogenic membrane glycoproteins may enhance vaccine potency [68].
Immunogold labeling of the unfixed NiV VLPs confirmed that the surface proteins in the VLPs of the present invention were functionally assembled and they were biologically active (
Neutralizing antibody response is the critical correlate of protection mediated by prophylactic vaccines [53,54] and native VLPs promise to be highly effective prophylactic vaccines for paramyxoviruses like NiV, and others like NDV and measles where neutralizing immune response is known to play a pivotal role in protection against disease [53,54,55,74]. The VLPs of the present invention were highly effective immunogens, and all, especially in the three higher treatment groups produced high levels of response by day 35 (
Immunogenicity to native VLPs has been reported previously for one other paramyxovirus namely NDV [49]. In that report, immune response to NDV VLPs was evaluated by primary inoculation of mice intraperitoneally with VLP concentrations ranging between 10 and 40 μg, and a booster dose of 10 μg, without adjuvant. NDV-specific titers by ELISA were high in each mouse in each treatment group. Neutralizing antibody response to 20 and 40 μg of these particles was also detected.
The nature of innate immune response dictates the type and duration of adaptive immune response [67,75]. The mechanism by which NiV VLPs are recognized by host cells and trigger the induction of innate immune response, and how this translates into effective adaptive immunity is not known. With the experimental conditions as described, VLP-induced activation of certain genes known to be involved in the induction of an effective innate immune response [75]. Results presented in the heat map in
Finally, as described above, the VLPs of the present invention were highly effective as immunogens, able to induce neutralizing antibody response in all animals with primary inoculation of as little as 7 μg VLP protein each. Fusogenic property of these VLPs may be critically relevant.
In conclusion, substantial quantities of NiV VLPs needed to characterize NiV VLPs were produced, their many virus-like properties and their effectiveness as immunogens were demonstrated.
The following references were cited herein:
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually incorporated by reference.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. It will be apparent to those skilled in the art that various modifications and variations can be made in practicing the present invention without departing from the spirit or scope of the invention.
This nonprovisional application claims benefit of priority under 35 U.S.C. §119(e) of provisional application U.S. Ser. No. 61/396,915, filed Jun. 4, 2010 now abandoned, the entirety of which is hereby incorporated by reference.
The invention was supported, in whole or in part, by Grant No. U54 AI057156-07 from the National Institutes of Health. Consequently, the Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61396915 | Jun 2010 | US |
