This invention pertains to a new, mutant equine herpesvirus that has reduced virulence compared to the parent virus and replicates in a variety of mammalian cell types, and that can be used as a live vaccine virus to immunize equines against equine herpesvirus-1 infection or as a viral vector to introduce exogenous genes or antigens in equine and other mammalian species.
Equine herpesvirus-1 (EHV-1) is a member of Genus Varicellovirus within the Alphaherpesvirinae subfamily. It is a major equine pathogen that causes severe diseases such as respiratory disease, epidemic abortion storms, and brain infections that lead to paralysis. Herpesvirus genomes are classified into groups A to F with regard to their structural properties, for example, the number and location of repeat and inverted sequences and the ability to exist in one, two, or four isomeric arrangements (Roizman, 1996; Roizman and Pellet, 2001). Herpesviruses, such as EHV-1 (Henry et al., 1981; O'Callaghan and Osterrieder, 2008; Whalley et al., 2007), with group D types of genomes have a fixed long region covalently linked to a short genomic region comprised of a pair of inverted repeat sequences that bracket the unique short segment.
Herpesviruses are currently being engineered for use as gene therapy vectors and for the development of recombinant vaccines (Rosas et al., 2008; Srinivasan et al., 2008; Yokoyama et al., 2008). Manipulation of the genome, such as the introduction or deletion of gene(s), can be carried out by homologous recombination utilizing full-length infectious genomes established as BACs in E. coli (Rudolph et al., 2002; Tischer et al., 2006). In herpesvirus genomes, the presence of repeat sequences makes manipulation of some genes difficult because deletion of a diploid gene may be rescued by the same gene that is located in the other repeat sequence (Ahn et al., 2010; Boldogkoi et al., 1998). This means that alterations of sequences within one repeat segment are repaired by homologous recombination events involving identical sequences within the other inverted repeat segment (Ahn et al., 2010; Boldogkoi et al., 1998). Therefore, for a potential vaccine, it would be preferable to manipulate viruses such that the viral genome presents a simpler structure and is less virulent, but it retains the ability to replicate and its other major biological properties.
The genomic sequence arrangement of EHV-1 (Henry et al., 1981; O'Callaghan and Osterrieder, 2008; Ruyechan et al., 1982; Whalley et al., 2007) is a group D type of genome and contains a short region with a central unique segment bracketed by a pair of inverted repeat sequences that allow the short region to invert relative to the long region. The group D type of genome of herpesviruses has sequences at one terminus that are repeated in an inverted orientation internally (Roizman, 1996; Roizman and Pellet, 2001). This type of structure is observed in the genomes of several members of the Alphaherpesvirinae subfamily, including human herpesvirus 3 (varicella-zoster virus), bovine herpesvirus 1, suid herpesvirus 1, gallid herpesvirus 1, equine herpesvirus 3, and equine herpesvirus 4 (Roizman, 1996; Roizman and Pellet, 2001).
Additionally, EHV-1 has a genome of 150,000 base pairs (bp) (Telford et al., 1992) and is comprised of a unique long (U
The IR1 gene encodes a sole IE protein that governs early and some late gene expression and downregulates its own expression (Caughman et al, 1985; Harty, 1990; Smith et al., 1992; 1993). The early IR2 gene is located within the IE (IR1) ORF and generates the IR2 protein (IR2P) that strongly downregulates expression of all genes as a potent negative regulator (Kim et al., 2006). The IR3 gene, unique to EHV-1, is trans-activated by the IE protein (IEP), EICP0 protein (EICPOP) and IR4 protein (IR4P), and produces a non-coding 1 kb late transcript (Ahn et al., 2007; Holden et al., 1992a) that downregulates expression of the IE gene in a luciferase reporter system (Ahn et al., 2010). The early regulatory IR4P cooperates with the IEP to enhance expression of early and late viral genes (Holden et al., 1995) and comprises the major portion of the IR4/U
U.S. Pat. No. 5,292,653 discloses a mutant equine herpesvirus type 1 that fails to produce any functional thymidine kinase and use of such mutants as vaccines and carriers for exogenous proteins.
U.S. Pat. No. 5,741,696 discloses recombinant equine herpesviruses using mutant equine herpesviruses with deletion of the DNA encoding the US2 gene and optionally further deletion or alteration of the DNA corresponding to one or more of the gpG, gpE, and TK genes.
U.S. Pat. No. 5,795,578 discloses the gene encoding the envelope glycoprotein of equine herpesvirus type 1, the glycoprotein D (gD) gene, and to antibodies against gD polypeptides.
U.S. Pat. Nos. 6,803,041 and 7,226,604 disclose a equine herpesvirus vaccine based on an inactivated EHV-1 (chemically inactivated EHV-1 KyA virus) and an adjuvant; and optionally includes antigens against other equine pathogens, such as inactivated EHV-4 and inactivated A1 and/or A2 strains of equine influenza virus.
U.S. Pat. No. 7,060,282 discloses equine herpesviruses (EHV) mutants involving changes to the immediate early gene of EHV.
We have constructed a mutant EHV-1 that is lacking the entire 12.7 kbp IR segment of the viral genome (vL11ΔIR) and found the mutant EHV-1 to be replication competent, to have the ability to replicate in mammalian cell types (including human cells) tested in cell culture assays, and to exhibit reduced virulence in the mouse model of EHV-1 virulence. We have showed that the IR segment is dispensable for EHV-1 replication, but that the vL11ΔIR mutant exhibits a smaller plaque size and delayed growth kinetics. We also restored the IR to the mutant virus (vL11ΔIRR).
Western blot analyses of cells infected with the mutant vL11ΔIR showed that the synthesis of viral proteins encoded by the immediate-early, early, and late genes was reduced at immediate-early and early times, but by late stages of replication, reached wild type levels. Intranasal infection of CBA mice revealed that the vL11ΔIR was significantly reduced, as mice with the vL11ΔIR showed a decreased lung viral titer and a greater ability to survive infection compared to mice that were infected with either parental or revertant virus.
This new EHV-1 mutant is the first known generation of a group D herpesvirus that lacks an entire internal inverted repeat sequence, and its genome cannot undergo inversion of the short region. In addition, the mutant has only one copy of the six viral genes found in each inverted repeat sequence. Therefore, the mutant virus can be used to generate additional mutant viruses that lack one or more genes in the inverted repeat segments. Because the mutant EHV-1 virus has a large section deleted, it can accept exogenous genes and carry those genes into host mammalian cells. The mutant virus can thus act as a carrier for exogenous genes. The exogenous genes could be known “antigens” of certain infectious diseases, and therefore, the mutant EHV-1 with the antigen could be a vaccine against the antigen-derived disease.
Since the mutant virus replicates in a variety of cell types and has a genome of reduced size, it would be a potential vector in gene therapy to accept and express as much as 13,000 bp of foreign DNA (several genes). Also, since the mutant has reduced virulence as compared to the parent virus used to make the mutant, it itself can be used as a live vaccine virus to immunize equines in order to protect them against wild type EHV-1 infection.
In addition, the mutant EHV-1 virus can be used to study the effects of mutations in the six genes found in the remaining copy of the inverted repeat sequence. In the mutant EHV-1 there is only one copy of each of these six genes, while in the parent EHV-1 there are two copies of each of these genes. Thus, the mutant EHV-1 lacking the entire IR would simplify approaches to mutate or delete any of the six genes that map with the short region repeat sequence.
We have made a mutant equine herpesvirus 1 (EHV-1) lacking the entire 12,700 base pairs (BP) internal repeat (IR) segment of the viral genome and found the mutant EHV-1 to be replication competent, to have the ability to replicate in mammalian cell types (including human cells) tested in cell culture assays, and to exhibit reduced virulence in the mouse model of EHV-1 virulence.
Since the mutant EHV-1 virus replicates in a variety of cell types and has a genome of reduced size, it can be used as a vector in gene therapy to accept and express as much as 13,000 bp of foreign DNA (several genes). Also, since the mutant EHV-1 has reduced virulence as compared to the parent virus used to make the mutant, it itself can be used as a live vaccine virus to immunize equines to protect them against wild type EHV-1 infection. This new EHV-1 mutant is the first known generation of a group D herpesvirus that lacks an entire internal inverted repeat, and thus cannot undergo inversion of the SHORT region. Also, because the mutant has only one copy of the six viral genes found in each inverted repeat, it will readily allow generation of additional mutant viruses that lack one or more genes in the inverted repeat segments.
The diagram in
We have created a mutant EHV-1 virus with a deletion of one of the two inverted repeat sequences. This virus thus has only one copy of the six genes found in the inverted repeat sequences—IR1, IR2, IR3, IR4, IR5, IR6, and a portion of U
The mutant EHV-1 virus with a deleted inverted repeat sequence was able to replicate in several types of mammalian cells, including mouse, equine, rabbit, monkey and human cells. The mutated virus was also shown to have lower virulence than the parental EHV-1 virus. Thus the virus can be used as a vaccine in horses to protect horses from EHV-1 infections. Equine herpesvirus type 1 is a major pathogen of equines worldwide with an enormous economic impact. EHV-1 causes respiratory symptoms through replication in epithelial cells of the upper respiratory tract, and causes fever, late-term abortions, and equine herpesvirus encephalomyelopathy (EHM or “equine stroke”). Thus the use of this mutant EHV-1 as a vaccine in horses should help prevent or decrease the symptoms associated with wildtype EHV-1 infections.
In addition, because the mutant EHV-1 virus has a large section deleted (over 12, 700 base pairs), it can accept exogenous genes and be used as a vector to deliver vaccine antigens and immunomodulatory genes into mammals. The mutant virus can thus act as viral vector to carry exogenous genes into mammals in which it replicates. We have shown the mutant EHV-1 has replicated in all five mammalian cells tested, cells from mice, horses, rabbits, monkeys, and human. The exogenous genes could be known “antigens” of certain infectious diseases, and thus the mutant EHV-1 with the antigen could be a vaccine against the antigen-derived disease. Examples of such exogenous genes that are antigens of other infectious diseases include, without limitation, genes expressing the Rabies G protein, equine infectious anemia ENV protein gp70-gp45, Eastern equine encephalitis virus E1 membrane protein, Eastern equine encephalitis virus E2 membrane protein, Venezuelan equine encephalitis virus E1 membrane protein, Venezuelan equine encephalitis virus E2 membrane protein, equine influenza virus hemagglutinin, equine influenza virus H3 protein, equine arteritis virus G1 membrane protein, equine arteritis virus G2 membrane protein, yellow fever virus prm-E protein, equine herpesvirus-4 glycoprotein gD, and other equine herpesviruses glycoproteins. The exogenous genes could also be known genes that encode proteins with known beneficial functions, e.g., proteins to increase or decrease the inflammatory response of the mammal. Examples include, without limitation, genes expressing interferon gamma (IFNγ), interleukin 12 (IL-12), and IL-2. To express the exogenous genes as proteins, these genes would need to be under the control of one or more promoters. Many such promoters are known in the literature, and examples, include without limitation, the EHV-1 immediate-early gene promoter, the EHV-1 tk gene promoter, the EHV-1 gp13 gene promoter, the EHV-1 gp 14 gene promoter, and the human cytomegalovirus immediate early gene promoter (See, for example, U.S. Pat. No. 5,292,653; and International Publication No. WO 2011/119925).
In addition, the mutant EHV-1 virus can be used to study the effects of mutations in the six genes found in the remaining copy of the inverted repeat sequence. In the mutant EHV-1 there is only one copy of each of these six genes, while in the parent EHV-1 there are two copies of each of these genes. The duplication of the six genes in the repeat segments of the short genomic region makes manipulation of these six genes quite problematic in the laboratory. Thus, the mutant EHV-1 lacking the entire IR would simplify approaches to mutate or delete any of the six genes that map with the short region repeat segment. For example, our previous work (Breitenbach, J. E., P. D. Ebner, and D. J. O'Callaghan 2009, Virology 383: 188-194) showed that the IR4 auxiliary regulatory protein is essential for EHV-1 pathogenesis and is a major factor in determining the host range of EHV-1. Thus, the delta-IR EHV-1 would be ideal to use as a parent virus to construct EHV-1 mutants with a deleted IR4 gene or with mutant forms of the IR4 gene; such IR4 mutants would be further attenuated and may have a limited tropism in the equine such as being incapable of replication in the lung or causing viremia that is an essential feature of the pathogenesis of outcomes such as abortion and infection of the central nervous system.
EHV-1 has been shown to exhibit a broad host range and replicates in a variety of cell types (O'Callaghan and Osterrieder, 2008; Trapp et al., 2005). Although closely related to EHV-1, EHV-4 has very limited cellular tropism that could be broadened when the EHV-4 gD gene was replaced with the EHV-1 homolog (Whalley et al., 2007). The fact that the tropism of vL11ΔIR was identical to that of the parental virus in the five cell types tested was interesting because recent studies with an EHV-1 mutant deleted of both copies of the IR4 gene showed a major change in its tropism as compared to that of the wild type EHV-1 (Breitenbach et al., 2009). Thus, a single copy of this auxiliary regulatory gene was sufficient for vL11ΔIR to replicate in the five cell types.
The virulence of EHV-1 in the CBA mouse model is well characterized by body weight loss and a significant mortality rate due to a massive inflammatory reaction in the lung mediated by the induction of cytokine/chemokine responses (Frampton et al., 2002; O'Callaghan and Osterrieder, 2008; Smith et al., 2005). We found that the vL11ΔIR was less virulent than the parental virus as judged by overall mortality and attribute this to the inability of this ΔIR mutant to replicate to high titers in the murine lung.
The term “vaccine” refers to a protein or any other biological agent, e.g., a virus with one or more antigens, in an administrable form capable of stimulating an immune response in a mammal given the vaccine and so confer resistance to the disease or infection in that mammal, including an ability of the immune system to remember the previously encountered antigen. For example, use of the mutant EHV-1 virus to stimulate an immune response in horses to confer resistance to equine herpesvirus-1 infection. Antibodies are produced as a result of the first exposure to an antigen and as a result of the initial immunization, a pool of memory B lymphocytes would be generated which could later produce antibodies. Thus in the event of subsequent exposure to the same antigen, the symptoms could be ameliorated, prevented, or decreased. In addition to the humoral immune response, the mutant EHV-1 virus would generate cell mediated immune responses (i.e., activation of T cells).
The term “adjuvant” refers to non-antigenic substance that, in combination with an antigen, enhances antibody production by inducing an inflammatory or other non-defined response, which leads to a local influx of antibody-forming cells. Adjuvants are used therapeutically in the preparation of vaccines, since they increase the production of antibodies against small quantities of antigen, lengthen the period of antibody production, and tend to induce memory cell responses. In the case of intranasal administration, the adjuvant may have bioadhesive properties to enhance exposure to the virus. Such adjuvants could include, but are not limited to, cross-linked polymers (e.g., as described in U.S. Pat. No. 6,803,041). Other adjuvants, particularly for administration by injection, include complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, dimethyldioctadecylammonium bromide, Adjuvax (Alpha-Beta Technology), Imject Alum (Pierce), Monophosphoryl Lipid A (Ribi Immunochem Research), MPL+TDM (Ribi Immunochem Research), Titermax (CytRx), vitamin E acetate solubilisate, aluminum phosphate, aluminum oxide, toxins, toxoids, glycoproteins, lipids or oils, squalene, glycolipids, bacterial cell walls, subunits (bacterial or viral), carbohydrate moieties (mono-, di-, tri- tetra-, oligo- and polysaccharide) various liposome formulations or saponins Alum is the adjuvant currently in use for human patients. However, for horses, incomplete Freund's adjuvant may be used.
The term “immune response” refers to the reaction of the body to an antigen, which is usually a foreign or potentially dangerous substance (antigen), particularly disease-producing microorganisms. For example, in the current technology, the mutant EHV-1 virus would carry the antigen. The response involves the production by specialized white blood cells (lymphocytes) of proteins known as antibodies, which react with the antigens to render them harmless. The antibody-antigen reaction is highly specific. Vaccines such as the mutant EHV-1 also stimulate immune responses.
The term “immunologically effective amount” refers to the quantity of an immune response inducing substance required to induce the necessary immunological memory required for an effective vaccine. A vaccine is often given in multiple doses, an initial treatment and a subsequent booster treatment to enhance the immune response and to increase the strength and longevity of the immune memory response.
Typically, such vaccines are prepared to be administered in a sterile manner, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation also may be emulsified. The active immunogenic ingredient is often mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, adjuvants or immunopotentiators that enhance the effectiveness of the vaccine.
The vaccines are conventionally administered intraperitoneally, intramuscularly, intradermally, subcutaneously, orally, intranasally, or parenterally. Vaccines to be injected are typically formulated with pharmacologically acceptable carriers that are suitable for injection, including sterile aqueous solutions or dispersions. The carrier can be, for example, water, ethanol, glycerol, propylene glycol, sugars or other stabilizers, and isotonic saline solutions. Additional formulations are suitable for other modes of administration and include oral formulations. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Additionally, the peptide can be encapsulated in a sustained release formulations or a coating that resist the acidic pH of the stomach. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.
The dose to be administered depends on a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle, and a particular treatment regimen. The quantity to be administered, both according to number of treatments and amount, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and degree of protection desired. The precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are on the order of 104 to 107 PFU, more preferably 105 to 106 of active live virus per individual subject. Suitable regimes for initial administration and booster shots also vary but are typified by an initial administration followed in one or two week intervals by one or more subsequent injections or other administration. Annual boosters may be used for continued protection.
Materials and Methods
Cell culture and viruses. Mouse L-M, rabbit RK13, equine NBL-6, monkey Vero, and human HeLa cells used for viral propagation were maintained with Eagle's minimal medium supplemented with 100 units of penicillin/ml, 100 μg of streptomycin/ml, nonessential amino acids, and 5% (or 10%) fetal bovine serum. All cells were obtained from the American Type Culture Collection (ATCC, Manassas, Va.). All routine chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.) or Fisher Scientific Company (Houston, Tex.). The pathogenic RacL11 EHV-1 strain (RacL11) (from Dr. Nikolaus Osterrieder) was used as the parental virus in our studies (Ahn et al., 2007; Ahn et al., 2010; Breitenbach et al., 2009).
Construction of Plasmids.
PCR products were amplified using Accuprime pfx polymerase (Invitrogen, Carlsbad, Calif.), pRacL11 EHV-1 BAC (pRacL11) template, and appropriate primers. GalK BAC technology was used in order to construct the IR-deleted EHV-1 (Ahn et al., 2010; Warming et al., 2005). pRacL11 (Rudolph et al., 2002) was transformed into SW106 E. coli (Warming et al., 2005). The purified PCR product of the GalK marker harboring the EHV-1 IR flanking sequences (Primers, 5′ ccg ggc cat atc tgg tca agg gtc acg ggc ccg cgc ccg aga gag agc ctg gcc cct gtt gac aat taa tca tcg gca 3′ (SEQ ID NO:1)/5′ aca ccg tag tgg gtg agt gtg ggt ttt cca aac ata gct cga att cat tag ttc agc act gtc ctg ctc ctt 3′ (SEQ ID NO:2)) was transfected into SW106 cells containing pRacL11. Positive colonies were selected on Gal positive selection agar plates (Warming et al., 2005), and confirmed by PCR amplification (left flanking region primers, 5′ atg atc ccg cag tta cag cct aca aac tgg 3′ (SEQ ID NO:3)/5′ tag cac acc taa cct cct gag tgt gag cg 3′ (SEQ ID NO:4); right flanking region primers, 5′ agt tga tgg ata ggc gag cat ctc aaa caa g 3′(SEQ ID NO:5)/5′ tga aac atc tgc aac tgc gta aca aca gct tcg g 3′ (SEQ ID NO:6)) of EHV-1 IR flanking regions (named pL11ΔIR-GalK). Counter selection was performed in order to remove the GalK marker from the intermediate (Ahn et al., 2010; Warming et al., 2005). Both flanking regions of the IR were combined by multiple rounds of PCR amplification (left flanking region primers, 5′ tag cac acc taa cct cct gag tgt gag cg 3′ (SEQ ID NO:4)/5′ aga tgt ata tct gcc agg ctc tct ctc ggg cg 3′(SEQ ID NO:7); right flanking region primers, 5′ aga tat aca tct act aat gaa ttc gag cta tgt ttg g 3′(SEQ ID NO:8)/5′ ttc tct ttg gat ggt ata aga caa tcg tcg 3′(SEQ ID NO:9); combined flanking region primers, 5′ tag cac acc taa cct cct gag tgt gag cg 3′(SEQ ID NO:4)/5′ ttc tct ttg gat ggt ata aga caa tcg tcg 3′(SEQ ID NO:9)).
Purified PCR amplification products of the IR flanking region were transfected into SW106 cells containing pL11ΔIR-GalK, and positive colonies were selected on the Gal counter selection plates (Ahn et al., 2010; Warming et al., 2005). To generate the revertant virus recovering the entire IR sequence, plasmid (pAYC177-XbaII/B1: harboring the entire IR sequence and IR flanking sequences of the EHV-1 genome) (Ahn et al., 2007) was electroplated into SW106 cells (from Dr. Lindsey Hutt-Fletcher, Louisiana State University Health Sciences Center, Shreveport, La.) containing pL11ΔIR-GalK (named pL11ΔIRR), and positive colonies were selected on the Gal counter selection plates (Ahn et al., 2010; Warming et al., 2005). The identity of the resulting final BAC clone, named pL11ΔIR and pL11ΔIRR, was confirmed by PCR targeting the flanking sequences of the IR-deleted BAC (primers, 5′ aca cat tga gtc ctt tct act ctc ctc ctc gg 3′ (SEQ ID NO:10)/5′ ttc tct ttg gat ggt ata aga caa tcg tcg 3′(SEQ ID NO:9)) and the flanking region of the revertant clone in which the IR had been restored (primers, 5′ ccg ttt gaa tgc gat tgg tgg g 3′(SEQ ID NO:11)/5′ gcg ttg tat cta gca gcc cac g 3′(SEQ ID NO:12) and 5′ aga gta ggc gtt cca tcc acg 3′(SEQ ID NO:13)/5′ gac cct acc aaa ggc gtg tag g 3′(SEQ ID NO:14)). The deletion and restoration of the entire IR was ultimately verified by sequence analysis of amplified PCR amplicons, BamHI digestion, and Southern blot analysis.
Generation of Recombinant EHV-1 from Cloned BAC DNA and DNA Isolation Form Virus-Infected RK13 Cells.
Purified pL11ΔIR DNA or pL11ΔIRR DNA and a plasmid DNA containing the EHV-1 U
Viruses were propagated in RK13 or NBL-6 cells (ATCC, Manassas, Va.), and titered according to standard procedures (Perdue et al., 1974). The deletion or restoration of the entire IR in the respective viruses was confirmed by the PCR amplification of the IR-flanking regions using virus-infected RK13 cell DNA as a template and primers (5′ ttc tct ttg gat ggt ata aga caa tcg tcg 3′ (SEQ ID NO:9), 5′ aca cat tga gtc ctt tct act ctc ctc ctc gg 3′(SEQ ID NO:10)). DNA from EHV-1-infected RK13 cells was prepared by using DNAzol reagent (Molecular Research Center, Inc., Cincinnati, Ohio) according to the manufacturer's instructions, and PCR was used as a template.
Southern and Western Blot Analyses.
To confirm the insertion of the GalK marker into pRacL11, the removal of the GalK marker from pL11ΔIR-GalK, and the replacement of the GalK marker from pL11ΔIR-GalK with the entire IR sequences, BamHI digested pRacL11, pL11ΔIR-GalK, pL11ΔIR, and pL11ΔIRR were separated on a 0.8% agarose gel and transferred onto a positively charged nylon membrane (Ambion, Austin, Tex.) by using a semi-dry electroblotter (Bio-Rad Laboratories, Hercules, Calif.). After DNA transfer, the membrane was placed on blot paper saturated with 0.5M NaOH for 15 min, briefly washed with 2× saline sodium citrate buffer (SSC), and incubated at 80° C. for 1 h. The PCR amplicon of the GalK marker (primers, 5′ cct gtt gac aat taa tca tcg gca tag 3′ (SEQ ID NO:15)/5′ act gtc ctg ctc ctt gtg atg g 3′ (SEQ ID NO:16)) was end-labeled with [γ-32P]ATP (New England Nuclear Corporation, Boston, Mass.) and T4 polynucleotide kinase (Promega, Madison, Wis.) according to the manufacturer's directions. Radiolabeled probe was denatured by adding 1/10 volume of 3M NaOH, incubated for 10 min at room temperature, and then neutralized by adding an equal volume of 1M Tris-HCl (pH 7). Prehybridization, hybridization, and washing were performed using a NorthernMax Kit (Ambion, Austin, Tex.) followed by autoradiography using a phosphorimage screen and the molecular imager FX system (Bio-Rad Laboratories). For protein detection, RK13 cells were infected with parental RacL11 virus or vL11ΔIR at a multiplicity of infection (moi) of 5, and cells were harvested at 4, 6, and 12 hours post infection (hpi). Whole cell lysates of virus-infected cells were separated by dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a nitrocellulose membrane (Ambion) by using a semi-dry electroblotter (Bio-Rad Laboratories). The IE, early (E; IR4, EICP0, U
Plaque Morphology, Growth Kinetics, and Cell Tropism.
For the plaque assays, RK13 cell monolayers were infected with serial 10-fold dilutions of the respective viruses and overlaid with medium containing 1.5% methylcellulose at 2 hours after infection. At 4 days post infection (dpi), plaques were fixed with 10% formalin, stained with 0.5% crystal violet, and then counted (Perdue et al., 1974). Plaque sizes were measured by using the ImageJ software program (http://rebweb.nih.gov/ij/). For single step growth kinetics, RK13 cells in 25 mm flasks were infected at a moi of 0.2 with the respective viruses. After 1 h of viral attachment at 4° C., cells were washed with PBS, 4 ml of growth medium was added, and viruses were harvested at designated time points. To determine the intracellular viral titer, virus infected cells were washed with PBS followed by adding 4 ml of growth medium, and freeze and thaw cycle, and the virus was titered. To determine the extracellular viral titer, supernatants were used. To determine the cellular tropism, five cell types (L-M, RK13, NBL-6, Vero, and HeLa cells) were infected at a moi of 1 with mutant, revertant, or parental viruses. After virus attachment for 1 h at 4° C., the virus-infected cells were washed with PBS followed by adding normal growth medium, and then the total viral titers were examined at 3 dpi.
Quantitative Real Time (RT)-PCR.
To compare the number of viruses attached to the host cells, quantitative real time PCR assays were performed using the DNAs from virus infected cells as the template, the EHV-1 U
Animal Experiments.
Animal experiments were also conducted using published procedures (Ahn et al., 2010; Frampton et al., 2002; Osterrieder et al., 1996b; von Einem et al., 2004). Groups of 4-week-old CBA female mice (Harlan Laboratories, Indianapolis, Ind.) were inoculated intranasally with sterile medium (mock infection) or 1×106 pfu of vL11ΔIR, vL11ΔIRR or RacL11. Mice were observed daily and weighed from prior to inoculation, and the weights were compared. Virus isolation from the lungs of mice infected with vL11ΔIR, vL11ΔIRR, or RacL11 (n=3/group) at 2, 3, and 4 dpi for live mice and at the time of death for dead mice was performed by using silica beads and BeadBeater (BioSpec Products, Inc., Bartlesville, Okla.) according to the manufacturer's directions, and viral titers were then determined. For statistical analyses, two-tailed Student's-t test was performed by using the Excel software program (Microsoft Corporation, Redman, Wash.). Virulence as judged by percent survival data was determined by the Log-rank (Mantel-Cox) test using GraphPad Prism software (GraphPad Software, Inc., La Jolla, Calif.).
We deleted the entire IR of the EHV-1 genome using GalK technology as previously described (Ahn et al., 2010; Rudolph et al., 2002; Warming et al., 2005), and characterized vL11ΔIR reconstituted from the recombinant BAC in cell culture. As shown in
The removal of the entire IR also resulted in deletion of 631 bp of the U
PCR analyses indicated that the expected sizes of amplicons were observed from pL11ΔIR-GalK (
Deletion and recovery of the IR were further examined by BamHI digestion and Southern blot analyses.
The BamHI digestion pattern showed that an additional band of approximately 10 kp in size was observed in the case of pL11ΔIR-GalK (
To confirm that the pL11ΔIR-GalK harbored the GalK marker in the proper location, that pL11ΔIR lacks the GalK marker, and that the GalK marker from pL11ΔIR-GalK was replaced with the entire IR sequence, Southern blot analyses were performed using BamHI digested BAC DNAs (pRacL11, pL11ΔIR-GalK, pL11ΔIR, and pL11ΔIRR) and a radiolabeled GalK marker PCR fragment as the probe. These analyses showed that the GalK marker probe bound only to one fragment of the BamHI digested pL11ΔIR-GalK DNA (
Once the deletion and restoration of the entire IR were confirmed, the recombinant vL11ΔIR and vL11ΔIRR viruses were generated by co-transfection of pL11ΔIR (or pL11ΔIRR) DNA and a plasmid containing the EHV-1 U
Successful reconstitution of vL11ΔIR cloned DNA indicated that the IR deletion virus was replication competent, but plaque assays showed that the plaque areas of vL11ΔIR were significantly reduced compared to those of parental RacL11 and vL11ΔIRR (p<0.0001;
To exclude the possibility that the entire IR was restored by the TR segment during serial virus passage in RK13 cells, the IR flanking region of the vL11ΔIR genome was PCR-amplified by a primer set specific for the IR flanking sequences. Characterization of the vL11ΔIR genome and IE protein expression in cells infected with vL11ΔIRR is shown in
To address whether the IR sequences restored in the revertant virus were functionally similar to the parental virus with respect to gene expression, synthesis of the IEP was examined in the various viruses.
Even though the IR was not essential for EHV-1 replication, there remained the possibility that the cellular tropism of vL11ΔIR may differ from that of the parental virus. Recent studies had revealed that a mutant EHV-1 in which both copies of the IR4 gene were absent was capable of replication in equine NBL-6 cells, but, unlike its parent virus, was not capable of replication in mouse, rabbit, monkey, or human cells (Breitenbach et al., 2009). These observations suggested that the deletion of the entire IR may affect the biological properties of EHV-1.
Investigation of the cellular tropism and replication of EHV-1 showed that vL11ΔIR, like the parental RacL11, was capable of replicating in all five cell types tested, but the vL11ΔIR replicated with significantly reduced titers when compared with the parental virus and the revertant virus (vL11ΔIRR) in all cell types examined (all p values were <0.05;
The growth kinetics of vL11ΔIR was analyzed in RK13 cells by examining intracellular and extracellular viral titers at various times after infection. The results are shown in
To examine whether the delayed growth of vL11ΔIR was due to an impaired ability of the mutant virus in entry/penetration, cell-associated viral DNA was quantified by real time PCR after the parental virus, vL11ΔIR, and the revertant virus were incubated with RK cells. RK13 cells were infected with RacL11, vL11ΔIR and vL11ΔIRR at a moi of 10 followed by incubation at 4° C. for 2 h and at 37° C. for 30 min, and PBS washing. Total DNAs were extracted from virus infected RK13 cells, and the relative number of viral genomes was quantified as described in Example 1. The results are shown in
Deletion of the EHV-1˜13 kbp IR revealed that one repeat is dispensable for virus replication, suggesting that construction of such a deleted virus is also possible for related herpesviruses with a genome that can assume one of two isomeric conformations. In addition, such a deletion mutant may be employed to accommodate the insertion and expression of foreign gene(s) that total to at least 13 kbp. The findings that the vL11ΔIR showed reduced plaque size and delayed growth in RK13 cells clearly suggest that the deletion of sequences including the genes within the IR affects the biological properties of EHV-1 in cell culture.
The change of phenotype and the delayed growth kinetics of vL11ΔIR suggested that the deletion of the IR may affect viral gene regulation such that proteins encoded by IR genes would be decreased in cells infected with the IR deleted virus. Therefore, the protein expression of the IR and representative early (IR4, EICP0, and U
Experiments were carried out to determine if the deletion of the IR affected virulence in the well-characterized CBA mouse model of EHV-1 pathogenesis (Frampton et al., 2002; O'Callaghan and Osterrieder, 2008; Osterrieder et al., 1996b; Smith et al., 2005; von Einem et al., 2004).
CBA mice infected intranasally with RacL11, vL11ΔIR, or vL11ΔIRR showed clinical signs of huddling, ruffled fur, lethargy, and significant loss of body weight from 2 dpi, whereas mock-infected control mice continued to gain weight and showed no clinical signs throughout the observation period (
Mortality was observed in all groups of mice, and 100% (9 of 9), 11% (1 of 9), and 89% (8 of 9) of mice infected with parental EHV-1, IR-deleted virus, and IR-restored virus, respectively, succumbed to infection. Differences in the virulence among RacL11, vL11ΔIR, and vL11ΔIRR were examined by monitoring the percent of survival as shown in
The finding that the vL11ΔIR was less virulent than the parental virus as judged by overall mortality was attributed to the inability of this ΔIR mutant to replicate to high titers in the murine lung. Whereas the EHV-1 mutant virus lacking both copies of the IR4 gene was completely avirulent, we showed above that the ΔIR virus that harbors and expresses one copy of the IR4 gene and one copy of the IR6 gene, a known determinant of virulence (Osterrieder et al., 1996b), could replicate in the mouse respiratory system and elicit a fatal outcome in a small percentage of the animals.
Tischer, B. K., von Einem, J., Kaufer, B., Osterrieder, N. (2006). Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40(2), 191-7.
The complete disclosures of all references cited in this application are hereby incorporated by reference. Specifically incorporated into this application are the following two documents: (1) Provisional application Ser. No. 61/521,131 filed Aug. 8, 2011; and (2) B. Ahn, Y. Zhung, N. Osterrieder, and D. J. O'Callaghan; “Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region,” Virology, vol. 410, pp. 327-335 (2011), available online 21 Dec. 2010. In the event of an otherwise irreconcilable conflict, however, the present specification shall control.
The benefit of the filing date of provisional U.S. application Ser. No. 61/521,131, filed 8 Aug. 2011, is claimed under 35 U.S.C. §119(e).
This invention was made with government support under grant number AI-22001 awarded by the National Institute of Allergy and Infectious Diseases and under grant number P20-RR018724 awarded by the National Center for Research Resources of the National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
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61521131 | Aug 2011 | US |