Herein-described are various methods for making a vaccine that are made of re-assembled virus like particles (VLP). First, the VLPs are disassembled into encapsidation intermediate populations. Each encapsidation intermediate population undergoes, for instance, chemical conjugation of unique peptide or nucleic moieties to form separate populations. Thereafter, a predetermined amount of each of the several (one or more) different encapsidation intermediates from the different populations is mixed and joined, forming intact VLPs, surrounding a nucleic acid core, that are composed of different encapsidation intermediate such that the reassembled VLP displays more than one peptide or nucleic acid. The nucleic acid can function either as a scaffold alone or can be engineered for the expression of an immunomodulatory protein in a eukaryotic cell.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention includes several unique solutions that address current limitations of VLP technology, while retaining all the positive characteristics of a successful VLP antigen scaffold. Applicant presents a method for generating VLP vaccines in adaptable, predictable, stable and scaleable manners. This work is highly innovative, and there is continuing development. The method includes generating multi-valent vaccines where different vaccine protein moieties are fused to the surface of a single VLP structure conferring a multi-functional effect--the availability of immune peptides (protein elements stimulating protective immunity) and peptides that either modulate the host immune response or facilitate efficient immune cell recognition or processing. The proposed vaccines will be also bi-functional, where the protein elements of the VLP, with or without a peptide fusion or series of fusions, encapsidate a modified RNA moiety. The modified RNA can carry an mRNA of interest and that protected RNA can then be used to carry nucleic acid content, along with protein, into an immune cell that takes up the vaccine. The RNA constituents works synergistically to generate strong, lasting immunological responses by encoding either an intact pathogen or oncology antigen, proteins that stimulate host immune responses or proteins that modulate either a type Th1 or Th2 immune response to the vaccine. The method alleviates problems associated with other VLP systems by having robust production potential, improved cellular uptake, and multi-epitope valency. A selection of structurally similar, yet immunologically distinct VLP carriers allows rotation of the coat backbone for prime-boost strategies that have proven unworkable in other VLP systems.

Vaccination with bi-functional RNAs presents an alternative to DNA vaccination, with some distinct advantages. In the first instance, there is little concern that an RNA-based vaccine could cause oncogenesis because it cannot incorporate into or transform the genome. Secondly, there is good evidence that one could deliver an RNA vaccine derived from an RNA virus (such as an alphavirus) as a safe self-amplifying vaccine vector. Alphavirus replicons are cytolytic for cells, and thus the replicating RNA vaccine is intrinsically transient and self-eliminating. Alphavirus "replicon" vaccines cause powerful immune responses--both antibody and cell-mediated--associated with both increases in the amount of antigen produced as well as the production of inflammatory cytokines induced by intracellular accumulation of the viral dsRNA replicative intermediate. These features indicate that the dosage of replicative RNA required for induction of effective immune responses would be orders of magnitude lower than that required by DNA immunization. However, the major drawback associated with naked RNA vaccines is the notoriously labile nature of the nucleic acid: this severely limits the application of RNA vaccines for mass immunizations.

Alphavirus replicon vaccines are currently delivered either as naked RNA transcribed in vitro, packaged in alphavirus-like particles (replicon particles), or as plasmids containing infectious cDNAs, driven by the cytomegalovirus immediate early promoter (CMV promoter). Replicon particles are very efficient as vehicles for carrying the replicon RNAs into cells, but production is complicated, inefficient and unreliable. An efficient packaging and RNA stabilization technology is therefore required to protect alphavirus-based RNA vaccines from degradation. Two viable options present themselves: (1) to deliver recombinant alphavirus constructs as infectious cDNA plasmids; (2) to package alphavirus RNA transcribed in vitro such that it is protected from nucleases and has good stability and storage properties. An approach for the latter option is presented below.

The inventors employ as a VLP carrier the well-characterized plant virus, tobacco mosaic virus (TMV), and exploit its unique abilities to reconstitute VLP structures in vitro onto various heterologous RNA sequences.

By introducing a cysteine in the solvent exposed sequences of TMV coat, we can introduce and fuse foreign antigen epitopes ex-vivo. Epitope sequences that are not amenable to in vitro synthesis will be fused in-frame genetically to the TMV coat protein. TMV VLPs will be reassembled in vitro decorated with a single epitope (monovalent), or with a collection of different epitopes (multivalent), derived from in vitro conjugation or expressed from a genetic fusion.

As a scaffold for reassembly, the present invention includes using an RNA that encodes a protein that will enhance vaccine potency, thereby creating a bi-functional antigen delivery system that derives its activity from both protein and nucleic acid. The RNA can also incorporate an alphavirus replicon to augment translation. Essential for the encapsidation of the RNA molecule by the TMV coat protein, to generate an RNA-containing VLP, is the presence of the 75 nucleotide sequence comprising loop 1 of the origin of assembly (OAS). By combining this 75 nucleotide sequence with foreign sequences encoding protein(s) or peptide(s) of therapeutic interest, the RNA molecule can function as an effective scaffold for the generation of a TMV-like VLP. The RNA can encode any number of immunomodulating factors (e.g. IL4, IL1.beta. or IFN.gamma.) that ensure a highly successful immune response to the vaccine, and help generate either protective or therapeutic immunity to the pathogen, or deliver inhibitory RNA signal (RNAi) for targeted gene inhibition. This VLP strategy can be applied to effectively target immune cells and stimulate Th1 type responses.

An important requirement to inducing a Th1 type immune response is getting VLPs into cells for processing and antigen presentation. Peptides with known cell targeting have been identified (Samuel 0., Shai, Y., 2001 Bichem. 40, 1340; Magnusson et al. 2001 J. Virol. 75 7280; Bushkin-Harav et al. 1998 FEBS L. 424 243) and can be tested in vitro by direct examination of cell entry, and in vivo for augmented antigen presentation by examining the type and speed of immune response to target antigens. Targeting and fusion peptides will be tested for their ability to augment cellular uptake of TMV, as well as their ability to deliver encapsidated RNA in vitro and in vivo.

A common method to improving vaccination is to co-administer an adjuvant or a specific T-helper peptide to stimulate T-cell help. CpG DNA has been shown to be an easily administered adjuvant that improves Th1 type immune responses when co-administered with an appropriate vaccine (Krieg. 2000 Vaccine 19, 618). Most CpG DNA adjuvants have been given mixed with the vaccine and administered subcutaneously (s.c.), although the single strand thiolated DNA can also be fused to a protein carrier through SPDP conjugation chemistry. Also, several universal T-helper peptides have been identified (Kulkarni, A. B., et al., 1995 J. Virol. 69,1261; Panina-Bordignon, 1989 Eu. J. Imm. 19, 2237; Boraschi, 1988 J Exp Med. 168,675; Weiner, G. et al., 1997 Proc. Nat. Acad. Sci 94 10833). Immunostimulatory peptides, usually fragments of cytokines, have also been identified that direct Th1 type immunity after vaccination in combination with pathogen or self-antigen peptides or subunit vaccines (IL1.beta., Boraschi, 1988 J Exp Med. 168,675). Coat fusions containing T-helper or adjuvant peptides or CpG DNA oligo will be used to augment the immunogenicity of co-expressed peptides, or encapsidated RNA.

Lastly, it is well established that cytokines play an important role in determining which arm of the immune system is activated after vaccine delivery. Interleukin 4 (IL4) has been implicated in directing Th2 type immune responses and interferon gamma (IFN.gamma.) is an important contributor to Th1 responses (Spellberg and Edwards. 2001 Clin Infect Dis 32, 76). By introducing IL4 and IFN.gamma. RNA into cells by encapsidation into a TMV VLP, we may be able to influence the type of immune response that is generated. Applicant can test both antibody isotype responses to antigen, which are a reflection of Th1 or Th2 antigen presentation, as well as assess CTL responses that are primarily a consequence of Th1 immunity.

Cell fusion peptides, T-help, adjuvants, pathogen antigens, tumor antigens and encapsidated cytokine RNA will be tested systematically in combination with antigens from Papillomavirus and melanoma murine disease models. Immunogencity and challenge models will establish incremental improvements over vaccination with single peptides, and define the best peptide/RNA combinations for generating Th1 or Th2 immune responses.

The availability of such a flexible and effective vaccine platform provides opportunities to apply non-live vaccines for humans and livestock thus reducing side effects and increasing effectiveness. New vistas of medical practice, including applications for breaking self-tolerance and driving immune responses against weak antigens, may be opened by the synergistic and high specific-activity of the disclosed vaccine platform.

The invention relates to a method where a specified virus, such as a tobacco mosaic virus (TMV), is disrupted into a plurality of subunits. Each subunit contains a genetically fused peptide or is subjected to a conjugation reaction in order to attach a predetermined epitope, peptide or nucleotide thereto. A plurality of subunits are processed in this manner to produce a plurality of subunit groups, where one subunit group has attached thereto a predetermined peptide; another subunit group has a second peptide; another subunit has a predetermined epitope attached there to; and another subunit group has a nucleotide attached thereto, and so on, for as many subunit groups necessary to provide the building blocks for a plurality of virus vaccines.

An alternative strategy is to employ TMV RNA modified to initiate internal ribosomal entry by introducing specific sequences known to cause such an effect. These internal ribosomal entry sites (IRES) are effective in causing internal translation products from a polycystronic RNA in mammalian cells (Yang et al., J Virol 1989 63(4):1651-60). Introduction of an IRES into a TMV genome in frame with an RNA encoding either a full length gene product or immunostimulatory cytokine or other kind of immunomodulatory protein allows for translation of that protein. Because these IRES are introduced into non-replicating RNA, the amount of TMV and proportional transcript taken up by a cell after vaccination is conceivably lower than with a self replicating RNA such as encoded by an alphavirus replicon, but the level of translation product should be sufficient to induce the correct response.

The present invention includes research and development of technological solutions to help the USA to produce and supply effective vaccine reagents in response to unanticipated pathogen threats. Applicant specifically addresses issues that limit bio-defense application of nucleic acid vaccines: poor environmental stability and high dosage requirements. In addressing these issues, we will draw upon the core of knowledge that the inventors possesses in the field of positive stranded RNA viruses and their applications in biotechnology to develop a set of molecular tools to improve nucleic acid vaccines. Applicant will also demonstrate our capacity to produce protein subunit vaccines that will-provide effective antibody responses. Production of protein subunit vaccines is inherently slower than nucleic acid vaccines and so, practically, will only be available within a delayed period following encounter with a new pathogen threat. However, the inventor's non-transgenic plant-based vaccine expression platform (GENEWARE.RTM.) has the capability to express a variety of proteins, including virus-like particles (VLP)--known to be potent inducers of antibodies in vaccinated individuals--rapidly. Applicant has recently used a modified TMV expression vector to produce 16 different human therapeutic vaccines in tobacco plants, and have shown excellent safety in a Phase I clinical trial (BB-IND #9283). Unlike other competing technologies, GENEWARE.RTM. does not require specialized fermentation facilities, and uses the efficient, rapid protein production strategy of the plant virus TMV to harness plant protein production machinery to produce vaccine proteins. A typical harvest time, post inoculation is less than 21 days. Since the same virus is used from pilot testing to large-scale manufacturing, there is little or no transition time between validation and manufacturing scale up. Most of the delay in delivery of vaccines via GENEWARE.RTM. technology would be in the growth of plants, and establishment of antigen-specific purification protocols. These aspects of the technology result in a low cost of production for plant-derived VLP vaccines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel method for for the colorimetric detection and quantitation of total protein.

The present invention relates to a novel method for construction of a plurality of vaccines and pharmaceuticals using viruses, such as the tobacco mosaic virus (TMV). In broad terms, the invention is practiced in a manner depicted generically in FIGS. 1-6 (see Original Patent), as described below.

The description of the present invention is first provided in general terms, followed by a more detailed description that includes many biochemical procedures.

Standard methodologies can produce a pseudo-multivalent vaccine product by a chemical conjugation process outlined in FIG. 1 (see Original Patent). VLP particles are produced (S1) and isolated (S2). Individual peptides are individually chemically conjugated to the surface of independent lots of VLPs (S3) to produce distinct populations of VLPs, each displaying a unique peptide adduct. It is possible to conceive that multiple peptides could be simultaneously conjugated on the surface of the same population of VLPs to produce VLPs with a random distribution of unique peptides. The distinct populations of immune particles are then mixed (S4) to produce a population of VLP particles with distinct peptides covalently attached to the surface (P1). The resulting product will display distinct peptides in its mixture, but each will be independently taken up by immune cells and independently used to stimulate the immune system. There will be a lack of synergy between the fused peptides since there is no special connection between different peptides; each functions independently.

The described invention exploits the unique properties of the tobacco mosaic virus (TMV) that is amenable to the procedures outlined in FIG. 1, but also new methods (FIG. 2 (see Original Patent)) with significant advantages. As represented by the box S5 in FIG. 1, a TMV virion is constructed with a surface associated amino acid allowing for improved chemical conjugation. This can be the presence of a unique, surface associated cysteine or lysine residue, although other methods can be employed. Large quantities of TMV are produced (S6) using, for instance, tobacco plants that are infected with the desired strain of TMV, then processed as described in co-pending patent application Ser. No. 09/962,527 filed Sep. 24, 2001, entitled PROCESS FOR ISOLATING AND PURIFYING VITAMINES AND SUGARS FROM PLANT SOURCES, and related U.S. Pat. Nos. 6,303,779, 6,033,895 and 6,037,456 all commonly assigned to Large Scale Biology Corporation, Vacaville, Calif., all of which are incorporated herein by reference in their entirety. Once large quantities of TMV are available, a process that is described in greater detail below disrupts the TMV in order to produce a large number of subunits (SU) or 20S disks, as represented by the box S7 and S8. The subunits are then separated at step S9 to form a plurality of subunits, each to be processed separately, as is described in greater detail below. As represented by step S9, each individual subunit group is subjected to a conjugation reaction in order to add predetermined components, such as a functional peptide, epitope, proteins or nucleic acid sequence to the subunits in that subunit group, in a manner that is described in greater detail below. As represented at step S10, pluralities of groups of subunits are now constructed into a single VLP structure where each subunit having specific epitopes, peptides, proteins or nucleotides attached thereto. TMV 20S disks naturally reassociate to form a rod-shaped virion surrounding an RNA molecule containing a unique sequence termed the TMV ori, or origin of assembly (OAS). This produces a multivalent vaccine (P2) that is not equivalent to a simple mixing reaction. Multifunctional peptide or nucleic acid adducts are linked physically to one another allowing each to synergistically enhance the cellular uptake of the VLP vaccine, immune processing, number of immune peptides presented to the immune system and the nature of the stimulated immune response. The simultaneous presentation of each peptide or nucleic acid component on the same VLP, rather than on distinct, unlinked VLP populations, is predicted to enhance the effectiveness of the VLP vaccine and lower the lower dose.

Further basic steps in the method of the present invention are depicted in FIG. 2 (see Original Patent). Specifically, at step S10 a specific recombinant RNA sequence is selected to be the scaffold for assembly of the TMV VLPs. The specific VLP subunits selected in step S10 are combined with the RNA selected to form a reassembled TMV via a process that is described in greater detail below. The RNA can act only as a structural scaffold and could represent only the TMV RNA itself, not offering any augmented function other than a building block of the new VLP vaccine. However, recombinant RNAs can be constructed containing the TMV ori (S10) that also encode proteins. Once the VLP is taken up in immune cells, the TMV virion has unique function. It is preferentially bound by ribosomes and disassembled by a co-translational mechanism (Mundry et al., J Gen Virol. April 1991; 72 (Pt 4):769-77). This would allow the efficient translation of this RNA so that the encoded protein is produced within the host immune cells. The encoded protein can either be an intact antigen to stimulate humoral or cellular immune responses against the targeted pathogen or cancer. Conversely, the RNA could encode immune stimulatory proteins (enhancing the amplitude of immune response) or modulatory proteins (insuring the direction, Th1 or Th2, of the immune response). This combination of protein elements that stimulate the immune response, as well as promoting the efficiency and effectiveness of the response, --in combination with an encoded nucleic acid component that is functional for augmenting the immune response, makes this vaccine truly bifunctional.

It should be noted that RNA is inherently unstable as a `naked` element, or one not coated with a protective protein coating. However, it has an advantage over DNA in nucleic acid vaccines since it promotes translation of the desired product within immune cells, but is degraded and does not risk the immunized host with DNA recombination and the associated oncologic events. `Naked` or uncoated nucleic acid vaccines of RNA or DNA types are very inefficient, where milligram (mg) quantities of DNA are required for any immune response in humans. Out of the mg of vaccine administered, picograms or less are taken up by immune cells. This results in expensive manufacturing and formulation costs, and very inefficient unpredictable immune responses. This invention allows the `naked` RNA encoding important antigens or immune enhancing proteins to be coated and protected within the VLP structure of TMV. Such coating enhances the stability of the RNA and improves the delivery efficiency.

VLP vaccines are not dependent only on chemical conjugation to add immune peptides to their surface. The art describes methods for generating VLP vaccine through the genetic fusion of immunologically relevant peptides to the surface of VLPs. This process is described in FIG. 3. In this case, individual (S11) or multiple (S14) peptides are fused to the surface of the VLP protein through recombinant DNA procedures where the protein coding sequence for the immune peptide is fused to that of the VLP structure. Each individual or multi-peptide displayed VLP structure is purified (S11) and then qualified for its properties (S12). A multivalent vaccine is constructed by mixing either individual VLP populations displaying one or more peptides by genetic fusion (S13) or simply using a single population of VLP that is displaying more than one peptide by genetic means (S14). These procedures produce a multivalent VLP immunogen composed of multiple separate VLP populations, each displaying a unique immune peptide (P3). This approach suffers from the same limitations of the vaccines produced in FIG. 1 where little to no synergistic activity can be predicted by the simple mixture of non-linked peptides. Further, the VLP vaccines lack a nucleic acid component and are simply single functional vaccines--only providing a protein-based signal to the immune system.

This invention overcomes these difficulties by allowing truly multi-valent and multi-functional vaccines to be derived. TMV is amenable to the same procedures described in FIG. 3 to produce mixtures of VLPs each with unique genetic fusions. However, its unique properties permit the procedure described in FIG. 4. Individual TMV virions can be prepared with single or multiple peptides by genetic means (S15). Each individual virion is isolated (S15) and qualified. Each TMV virion is separately disassembled (S16) and SU are prepared (S17) composed of 20S disks displaying a unique array of immune peptides. This plurality of SU are then reassembled surrounding a RNA containing the TMV ori to produce TMV VLP (S18). The final product is indeed a VLP vaccine that displays multiple immune peptides simultaneously on the surface of each VLP (P4) and contains RNA that functions both as a scaffold for VLP assembly and as a separate immune stimulus. The advantages of this approach are the same as described above in that the particle is multi-functional in terms of the plurality of immune, immune modulatory, immune stimulatory or cell uptake facilitating peptides simultaneously displayed on the surface of the VLP. This allows more efficient cellular uptake, processing and immune stimulation resulting in reduced dose and improved immune protection. The RNA again contributes essential functions beyond a scaffolding device. It can encode intact antigens, immune modulatory, immune stimulatory proteins to further augment the immune response. The RNA is protected within the VLP and is delivered efficiently to the cellular translation apparatus by the natural functions of TMV VLPs.

It should be understood that the above description is only a basic framework of steps upon which the present invention functions, and a basic understanding of the platform for constructing vaccines and pharmaceutical products in accordance with the present invention. The steps outlined in the flow diagrams in FIG. 2 and FIG. 4 are illustrated visually in FIGS. 5 and 6. Two further points should be noted with regard to the basic frameworks outlined in FIGS. 1 to 4. Firstly these figures indicate that various vaccine compositions contain 3 unique epitopes either displayed on separate VLPs or virions or all reassembled onto one VLP or virion. The number three was chosen purely for illustrative purposes and it should be understood that any number of epitopes can be recombined to form a multivalent vaccine. Secondly the entity displayed on the surface of the VLP or virion need not be limited to a peptide epitope as indicated in FIGS. 3 and 4. The displayed entity can also be a nucleotide, introduced by chemical fusion, or a complete protein, introduced by either chemical or genetic fusion. Furthermore all possible combinations of nucleotide, peptide epitope and complete protein, in terms of both number and ratio, can be envisioned for multivalent vaccine reassembly. For example peptide 1, nucleotide A and complete protein X, each displayed on separate virions or VLPs can be combined to yield a multivalent VLP vaccine similar to P3 in FIG. 3. Alternatively separate pools of 20S disks each displaying peptide 1, nucleotide A and complete protein X can be reassembled in vitro to generate a multivalent vaccine similar to P4 in FIG. 4, where all entities reside on a single VLP or virion.
 

Claim 1 of 3 Claims

1. A method for making a virus-like particle (VLP) containing multiple, different composition peptides or proteins displayed by a process comprising the steps of: a) disassembling separate VLP populations, each displaying a distinct peptide or protein via genetic fusion; b) disassembling a separate VLP population that has a surface residue for chemical conjugation, provided by genetic fusion; c) forming encapsidation intermediate populations such that: i) each displays a distinct peptide or protein and ii) each displays a surface residue for chemical conjugation; d) effecting chemical conjugation of unique peptide, protein or nucleic acid moieties to separate populations of the encapsidation intermediate displaying surface residue for chemical conjugation; e) mixing encapsidation intermediates from different populations displaying peptides or proteins by genetic fusion or displaying peptides, proteins or nucleic acids by chemical conjugation; f) forming intact VLP surrounding a nucleic acid core that is composed of different encapsidation intermediates such that the VLP displays more than one moiety, be it peptide, protein or nucleic acid, or some combination of these moieties.
 

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