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Virus expression vectors
For many years now, virus expression vectors have been explored as a mechanism for gene delivery, cancer therapy and vaccine development. More recently, the next generation of virus vectors have been generated that possess greater attributes such as tissue specificity and improved expression levels, while at the same time lack many of the shortcomings of their predecessors, such as issues regarding immunogenicity and safety. This review article describes several of the recent patents that have been issued in the field of virus expression vector development. Innovations in both plant and animal virus expression vectors are covered. The review concludes with a discussion of future prospects of virus expression vectors as tools in medical research.
Background Recent innovations in the field of virus expression vector development offer great promise. From adenoviruses to lentiviruses, expression vectors are constantly being improved upon and redesigned to perform better in disciplines including gene therapy and vaccine development. Plant virus expression vectors are also rapidly gaining a role in medicine by providing new ways to generate adjuvants and pharmaceutical proteins. Recent innovations with respect to the construction and application of expression vectors and other virus sequences are the subject of this review article. The material presented here has been provided from US patents that have been issued over the past 2 years and the complete list is presented in Table 1. In a number of cases, advances have been made with regard to vector design, so that issues such as host safety and tissue specificity are better addressed. Other examples provide the initial development of novel expression vectors based on viruses that have not previously been studied in great detail. The review concludes with a discussion of the future prospects of virus expression vectors as tools in medical research and in the design of pharmaceuticals.
10.4155/PPA.14.17 © 2014 Future Science Ltd
Kathleen L Hefferon1 Cornell University, Ithaca, NY, 14853, USA [email protected] 1
Patents involved in animal/human virus expression vectors While many different animal virus expression vectors currently exist, there is always a need to improve upon them so that their direct applications can be expanded. Many virus expression systems have drawbacks ranging from poor expression levels to tissue trophism constraints and even to biosafety concerns. Expression systems based on new unexplored virus isolates are also under development. The following section describes patents recently issued that concern improvements upon adenovirus, lentivirus and vaccinia virus expression vectors, as well as other animal viruses. Adenovirus expression vectors The first example of a recently patented virus expression vector concerns adenovirus, a double-stranded DNA virus with a genome approximately 36 kilobases in size. Adenoviruses have been developed as expression vector systems for many years [17] . Adenoviruses are highly desirable as expression vectors since they can achieve highly efficient gene transfer in a variety of target tissues and maintain the ability to accommodate large transgenes [2,3] . In many cases, the adenovirus E1 gene has been replaced with the foreign gene of
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Table 1. Patents discussed in this review.
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Patent number Title
Inventors
Source
Date
US8394386
Sequential delivery of immunogenic molecules via adenovirus and adenoassociated virus-mediated administrations
Wilson, James
University of Pennsylvania
March 12, 2013
[1]
US6083716
Chimpanzee Adenovirus Vectors
Wilson, James and Farina, Steven
University of Pennsylvania
October 20, 2011
[2]
WO02/33645
Adenovirus Hexon Protein and Uses Thereof
Soumitra Roy and Wilson, James
University of Pennsylvania
January 14, 2009
[3]
WO05080575A1 Nucleic acid containing chimeric gene derived from hepatitis type-c virus
Daisuke Akazawa, and Takaji Wakita,
Toray Industries, Inc., Tokyo, Japan
February 9, 2011
[4]
US8604179
Nucleic acid comprising chimeric gene derived from hepatitis C virus
Akazawa, Daisuke, Wikita, Takaji,
Tokyo December Metropolitan 10, 2013 Organization for Medical Research Tokyo, Japan
[5]
US8551773
Methods and compositions relating to improved lentiviral vectors and their applications
Trono, Didier, Salmon, Patrick
Research Development Foundation, Carson City, Nevada
October 8, 2013
[6]
US8506947
Vaccinia virus expression McCart, J. Andrea, vector for selective Bartolett, David, replication in a tumor cell and Moss, Bernard introduction of exogenous nucleotide sequence into a tumor cell
US Department August 13, of Health and 2013 Human Services
[7]
US8609827
Infectious cDNA clone of North American porcine reproductive and respiratory syndrome (PRRS) virus and uses thereof
Calvert, Jay, Sheppard, Michael, Welch, Siao-Kun, Rowland, Raymond, Kim, Dal-Young
Zoetis Llc
December 17, 2013
[8]
US8067535
Identification of gene sequences and proteins involved in vaccinia virus dominant T cell epitopes
Terajima; Masanori, Cruz; John Ennis; Francis A.
University of Massachusetts
November 29, 2011
[9]
US8597950
Two-component RNA virusderived plant expression system
Marillonnet, Sylvestre, Engler, Carola, Muhlbauer, Stefan, Hertz, Stefan, Klimyuk, Victor, Gleba, Yuri
Icon Genetics GmbH
December 3, 2013
[10]
US8101189
Vaccines and immunopotentiating compositions and methods for making and using them
Leclerc, Denis, Majeau, Nathalie, Lopez-Macias, Constantino, Lamarre, Alain
Laval University January 24, 2012
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Ref.
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[11]
Virus expression vectors
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Table 1. Patents discussed in this review (cont.). Patent number Title
Inventors
Source
US8282940
Adjuvant viral particle
Leclerc Deinis, Lopez-Macias, Constantino,
Laval University October 9, 2012
[12]
US8586364
Cells and Methodology to generate non-segmented negative-strand RNA viruses
Tangy, Frederic, Charneau, Pierre, Jacob, Yves,
Institute Pasteur
November 19, 2013
[13]
US8168200
Vaccine against african horse sickness virus
Minke, Jules, Audonnet, JeanChristoph, Guthrie, Alan, MacLachlan, Nigel, Yao, Jiansheng
Meriel Ltd, Deluth, GA, University of California, USA and University of Pretoria, ZA
May 1, 2012
[14]
US8574590
Lipoparticles comprising Doranz Benjamin, proteins, methods of making, Willis, Sharon, Ross, and using the same Eric, Greene, Tiffani
Integral Molecular, Inc., Philadelphia, PA
November 5, 2013
[15]
US8282939
Attenuated live triple G protein recombinant rabies virus vaccine for pre- and post-exposure prophylaxis of rabies
Thomas Jefferson University
October 9, 2012
[16]
Faber, Milosz, Dietzschold, Bernhard, Hooper, Douglas
interest, under control of a desired promoter. These recombinant virus vectors are constructed in a manner that renders them replication defective. A principal problem encountered with the use of adenovirus vectors is the fact that the vector carrier itself can induce an unwanted immune response. These adverse effects make it difficult to utilize adenovirus vectors to express heterologous proteins such as potential vaccine antigens. Therefore, a new method of immunization using an adenovirus vector that is capable of inducing a strong immune response, but with minimal adverse reactions to the vaccine carrier, is urgently required. In the patent US8394386 [1] , the authors have developed a technique by which they can sequentially deliver one or more selected heterologous gene(s) to a patient using both an adenoviral (Ad) vector as well as an adeno-associated virus (AAV) vector [1] . Each of these vectors is designed to express the same immunogenic or antigenic product or alternatively, different but cross-reactive products. The result of this is a boosting of the immune response to the product carried by both viral vectors [18] . This boost of an antigen-specific humoral response was a phenomenon that was found by the authors unexpectedly, when they provided a regimen involving sequential rAd-mediated and rAAVmediated administration of the antigen. In addition to this, it is speculated that an enhanced T-cell response may also be observed in certain instances using this
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sequential administration (Figure 1) . The authors use as an example the design of vaccine expression vectors against Ebola virus using AAV and Ad expressing the glycoprotein of Ebola virus (Ebo GP). Undesired immune responses related to the vectors themselves were not observed. The results suggest that this expression vector system should be a preferred genetic vaccine carrier, and can generate robust T- and B-cell responses. Hepatitis C Virus expression vectors Hepatitis C virus (HCV) was originally identified as the causative agent of non-A and non-B hepatitis by Choo et al., and is a major cause of cirrhosis and hepatic cancer [20] . Approximately 170 million HCV patients can be found globally. Few animal model systems can support HCV infection and the lack of an effective in vitro culture system has delayed research in this field. More recently, HCV replicon systems have been developed and have hastened the search for anti-viral inhibitors [21] . Recently, the HCV JFH-1 strain of genotype 2a has been isolated from a patient with Key term fulminant hepatitis by Wakita et al. and the strain was released in the Virus expression vector: Virus that has been designed by genetic form of infectious virus particles engineering to infect specific into a culture medium of Huh-7 target cells and express large cells [4,22] . The HCV genome amounts of a desired protein. that was capable of virus particle
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MHCI
MHCII GV
ER
P
A
B
Figure 1. Processing and presentation of adeno-associated virus-encoded antigens in the antigen-presenting cell. (A) AAV vector transduces the APC by receptor-mediated endocytosis. Endosomal escape and nuclear uncoating lead to production of the endogenous transgene product. In the cytosol, endogenously produced antigen is processed through the proteosome (P), enters the ER via TAP, where it is loaded onto MHCI and shuttled through the Golgi apparatus to the cell surface by ‘direct presentation’. Capsid fragments can be processed within the endosome for direct presentation onto MHCII; capsid can also escape the endosome and get fed into the ER to be ‘cross-presented’ onto MHCI. (B) Phagocytosis of transduced cells or secreted transgene products enter the APC. Exogenous antigen is processed in the endosome and presented onto MHCII. Conversely, exogenous antigen can escape the endosome and be cross-presented onto MHCI. Purple solid line: MHCI direct presentation; purple dashed line: MHCI cross-presentation; purple receptor: MHCI; blue line: MHCII presentation; blue receptor: MHCII. AAV: Adeno-associated virus; APC: Antigen-presenting cell; ER: Endoplasmic reticulum; GV: Golgi vesicle; MHC: Major histocompatibility complex. Please see color figure at www.future-science.com/doi/full/10.4155/PPA.14.17 Reproduced from [19] © Rights Managed by Nature Publishing Group (2010).
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production was identified as chimeric HCV of the JFH-1 strain and another non-JFH-1 strain. Such chimeric HCV can be prepared by recombining the structural genes of the JFH-1 genome with the structural genes of other HCV strains. Regardless, it is difficult to generate large quantities of infectious virus particles with the structure of genotype 1b that can be cultured in a persistent infection system. In the patent US20138604179 [5] , the authors Akazawa et al. generated a chimeric gene derived from HCVs, in addition to a chimeric HCV particle consisting of the JFH-1 strain and a strain other than JFH1. The chimeric virus particle can then be utilized to screen for anti-HCV drugs, as a vaccine (by inactivating or attenuating the virus particle) or for the production of anti-HCV antibodies (Figure 2) . The inventors examined the ability of HCV particles to be produced via cell culture, and discovered an adaptive mutation that appears during HCV proliferation and yields a significantly enhanced ability to produce high levels of HCV particles. The mutation is at amino acid 328 at the N terminus of the core protein. The increased HCV particle yield will enable researchers to more easily generate attenuated virions for vaccines or for increased antigens for antibody production [5] . Lentivirus expression vectors Gene therapy, through the transduction of human hematopoietic stem cells, offers an attractive approach to treat a number of inherited and acquired lymphohematological disorders. It has been unrealistic to provide a stable population of human hematopoietic stem cells with existing gene delivery systems such as oncoretroviral vectors derived from Moloney murine leukemia virus, because these vectors require the breakdown of the cell’s nuclear membrane in order to transport the murine leukemia virus preintegration complex into the chromosome. Human stem cells are quiescent and lose their pluripotentiality after stimulation and proliferation. However, lentiviruses, a subgroup of retroviruses that can infect nondividing cells and actively import their preintegration complexes through the nucleopore, can be designed as delivery vectors for gene therapy [23] . Lentiviral vectors derived from human immunodeficiency virus type 1 have been developed for the delivery, integration and long-term expression of transgenes into nondividing cells such as human CD34+ hematopoietic cells [24,25] . The patent US20138551773 discusses the construction of a lentiviral vector with promoters that activate transgene expression in hematopoietic cells (Figure 3A) [6] . These cells are then able to be long-term engrafted into NOD/SCID mice and bone marrow from these primary recipients can repopulate secondary mice [26] . Lentiviral vectors offer potential
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for the gene therapy of inherited and acquired lympho-hematopoietic disorders via the genetic modification of hematopoietic stem cells (Figure 3B).
Patent Review
Key terms Chimeric virus: Recombinant virus that is composed of two or more different virus strains. CD8+ T-cell: Type of T lymphocyte that can kill cancer cells or cells infected with virus.
Vaccinia virus expression vectors The patent US20138506947 relates to an expression vector based on vaccinia virus that carries a novel mutation [7] . In the past, vaccinia virus has been used extensively as an expression vector and is perhaps best known for its use as a vaccine against smallpox. The mutant expression vectors of the present invention are unable to replicate in nondividing cells and as a result are well suited for use as vaccines and cancer therapies, as well as for gene delivery vectors. Recombinant vaccinia virus has been successfully used to express vaccine proteins including hepatitis B virus surface antigen, influenza A virus hemagglutinin, herpes simplex virus type 1 D glycoprotein, rabies virus (RV) G glycoprotein and vesicular stomatitis virus G glycoprotein. Animals immunized using vaccinia expression vectors and challenged with these viruses were protected against infection. However, the propensity of vaccinia virus to induce hyperplastic responses and even tumors in the skin of infected subjects is one adverse condition that must be dealt with. Since the vaccinia virus growth factor (VGF) is believed to play a role in these complications, both copies of the VGF gene were deleted from a recombinant vaccinia virus. No difference was observed between the wild-type and mutant viruses in cell culture, however, infection of eggs with wild type virus resulted in a rapid proliferation of chicken embryo cells. This was not the case with eggs infected with the VGF double mutant, although this mutant virus retained its ability to undergo replication. This double mutant was used as a gene therapy vehicle that expressed suicide genes, for example, to prevent the growth of cancer cells or tumours. The patent US20118067535 describes the identification of CD8+ T-cell epitopes to vaccinia virus [9] . The authors describe the identification of five T-cell epitopes that are conserved among different strains of vaccinia and variola viruses that were capable of eliciting the desired responses. These include peptide 74A, peptide 165, peptide 029D, peptide B7080, peptide B7034 and immunogenic fragments or mutants thereof [28,29] . This patent also includes the vaccinia virus-specific CD8+ cytotoxic T lymphocyte lines that were established from the isolation of these epitopes. The cell lines were developed by limiting dilution cloning from the peripheral blood mononuclear cells isolated from donors who received primary immunization with smallpox vaccine.
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(1) In vitro transcription Plasmid containing HCV replicon
Replicon RNAs (2) Transfection into cells
(4) Release of infectious virus particles
(3) Select for transfected cells with antibiotic
Figure 2. Steps involved in production of hepatitis C virus particles from cell culture. (1) Multiple RNA transcripts encoding the HCV replicon are produced via in vitro transcription and (2) transfected into Huh-7 cells. (3) Cells that were successfully transfected are then selected for using media containing an antibiotic. (4) Upon virus replication, infectious virus particles are released into the surrounding media. HCV: Hepatitis C virus.
The epitopes derived from this invention can be utilized for the design of new vaccinia vaccines, as well as for basic research into human T-cell memory. Measles virus-based vaccine The patent US20138586364 describes a means for development of a live attenuated measles virus based expression vector [13] . The measles vaccine has been used safely and effectively since the 1960s for hundreds of millions of children, and induces life-long immunity after one or two injections. Since they can be easily produced inexpensively and at a large scale, attenuated measles vaccine strains represent good candidates as vectors to deliver other infectious disease antigens, including as HIV (retroviruses), flavivirus or coronavirus (SARS) diseases. Measles infects approximately 45 million people each year and is responsible for the deaths of 700,000 children per year. To address this, the World Health Organization has expanded its global vaccination program for the next two decades. By using virus expression vectors that have already been constructed from the measles virus and used extensively, vaccines against other infectious diseases based on this same vector could also be constructed and simultaneously applied to children. To this end, the authors of this invention have previously developed a vector using the Schwarz MV, the most commonly used measles vaccine in the world [30] . This vector was shown to stably express a variety of genes and could be produced in cell culture at titers comparable to standard MV. The authors developed a pTM-MVSchw plasmid that they modified for the expression of foreign genes by the introduction of additional transcriptional units containing multiple
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cloning cassettes at different positions of the genome (Figure 4) . This new recombinant version of the original vaccine vector allows for the design of combined vaccines that are based on a live attenuated vaccine strain that is approved and currently globally in use. RV-based vaccine In the patent US20128282939, the authors of the invention constructed a nonpathogenic recombinant RV that consists of multiple copies of a modified external surface glycoprotein (G) gene [16] . This recombinant virus can be used not only as a vaccine to protect against RV infection but also to clear RV from nervous tissues postinfection. Rabies causes an approximately 55,000 human deaths globally each year, with half of these deaths taking place in Africa [31] . In addition to this, 11 million people around the world must undergo rabies postexposure prophylaxis each year. Rabies carried by dogs remains the major cause of human cases and a significant proportion of patients are children under the age of 15 [32] . Major hurdles to preventing the spread of rabies in developing countries are poorly established dog vaccination programs and limited postexposure treatment for individuals who have been exposed to rabid dogs. RV is a negative-stranded RNA virus of the rhabdoviridae family and contains genes encoding five structural proteins: an RNA-dependent RNA polymerase (L), a nucleoprotein (N), a phosphorylated protein (P), a matrix protein (M) and an external surface glycoprotein (G). RV is neuroinvasive, meaning that it possesses the unique ability to invade the CNS [33] . Attenuated strains of rabies replicate quickly and express G protein in large quantities, thereby inducing strong adaptive immune responses that result in
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Virus expression vectors
Patent Review
A 5’LTR
Promoter
EGFP
3’LTR
WPRE
SIN
B ii. Lentiviralvector particles
HSC
iv. Genetically modified HSCs
i. HSCs iii. Chemotherapy
Figure 3. Gene therapy using lentivirus expression vector. (A) Simplified schematic diagram of a lentivirus expression vector. (B) Ex vivo gene transfer to bone marrow-derived CD34+ hematopoietic stem cells of a patient with inherited severe immune deficiency. Autologous hematopoietic stem cells are cultured and infected with a recombinant retroviral or lentiviral vector carrying a functional copy of the defective gene (for example, ADA, gamma-c or ABCD1). The gene-corrected cells are then injected back into the patient. For some protocols, the patients may receive mild myeloablation prior to infusion of the hematopoietic stem cells. EGFP: Enhanced green fluorescent protein; HSC: Hematopoietic stem cell; LTR: Long terminal repeat; SIN: Selfinactivating vector; WPRE: Woodchuck hepatitis virus post-ranscriptional regulatory element. Reproduced with permission from [27] © Macmillan Publishers Ltd (2000).
virus clearance. As a result, a live-attenuated RV vaccine is likely to provide effective immunization with a single dose and can elicit an immune response that can clear virulent RVs from the CNS [34,35] . Thus, attenuated strains of RV can serve as vaccines and also provide effective treatment for early stage rabies infection. Vaccines produced using tissue culture are expensive and, as a result, are unaffordable to most people in developing countries. It is clear that T7
N
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efficacious and affordable RV vaccines are greatly needed. The invention described in this patent provides a nonpathogenic recombinant RV that contains at least three copies of a mutated G gene, encoding the glycoprotein of the virus. The G gene is mutated at amino acid serine 194 and amino acid glutamic acid 333 in the glycoprotein. The presence of two mutated G genes also substantially limits any possibility of virus reversion F
H
L
P
Figure 4. Simplified schematic diagram depicting plasmid containing recombinant measles virus vaccine. Arrows represent sites of additional transcription units. F: Fusion protein; H: Hemagglutinin; L: Polymerase; N: Nucleoprotein; P/C/V: Phosphoprotein; T7: T7 promoter.
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Figure 5. Schematic of the construction of recombinant rabies viruses containing 1, 2 or 3 modified G genes. To abolish the pathogenicity, two amino acid substitutions were introduced into rabies virus G (Arg333 → Glu333 and Asn194 →Ser194 ) resulting in GAS. In SPBAANGAS-GAS(−)-GAS(−), all ATG codons of the last two GAS genes were scrambled. G: Glycoprotein; L: RNA-dependent RNA polymerase; LS: Leader sequence; M: Matrix protein; N: Nucleoprotein; S: Scrambled ATG codons; TS: Terminal sequence. Reproduced with permission from [37] © National Academy of Sciences (2009).
to wild type (Figure 5) [36] . In one embodiment, the recombinant RV may also express a gene encoding an immune-stimulatory protein as a means by which an immune response to RV can be elicited in a mammal. African horse sickness virus-based vaccine The patent US20128168200 discusses the construction and use of recombinant vectors expressing, in a host, one or more immunogenic proteins of African horse sickness (AHS) virus, in order to confer protective immunity against infection by this virus [14] . AHS is a serious viral disease of horses, mules, donkeys and zebras that is carried by insects and can cause mortality as high as 95%. The insect Culicoides imicola, the principal vector for this disease, as well as other potential arthropod vectors, exist throughout virtually all regions of the world, including the USA. There are nine serotypes of AHS virus. AHS virus belongs to the family Reoviridae and the genome is composed of ten double-stranded RNA segments (Figure 6). Seven of the proteins produced from these gene products are structural proteins involved in virus particle formation. Several of these, including virus proteins VP2 and VP5, have been shown to be immunogenic. The invention describes the use of a recombinant virus vector to express antigens derived from these immunogenic proteins to generate an immune response against AHS virus. The virus expression vector is preferentially a poxvirus, such as canarypox virus [14] . The invention further provides for methods of inducing an immune response and protecting against AHS virus [38,39] . Porcine reproductive & respiratory syndrome virus vaccine The patent US20138609827 pertains to the field of animal health [8] . This patent is directed to infectious cDNA clones of positive polarity RNA viruses and
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concerns the use of these cDNA clones to construct vaccines for swine. Specifically, the patent deals with Porcine reproductive and respiratory syndrome (PRRS), a swine disease first described in 1987 in North America and in 1990 in Europe. The PRRS virus belongs to the Arteriviridae family. Today, PRRS affects the major swine producing countries of the world. Symptoms include reproductive problems, litters of small weak pigs and respiratory disease in piglets. The PRRS virus is difficult to control and is one of the most economically damaging diseases to the swine industry. To date, commercial vaccines that exist against PRRS are either attenuated live virus or inactivated cell culture preparations of the virus. These vaccines have been problems with regard to both safety and efficacy. Recently, a full-length infectious cDNA clone has been constructed of the European PRRS virus [41] . The authors of the current patent were able to generate an infectious RNA molecule that encodes the North American PRRS virus ( Figure 7A). This cDNA clone has been genetically modified in such a way that it is no longer pathogenic and cannot produce symptoms, yet is able to elicit an effective immunoprotective response against the PRRS virus in swine [42] . Patents involved in plant virus expression systems Plant viruses have also been utilized to act as expression systems. The following section describes recent developments pertaining to a RNA plant virus such as Tobacco mosaic virus, which are used to generate high yields of industrial and pharmaceutical proteins in plants, as well as a novel plant potexvirus that can act as both a carrier and adjuvant for vaccine proteins. The patent US20138597950, by Marillonnet et al.,
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Figure 6. Genomic organization of African horse sickness virus. NS: Nonstructural protein; VP: Viral protein. Reproduced with permission from [40] © Oxford University Press (2011).
describes virus-based expression systems used to produce proteins in plants [10] . This invention concerns an RNA replicon based on a plant RNA virus such as tobacco mosaic virus, along with a helper replicon [43] . The replicons are incapable of systemic movement in the plant unless the proteins included for virus movement are included. This viral vector system can be used to express a protein of interest in a plant, such as an industrial enzyme or pharmaceutical protein (Figure 7B). This expression system increases biological safety and reduces environmental risk by preventing the formation of virus particles that can escape the host plant and infect other plants [44,45] In the patent US20128101189 , the inventors develop a means by which to stimulate the immune system using the potexvirus papaya mosaic virus (PapMV), or a virus-like particle derived from PapMV coat protein (Figure 7C) [11] . PapMV has been demonstrated to as an adjuvant and thus elicit an immune response in an animal. An immunogen can then be fused to or otherwise associated with this virus or virus-like particle and a humoral and/or a cellular response will be potentiated, making this system suitable for use as an adjuvant or vaccine. A second invention [12] , relates further to a viral particle that bears immunogens and has adjuvant activity. This invention particularly relates to recombinant virus-like particles and a way to enhance the immune response in animals or humans by means of these particles, using a fusion event with an epitope derived from a viral, bacterial or parasital pathogen (Figure 7D). Other virus-related patents This section describes two HIV-related patents that have been issued over the past 2 years. These relate to portions of the virus that can be used for research purposes. The
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first can be applied as a means to determine novel ways to block HIV infection and the second involves the use of lipoparticles as tools for assessing the binding properties of host cell membrane proteins by using the HIV protein as a probe in an optical biosensor array. Optical biosensor analysis based on lipoparticles to analyze retrovirus host protein interactions Lipoparticles are readily used for many research applications. A lipoparticle based on the retrovirus structure enables structurally intact cellular proteins to be purified away from the cell. When a retrovirus is produced from a cell, the protein core of the virus buds through the membrane of the cell. As a consequence, the virus becomes enwrapped by the cellular membrane. Once the membrane ‘pinches’ off, the virus particle is free to diffuse. Normally, the virus also produces its own membrane protein (envelope) that is expressed on the cell surface and that becomes incorporated into the virus, but even if the gene for the viral membrane protein is deleted, virus assembly and budding can still occur. Under these conditions, the membrane enwrapping the virus contains a number of cellular proteins [46,47] . The invention described in the patent US20138574590 allows for the rapid purification of a wide spectrum of membrane proteins and their analysis with optical biosensors [15] . Host cellular proteins can act as receptors for virus entry, as well as for other functions in viral pathogenesis. While it is often slow and laborious to identify and analyze virus host membrane protein interactions, optical biosensors can now be employed to detect small levels of intramolecular interactions, and can be used to study interactions between virus and host membrane proteins. One way that the inventors allow for
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Figure 7. RNA virus expression vectors. (A) Genomic organization of PRRSV. (B) TMV expression vector construct. (C) Genomic organization of PapMV. (D) Plant virus nanoparticle vaccine system. (1) Recombinant virus displays disease epitope on surface of virus particle. (2) Nanoparticles are injected into mice. (3) An immune response is induced that is specific to the epitope and can protect against infection. 35SP: Cauliflower mosaic virus 35S promoter; 35ST: Cauliflower mosaic virus 35S terminator; CP: Coat protein; E: Envelope protein; GOI: Gene of interest; GP: Gene product; LB: Left border; M: Matrix protein; MP: Movement protein; N: Nucleoprotein; ORF: Open reading frame; PapMV: Papaya mosaic virus; PRRSV: Porcine reproductive and respiratory syndrome virus; RB: Right border; TGB: Triple gene block (movement proteins); TMV: Tobacco mosaic virus. (A) Reproduced with permission from [40] © Oxford University Press (2011).
this is to recreate the cell surface in vitro by using arrays of membrane proteins in conjunction with optical biosensors. This can result in the formation of a biosensor chip containing thousands of membrane proteins [15,48] . Retroviruses have been designed to harbour incorporated proteins of desired specificity and function, and used as probes. These will have applications in the field of cancer biology, cell biology and gene therapy. The invention comprises assessing the interactions between the binding of a protein comprised in a lipoparticle and a ligand, as well as eliciting an immune response to a
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protein by introducing the lipoparticle to an animal. The invention can be used as well as to identify inhibitors to this binding activity and to identify binding partners to lipoparticles, viruses or virus like particles [12,15,46–49] . Conclusion & future perspective This article has discussed the use of virus expression vectors for applications ranging from gene therapy to vaccine development. Many of these accomplishments serve to overcome basic shortcomings with respect to previous vector systems. Understanding the factors
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involved in developing immunogenicity of virus expression vectors remains a key component for their successful design and utilization. Potential toxicity or adverse reactions on the cellular level as well as in terms of a patient are still major hurdles to triumph over in vector development. Poor replication efficiency and low expression levels of foreign proteins are other problems that must be addressed. As described in several aspects of this review, one result of this has initiated the development of novel hybrid vectors that incorporate more favorable attributes while eliminating others that are undesirable. Based on clinical trial studies, the inherent safety of virus expression vectors will continue to be a significant challenge that delays their routine use in medicine. Nonetheless, the lineage of new virus vectors that have been described in this review would imply that superior performance with respect to safety and
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efficacy are currently underway. The development of novel vectors based on viruses that have not been sufficiently investigated, including PRRS virus and AHS virus, in addition to the application of virus sequences for the development of new strategies to combat important illnesses such as HIV/AIDS, will make virus expression vectors an integral component of modern medicine for many years to come. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary • New developments in virus expression vector design have led to a number of patents being issued over the last 2 years. This review discusses a number of these expression vector systems and details their uses. • Established virus vectors with improved immunogenicity profiles, tissue trophism and issues regarding biosafety are discussed. • Newly isolated viruses that have been recently constructed as vaccine vectors are presented. • Patents pertaining to plant virus expression vectors that are used as vaccines are described. • The review concludes with a broad discussion of the current trends in the patent literature with regard to virus expression vectors.
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Virus expression vectors
For many years now, virus expression vectors have been explored as a mechanism for gene delivery, cancer therapy and vaccine development. More recently, the next generation of virus vectors have been generated that possess greater attributes such as tissue specificity and improved expression levels, while at the same time lack many of the shortcomings of their predecessors, such as issues regarding immunogenicity and safety. This review article describes several of the recent patents that have been issued in the field of virus expression vector development. Innovations in both plant and animal virus expression vectors are covered. The review concludes with a discussion of future prospects of virus expression vectors as tools in medical research.
Background Recent innovations in the field of virus expression vector development offer great promise. From adenoviruses to lentiviruses, expression vectors are constantly being improved upon and redesigned to perform better in disciplines including gene therapy and vaccine development. Plant virus expression vectors are also rapidly gaining a role in medicine by providing new ways to generate adjuvants and pharmaceutical proteins. Recent innovations with respect to the construction and application of expression vectors and other virus sequences are the subject of this review article. The material presented here has been provided from US patents that have been issued over the past 2 years and the complete list is presented in Table 1. In a number of cases, advances have been made with regard to vector design, so that issues such as host safety and tissue specificity are better addressed. Other examples provide the initial development of novel expression vectors based on viruses that have not previously been studied in great detail. The review concludes with a discussion of the future prospects of virus expression vectors as tools in medical research and in the design of pharmaceuticals.
10.4155/PPA.14.17 © 2014 Future Science Ltd
Kathleen L Hefferon1 Cornell University, Ithaca, NY, 14853, USA [email protected] 1
Patents involved in animal/human virus expression vectors While many different animal virus expression vectors currently exist, there is always a need to improve upon them so that their direct applications can be expanded. Many virus expression systems have drawbacks ranging from poor expression levels to tissue trophism constraints and even to biosafety concerns. Expression systems based on new unexplored virus isolates are also under development. The following section describes patents recently issued that concern improvements upon adenovirus, lentivirus and vaccinia virus expression vectors, as well as other animal viruses. Adenovirus expression vectors The first example of a recently patented virus expression vector concerns adenovirus, a double-stranded DNA virus with a genome approximately 36 kilobases in size. Adenoviruses have been developed as expression vector systems for many years [17] . Adenoviruses are highly desirable as expression vectors since they can achieve highly efficient gene transfer in a variety of target tissues and maintain the ability to accommodate large transgenes [2,3] . In many cases, the adenovirus E1 gene has been replaced with the foreign gene of
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Table 1. Patents discussed in this review.
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Patent number Title
Inventors
Source
Date
US8394386
Sequential delivery of immunogenic molecules via adenovirus and adenoassociated virus-mediated administrations
Wilson, James
University of Pennsylvania
March 12, 2013
[1]
US6083716
Chimpanzee Adenovirus Vectors
Wilson, James and Farina, Steven
University of Pennsylvania
October 20, 2011
[2]
WO02/33645
Adenovirus Hexon Protein and Uses Thereof
Soumitra Roy and Wilson, James
University of Pennsylvania
January 14, 2009
[3]
WO05080575A1 Nucleic acid containing chimeric gene derived from hepatitis type-c virus
Daisuke Akazawa, and Takaji Wakita,
Toray Industries, Inc., Tokyo, Japan
February 9, 2011
[4]
US8604179
Nucleic acid comprising chimeric gene derived from hepatitis C virus
Akazawa, Daisuke, Wikita, Takaji,
Tokyo December Metropolitan 10, 2013 Organization for Medical Research Tokyo, Japan
[5]
US8551773
Methods and compositions relating to improved lentiviral vectors and their applications
Trono, Didier, Salmon, Patrick
Research Development Foundation, Carson City, Nevada
October 8, 2013
[6]
US8506947
Vaccinia virus expression McCart, J. Andrea, vector for selective Bartolett, David, replication in a tumor cell and Moss, Bernard introduction of exogenous nucleotide sequence into a tumor cell
US Department August 13, of Health and 2013 Human Services
[7]
US8609827
Infectious cDNA clone of North American porcine reproductive and respiratory syndrome (PRRS) virus and uses thereof
Calvert, Jay, Sheppard, Michael, Welch, Siao-Kun, Rowland, Raymond, Kim, Dal-Young
Zoetis Llc
December 17, 2013
[8]
US8067535
Identification of gene sequences and proteins involved in vaccinia virus dominant T cell epitopes
Terajima; Masanori, Cruz; John Ennis; Francis A.
University of Massachusetts
November 29, 2011
[9]
US8597950
Two-component RNA virusderived plant expression system
Marillonnet, Sylvestre, Engler, Carola, Muhlbauer, Stefan, Hertz, Stefan, Klimyuk, Victor, Gleba, Yuri
Icon Genetics GmbH
December 3, 2013
[10]
US8101189
Vaccines and immunopotentiating compositions and methods for making and using them
Leclerc, Denis, Majeau, Nathalie, Lopez-Macias, Constantino, Lamarre, Alain
Laval University January 24, 2012
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Ref.
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[11]
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Table 1. Patents discussed in this review (cont.). Patent number Title
Inventors
Source
US8282940
Adjuvant viral particle
Leclerc Deinis, Lopez-Macias, Constantino,
Laval University October 9, 2012
[12]
US8586364
Cells and Methodology to generate non-segmented negative-strand RNA viruses
Tangy, Frederic, Charneau, Pierre, Jacob, Yves,
Institute Pasteur
November 19, 2013
[13]
US8168200
Vaccine against african horse sickness virus
Minke, Jules, Audonnet, JeanChristoph, Guthrie, Alan, MacLachlan, Nigel, Yao, Jiansheng
Meriel Ltd, Deluth, GA, University of California, USA and University of Pretoria, ZA
May 1, 2012
[14]
US8574590
Lipoparticles comprising Doranz Benjamin, proteins, methods of making, Willis, Sharon, Ross, and using the same Eric, Greene, Tiffani
Integral Molecular, Inc., Philadelphia, PA
November 5, 2013
[15]
US8282939
Attenuated live triple G protein recombinant rabies virus vaccine for pre- and post-exposure prophylaxis of rabies
Thomas Jefferson University
October 9, 2012
[16]
Faber, Milosz, Dietzschold, Bernhard, Hooper, Douglas
interest, under control of a desired promoter. These recombinant virus vectors are constructed in a manner that renders them replication defective. A principal problem encountered with the use of adenovirus vectors is the fact that the vector carrier itself can induce an unwanted immune response. These adverse effects make it difficult to utilize adenovirus vectors to express heterologous proteins such as potential vaccine antigens. Therefore, a new method of immunization using an adenovirus vector that is capable of inducing a strong immune response, but with minimal adverse reactions to the vaccine carrier, is urgently required. In the patent US8394386 [1] , the authors have developed a technique by which they can sequentially deliver one or more selected heterologous gene(s) to a patient using both an adenoviral (Ad) vector as well as an adeno-associated virus (AAV) vector [1] . Each of these vectors is designed to express the same immunogenic or antigenic product or alternatively, different but cross-reactive products. The result of this is a boosting of the immune response to the product carried by both viral vectors [18] . This boost of an antigen-specific humoral response was a phenomenon that was found by the authors unexpectedly, when they provided a regimen involving sequential rAd-mediated and rAAVmediated administration of the antigen. In addition to this, it is speculated that an enhanced T-cell response may also be observed in certain instances using this
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Date
Ref.
sequential administration (Figure 1) . The authors use as an example the design of vaccine expression vectors against Ebola virus using AAV and Ad expressing the glycoprotein of Ebola virus (Ebo GP). Undesired immune responses related to the vectors themselves were not observed. The results suggest that this expression vector system should be a preferred genetic vaccine carrier, and can generate robust T- and B-cell responses. Hepatitis C Virus expression vectors Hepatitis C virus (HCV) was originally identified as the causative agent of non-A and non-B hepatitis by Choo et al., and is a major cause of cirrhosis and hepatic cancer [20] . Approximately 170 million HCV patients can be found globally. Few animal model systems can support HCV infection and the lack of an effective in vitro culture system has delayed research in this field. More recently, HCV replicon systems have been developed and have hastened the search for anti-viral inhibitors [21] . Recently, the HCV JFH-1 strain of genotype 2a has been isolated from a patient with Key term fulminant hepatitis by Wakita et al. and the strain was released in the Virus expression vector: Virus that has been designed by genetic form of infectious virus particles engineering to infect specific into a culture medium of Huh-7 target cells and express large cells [4,22] . The HCV genome amounts of a desired protein. that was capable of virus particle
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MHCI
MHCII GV
ER
P
A
B
Figure 1. Processing and presentation of adeno-associated virus-encoded antigens in the antigen-presenting cell. (A) AAV vector transduces the APC by receptor-mediated endocytosis. Endosomal escape and nuclear uncoating lead to production of the endogenous transgene product. In the cytosol, endogenously produced antigen is processed through the proteosome (P), enters the ER via TAP, where it is loaded onto MHCI and shuttled through the Golgi apparatus to the cell surface by ‘direct presentation’. Capsid fragments can be processed within the endosome for direct presentation onto MHCII; capsid can also escape the endosome and get fed into the ER to be ‘cross-presented’ onto MHCI. (B) Phagocytosis of transduced cells or secreted transgene products enter the APC. Exogenous antigen is processed in the endosome and presented onto MHCII. Conversely, exogenous antigen can escape the endosome and be cross-presented onto MHCI. Purple solid line: MHCI direct presentation; purple dashed line: MHCI cross-presentation; purple receptor: MHCI; blue line: MHCII presentation; blue receptor: MHCII. AAV: Adeno-associated virus; APC: Antigen-presenting cell; ER: Endoplasmic reticulum; GV: Golgi vesicle; MHC: Major histocompatibility complex. Please see color figure at www.future-science.com/doi/full/10.4155/PPA.14.17 Reproduced from [19] © Rights Managed by Nature Publishing Group (2010).
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production was identified as chimeric HCV of the JFH-1 strain and another non-JFH-1 strain. Such chimeric HCV can be prepared by recombining the structural genes of the JFH-1 genome with the structural genes of other HCV strains. Regardless, it is difficult to generate large quantities of infectious virus particles with the structure of genotype 1b that can be cultured in a persistent infection system. In the patent US20138604179 [5] , the authors Akazawa et al. generated a chimeric gene derived from HCVs, in addition to a chimeric HCV particle consisting of the JFH-1 strain and a strain other than JFH1. The chimeric virus particle can then be utilized to screen for anti-HCV drugs, as a vaccine (by inactivating or attenuating the virus particle) or for the production of anti-HCV antibodies (Figure 2) . The inventors examined the ability of HCV particles to be produced via cell culture, and discovered an adaptive mutation that appears during HCV proliferation and yields a significantly enhanced ability to produce high levels of HCV particles. The mutation is at amino acid 328 at the N terminus of the core protein. The increased HCV particle yield will enable researchers to more easily generate attenuated virions for vaccines or for increased antigens for antibody production [5] . Lentivirus expression vectors Gene therapy, through the transduction of human hematopoietic stem cells, offers an attractive approach to treat a number of inherited and acquired lymphohematological disorders. It has been unrealistic to provide a stable population of human hematopoietic stem cells with existing gene delivery systems such as oncoretroviral vectors derived from Moloney murine leukemia virus, because these vectors require the breakdown of the cell’s nuclear membrane in order to transport the murine leukemia virus preintegration complex into the chromosome. Human stem cells are quiescent and lose their pluripotentiality after stimulation and proliferation. However, lentiviruses, a subgroup of retroviruses that can infect nondividing cells and actively import their preintegration complexes through the nucleopore, can be designed as delivery vectors for gene therapy [23] . Lentiviral vectors derived from human immunodeficiency virus type 1 have been developed for the delivery, integration and long-term expression of transgenes into nondividing cells such as human CD34+ hematopoietic cells [24,25] . The patent US20138551773 discusses the construction of a lentiviral vector with promoters that activate transgene expression in hematopoietic cells (Figure 3A) [6] . These cells are then able to be long-term engrafted into NOD/SCID mice and bone marrow from these primary recipients can repopulate secondary mice [26] . Lentiviral vectors offer potential
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for the gene therapy of inherited and acquired lympho-hematopoietic disorders via the genetic modification of hematopoietic stem cells (Figure 3B).
Patent Review
Key terms Chimeric virus: Recombinant virus that is composed of two or more different virus strains. CD8+ T-cell: Type of T lymphocyte that can kill cancer cells or cells infected with virus.
Vaccinia virus expression vectors The patent US20138506947 relates to an expression vector based on vaccinia virus that carries a novel mutation [7] . In the past, vaccinia virus has been used extensively as an expression vector and is perhaps best known for its use as a vaccine against smallpox. The mutant expression vectors of the present invention are unable to replicate in nondividing cells and as a result are well suited for use as vaccines and cancer therapies, as well as for gene delivery vectors. Recombinant vaccinia virus has been successfully used to express vaccine proteins including hepatitis B virus surface antigen, influenza A virus hemagglutinin, herpes simplex virus type 1 D glycoprotein, rabies virus (RV) G glycoprotein and vesicular stomatitis virus G glycoprotein. Animals immunized using vaccinia expression vectors and challenged with these viruses were protected against infection. However, the propensity of vaccinia virus to induce hyperplastic responses and even tumors in the skin of infected subjects is one adverse condition that must be dealt with. Since the vaccinia virus growth factor (VGF) is believed to play a role in these complications, both copies of the VGF gene were deleted from a recombinant vaccinia virus. No difference was observed between the wild-type and mutant viruses in cell culture, however, infection of eggs with wild type virus resulted in a rapid proliferation of chicken embryo cells. This was not the case with eggs infected with the VGF double mutant, although this mutant virus retained its ability to undergo replication. This double mutant was used as a gene therapy vehicle that expressed suicide genes, for example, to prevent the growth of cancer cells or tumours. The patent US20118067535 describes the identification of CD8+ T-cell epitopes to vaccinia virus [9] . The authors describe the identification of five T-cell epitopes that are conserved among different strains of vaccinia and variola viruses that were capable of eliciting the desired responses. These include peptide 74A, peptide 165, peptide 029D, peptide B7080, peptide B7034 and immunogenic fragments or mutants thereof [28,29] . This patent also includes the vaccinia virus-specific CD8+ cytotoxic T lymphocyte lines that were established from the isolation of these epitopes. The cell lines were developed by limiting dilution cloning from the peripheral blood mononuclear cells isolated from donors who received primary immunization with smallpox vaccine.
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(1) In vitro transcription Plasmid containing HCV replicon
Replicon RNAs (2) Transfection into cells
(4) Release of infectious virus particles
(3) Select for transfected cells with antibiotic
Figure 2. Steps involved in production of hepatitis C virus particles from cell culture. (1) Multiple RNA transcripts encoding the HCV replicon are produced via in vitro transcription and (2) transfected into Huh-7 cells. (3) Cells that were successfully transfected are then selected for using media containing an antibiotic. (4) Upon virus replication, infectious virus particles are released into the surrounding media. HCV: Hepatitis C virus.
The epitopes derived from this invention can be utilized for the design of new vaccinia vaccines, as well as for basic research into human T-cell memory. Measles virus-based vaccine The patent US20138586364 describes a means for development of a live attenuated measles virus based expression vector [13] . The measles vaccine has been used safely and effectively since the 1960s for hundreds of millions of children, and induces life-long immunity after one or two injections. Since they can be easily produced inexpensively and at a large scale, attenuated measles vaccine strains represent good candidates as vectors to deliver other infectious disease antigens, including as HIV (retroviruses), flavivirus or coronavirus (SARS) diseases. Measles infects approximately 45 million people each year and is responsible for the deaths of 700,000 children per year. To address this, the World Health Organization has expanded its global vaccination program for the next two decades. By using virus expression vectors that have already been constructed from the measles virus and used extensively, vaccines against other infectious diseases based on this same vector could also be constructed and simultaneously applied to children. To this end, the authors of this invention have previously developed a vector using the Schwarz MV, the most commonly used measles vaccine in the world [30] . This vector was shown to stably express a variety of genes and could be produced in cell culture at titers comparable to standard MV. The authors developed a pTM-MVSchw plasmid that they modified for the expression of foreign genes by the introduction of additional transcriptional units containing multiple
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cloning cassettes at different positions of the genome (Figure 4) . This new recombinant version of the original vaccine vector allows for the design of combined vaccines that are based on a live attenuated vaccine strain that is approved and currently globally in use. RV-based vaccine In the patent US20128282939, the authors of the invention constructed a nonpathogenic recombinant RV that consists of multiple copies of a modified external surface glycoprotein (G) gene [16] . This recombinant virus can be used not only as a vaccine to protect against RV infection but also to clear RV from nervous tissues postinfection. Rabies causes an approximately 55,000 human deaths globally each year, with half of these deaths taking place in Africa [31] . In addition to this, 11 million people around the world must undergo rabies postexposure prophylaxis each year. Rabies carried by dogs remains the major cause of human cases and a significant proportion of patients are children under the age of 15 [32] . Major hurdles to preventing the spread of rabies in developing countries are poorly established dog vaccination programs and limited postexposure treatment for individuals who have been exposed to rabid dogs. RV is a negative-stranded RNA virus of the rhabdoviridae family and contains genes encoding five structural proteins: an RNA-dependent RNA polymerase (L), a nucleoprotein (N), a phosphorylated protein (P), a matrix protein (M) and an external surface glycoprotein (G). RV is neuroinvasive, meaning that it possesses the unique ability to invade the CNS [33] . Attenuated strains of rabies replicate quickly and express G protein in large quantities, thereby inducing strong adaptive immune responses that result in
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Virus expression vectors
Patent Review
A 5’LTR
Promoter
EGFP
3’LTR
WPRE
SIN
B ii. Lentiviralvector particles
HSC
iv. Genetically modified HSCs
i. HSCs iii. Chemotherapy
Figure 3. Gene therapy using lentivirus expression vector. (A) Simplified schematic diagram of a lentivirus expression vector. (B) Ex vivo gene transfer to bone marrow-derived CD34+ hematopoietic stem cells of a patient with inherited severe immune deficiency. Autologous hematopoietic stem cells are cultured and infected with a recombinant retroviral or lentiviral vector carrying a functional copy of the defective gene (for example, ADA, gamma-c or ABCD1). The gene-corrected cells are then injected back into the patient. For some protocols, the patients may receive mild myeloablation prior to infusion of the hematopoietic stem cells. EGFP: Enhanced green fluorescent protein; HSC: Hematopoietic stem cell; LTR: Long terminal repeat; SIN: Selfinactivating vector; WPRE: Woodchuck hepatitis virus post-ranscriptional regulatory element. Reproduced with permission from [27] © Macmillan Publishers Ltd (2000).
virus clearance. As a result, a live-attenuated RV vaccine is likely to provide effective immunization with a single dose and can elicit an immune response that can clear virulent RVs from the CNS [34,35] . Thus, attenuated strains of RV can serve as vaccines and also provide effective treatment for early stage rabies infection. Vaccines produced using tissue culture are expensive and, as a result, are unaffordable to most people in developing countries. It is clear that T7
N
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efficacious and affordable RV vaccines are greatly needed. The invention described in this patent provides a nonpathogenic recombinant RV that contains at least three copies of a mutated G gene, encoding the glycoprotein of the virus. The G gene is mutated at amino acid serine 194 and amino acid glutamic acid 333 in the glycoprotein. The presence of two mutated G genes also substantially limits any possibility of virus reversion F
H
L
P
Figure 4. Simplified schematic diagram depicting plasmid containing recombinant measles virus vaccine. Arrows represent sites of additional transcription units. F: Fusion protein; H: Hemagglutinin; L: Polymerase; N: Nucleoprotein; P/C/V: Phosphoprotein; T7: T7 promoter.
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pSPBAANGAS-GAS-GAS
pSPBAANGAS-GAS(-)-GAS(-)
Pac/
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Figure 5. Schematic of the construction of recombinant rabies viruses containing 1, 2 or 3 modified G genes. To abolish the pathogenicity, two amino acid substitutions were introduced into rabies virus G (Arg333 → Glu333 and Asn194 →Ser194 ) resulting in GAS. In SPBAANGAS-GAS(−)-GAS(−), all ATG codons of the last two GAS genes were scrambled. G: Glycoprotein; L: RNA-dependent RNA polymerase; LS: Leader sequence; M: Matrix protein; N: Nucleoprotein; S: Scrambled ATG codons; TS: Terminal sequence. Reproduced with permission from [37] © National Academy of Sciences (2009).
to wild type (Figure 5) [36] . In one embodiment, the recombinant RV may also express a gene encoding an immune-stimulatory protein as a means by which an immune response to RV can be elicited in a mammal. African horse sickness virus-based vaccine The patent US20128168200 discusses the construction and use of recombinant vectors expressing, in a host, one or more immunogenic proteins of African horse sickness (AHS) virus, in order to confer protective immunity against infection by this virus [14] . AHS is a serious viral disease of horses, mules, donkeys and zebras that is carried by insects and can cause mortality as high as 95%. The insect Culicoides imicola, the principal vector for this disease, as well as other potential arthropod vectors, exist throughout virtually all regions of the world, including the USA. There are nine serotypes of AHS virus. AHS virus belongs to the family Reoviridae and the genome is composed of ten double-stranded RNA segments (Figure 6). Seven of the proteins produced from these gene products are structural proteins involved in virus particle formation. Several of these, including virus proteins VP2 and VP5, have been shown to be immunogenic. The invention describes the use of a recombinant virus vector to express antigens derived from these immunogenic proteins to generate an immune response against AHS virus. The virus expression vector is preferentially a poxvirus, such as canarypox virus [14] . The invention further provides for methods of inducing an immune response and protecting against AHS virus [38,39] . Porcine reproductive & respiratory syndrome virus vaccine The patent US20138609827 pertains to the field of animal health [8] . This patent is directed to infectious cDNA clones of positive polarity RNA viruses and
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concerns the use of these cDNA clones to construct vaccines for swine. Specifically, the patent deals with Porcine reproductive and respiratory syndrome (PRRS), a swine disease first described in 1987 in North America and in 1990 in Europe. The PRRS virus belongs to the Arteriviridae family. Today, PRRS affects the major swine producing countries of the world. Symptoms include reproductive problems, litters of small weak pigs and respiratory disease in piglets. The PRRS virus is difficult to control and is one of the most economically damaging diseases to the swine industry. To date, commercial vaccines that exist against PRRS are either attenuated live virus or inactivated cell culture preparations of the virus. These vaccines have been problems with regard to both safety and efficacy. Recently, a full-length infectious cDNA clone has been constructed of the European PRRS virus [41] . The authors of the current patent were able to generate an infectious RNA molecule that encodes the North American PRRS virus ( Figure 7A). This cDNA clone has been genetically modified in such a way that it is no longer pathogenic and cannot produce symptoms, yet is able to elicit an effective immunoprotective response against the PRRS virus in swine [42] . Patents involved in plant virus expression systems Plant viruses have also been utilized to act as expression systems. The following section describes recent developments pertaining to a RNA plant virus such as Tobacco mosaic virus, which are used to generate high yields of industrial and pharmaceutical proteins in plants, as well as a novel plant potexvirus that can act as both a carrier and adjuvant for vaccine proteins. The patent US20138597950, by Marillonnet et al.,
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Virus expression vectors
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Figure 6. Genomic organization of African horse sickness virus. NS: Nonstructural protein; VP: Viral protein. Reproduced with permission from [40] © Oxford University Press (2011).
describes virus-based expression systems used to produce proteins in plants [10] . This invention concerns an RNA replicon based on a plant RNA virus such as tobacco mosaic virus, along with a helper replicon [43] . The replicons are incapable of systemic movement in the plant unless the proteins included for virus movement are included. This viral vector system can be used to express a protein of interest in a plant, such as an industrial enzyme or pharmaceutical protein (Figure 7B). This expression system increases biological safety and reduces environmental risk by preventing the formation of virus particles that can escape the host plant and infect other plants [44,45] In the patent US20128101189 , the inventors develop a means by which to stimulate the immune system using the potexvirus papaya mosaic virus (PapMV), or a virus-like particle derived from PapMV coat protein (Figure 7C) [11] . PapMV has been demonstrated to as an adjuvant and thus elicit an immune response in an animal. An immunogen can then be fused to or otherwise associated with this virus or virus-like particle and a humoral and/or a cellular response will be potentiated, making this system suitable for use as an adjuvant or vaccine. A second invention [12] , relates further to a viral particle that bears immunogens and has adjuvant activity. This invention particularly relates to recombinant virus-like particles and a way to enhance the immune response in animals or humans by means of these particles, using a fusion event with an epitope derived from a viral, bacterial or parasital pathogen (Figure 7D). Other virus-related patents This section describes two HIV-related patents that have been issued over the past 2 years. These relate to portions of the virus that can be used for research purposes. The
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first can be applied as a means to determine novel ways to block HIV infection and the second involves the use of lipoparticles as tools for assessing the binding properties of host cell membrane proteins by using the HIV protein as a probe in an optical biosensor array. Optical biosensor analysis based on lipoparticles to analyze retrovirus host protein interactions Lipoparticles are readily used for many research applications. A lipoparticle based on the retrovirus structure enables structurally intact cellular proteins to be purified away from the cell. When a retrovirus is produced from a cell, the protein core of the virus buds through the membrane of the cell. As a consequence, the virus becomes enwrapped by the cellular membrane. Once the membrane ‘pinches’ off, the virus particle is free to diffuse. Normally, the virus also produces its own membrane protein (envelope) that is expressed on the cell surface and that becomes incorporated into the virus, but even if the gene for the viral membrane protein is deleted, virus assembly and budding can still occur. Under these conditions, the membrane enwrapping the virus contains a number of cellular proteins [46,47] . The invention described in the patent US20138574590 allows for the rapid purification of a wide spectrum of membrane proteins and their analysis with optical biosensors [15] . Host cellular proteins can act as receptors for virus entry, as well as for other functions in viral pathogenesis. While it is often slow and laborious to identify and analyze virus host membrane protein interactions, optical biosensors can now be employed to detect small levels of intramolecular interactions, and can be used to study interactions between virus and host membrane proteins. One way that the inventors allow for
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Figure 7. RNA virus expression vectors. (A) Genomic organization of PRRSV. (B) TMV expression vector construct. (C) Genomic organization of PapMV. (D) Plant virus nanoparticle vaccine system. (1) Recombinant virus displays disease epitope on surface of virus particle. (2) Nanoparticles are injected into mice. (3) An immune response is induced that is specific to the epitope and can protect against infection. 35SP: Cauliflower mosaic virus 35S promoter; 35ST: Cauliflower mosaic virus 35S terminator; CP: Coat protein; E: Envelope protein; GOI: Gene of interest; GP: Gene product; LB: Left border; M: Matrix protein; MP: Movement protein; N: Nucleoprotein; ORF: Open reading frame; PapMV: Papaya mosaic virus; PRRSV: Porcine reproductive and respiratory syndrome virus; RB: Right border; TGB: Triple gene block (movement proteins); TMV: Tobacco mosaic virus. (A) Reproduced with permission from [40] © Oxford University Press (2011).
this is to recreate the cell surface in vitro by using arrays of membrane proteins in conjunction with optical biosensors. This can result in the formation of a biosensor chip containing thousands of membrane proteins [15,48] . Retroviruses have been designed to harbour incorporated proteins of desired specificity and function, and used as probes. These will have applications in the field of cancer biology, cell biology and gene therapy. The invention comprises assessing the interactions between the binding of a protein comprised in a lipoparticle and a ligand, as well as eliciting an immune response to a
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protein by introducing the lipoparticle to an animal. The invention can be used as well as to identify inhibitors to this binding activity and to identify binding partners to lipoparticles, viruses or virus like particles [12,15,46–49] . Conclusion & future perspective This article has discussed the use of virus expression vectors for applications ranging from gene therapy to vaccine development. Many of these accomplishments serve to overcome basic shortcomings with respect to previous vector systems. Understanding the factors
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Virus expression vectors
involved in developing immunogenicity of virus expression vectors remains a key component for their successful design and utilization. Potential toxicity or adverse reactions on the cellular level as well as in terms of a patient are still major hurdles to triumph over in vector development. Poor replication efficiency and low expression levels of foreign proteins are other problems that must be addressed. As described in several aspects of this review, one result of this has initiated the development of novel hybrid vectors that incorporate more favorable attributes while eliminating others that are undesirable. Based on clinical trial studies, the inherent safety of virus expression vectors will continue to be a significant challenge that delays their routine use in medicine. Nonetheless, the lineage of new virus vectors that have been described in this review would imply that superior performance with respect to safety and
Patent Review
efficacy are currently underway. The development of novel vectors based on viruses that have not been sufficiently investigated, including PRRS virus and AHS virus, in addition to the application of virus sequences for the development of new strategies to combat important illnesses such as HIV/AIDS, will make virus expression vectors an integral component of modern medicine for many years to come. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary • New developments in virus expression vector design have led to a number of patents being issued over the last 2 years. This review discusses a number of these expression vector systems and details their uses. • Established virus vectors with improved immunogenicity profiles, tissue trophism and issues regarding biosafety are discussed. • Newly isolated viruses that have been recently constructed as vaccine vectors are presented. • Patents pertaining to plant virus expression vectors that are used as vaccines are described. • The review concludes with a broad discussion of the current trends in the patent literature with regard to virus expression vectors.
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