- Open Access
Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization
© Shinoda et al; licensee BioMed Central Ltd. 2009
- Received: 10 February 2009
- Accepted: 03 March 2009
- Published: 03 March 2009
The licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun.
The L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus.
Modifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine.
- Signal Peptide Sequence
- Neutralize Antibody Response
- Mature Virion
- Smallpox Vaccine
- Variola Virus
Since the eradication of smallpox and the cessation of vaccination three decades ago, large segments of the population have become susceptible to infection with variola virus . This vulnerability coupled with fears of variola virus dissemination for nefarious purposes have led to a resurgence of interest in smallpox vaccination [2, 3]. The current smallpox vaccine consists of infectious vaccinia virus (VACV), which is closely related to variola virus, and provides complete and long lasting immunity . Nevertheless, the live vaccine can produce serious side effects particularly in individuals with an immunodeficiency or eczema . Consequently, alternative vaccination strategies including administration of attenuated strains of VACV, recombinant proteins and DNA are being evaluated .
Orthopoxviruses, including VACV and variola virus, have two major infectious forms known as the mature virion (MV) and the enveloped virion (EV) . The precursor MV membrane is formed at the initial stage of morphogenesis within specialized areas of the cytoplasm, whereas the EV membrane is derived from modified Golgi or endosomal membranes and encloses the MV . The EV membrane has a role in intracellular trafficking and extracellular spread, whereas the MV membrane fuses with the cell membrane to allow entry of the core into the cytoplasm [9, 10]. The viral protein compositions of the two membranes are entirely different and the most effective protein and DNA vaccines induce antibodies to components of both [11–14]. Several MV membrane proteins are known targets of neutralizing antibody: A27 [15, 16], A28 , D8 , H3 [19, 20] and L1 . Of these proteins, A27 [22–24], H3  and D8  are involved in virus attachment and A28  and L1  in membrane fusion and virus entry. The MV proteins do not traffic through the secretory pathway of the cell, creating obstacles to their isolation for protein vaccines and presentation for DNA vaccines.
The L1 protein lacks a signal peptide but is myristoylated at the N-terminus and has a C-terminal transmembrane domain . The ectodomain of L1 faces the cytoplasm in intracellular virions and contains three intramolecular disulfide bonds that are formed by VACV encoded redox system . A soluble, recombinant form of L1 was made by attaching a signal peptide to the N-terminus and removing the C-terminal transmembrane domain [13, 30]. When expressed in insect cells, the secreted protein was correctly folded and capable of inducing neutralizing antibody. Having shown that L1 could be engineered to traffic through the secretory pathway, we investigated a related approach to improve DNA vaccination. Modifications of the gene encoding L1 included codon optimization for mammalian expression, mutation of glycosylation sites since the viral protein is not glycosylated, addition of a signal peptide for traffic through the endoplasmic reticulum and Golgi apparatus to the plasma membrane, and the further truncation of the C-terminus to remove the transmembrane domain and allow secretion. As shown here, these modifications achieved the goal of increasing surface presentation and secretion and increased the production of neutralizing antibody in mice. Mice inoculated with plasmids expressing any of the recombinant L1 proteins partially protected mice against disease. The present work complements and extends recent reports of Golden and coworkers [31, 32] on immunization with an L1 gene that contains an added signal peptide.
Addition of a heterologous signal peptide sequence to L1 increases cell surface expression
Increased binding and neutralizing antibodies generated by addition of signal sequence
Neutralizing antibody was determined with a well-documented flow cytometry assay using a recombinant VACV that expresses green fluorescent protein . After the third immunization, the neutralizing antibody titer was more than three times higher in those mice that received plasmids expressing sL1op than L1op (Figure 3B). Thus, there was a correlation between increased surface expression and neutralizing antibody titer.
Expression and immunogenicity of C-terminal truncated forms of L1op and sL1op
Protection of mice by gene gun immunization with plasmids expressing recombinant L1
The proteins comprising the outer membranes of most viruses traffic through the secretory pathway of the cell where their extracellular domains are glycosylated and disulfide bonds form. Poxviruses are exceptional in that the MV membrane is formed within the cytoplasm and as a consequence the proteins are not normally glycosylated and a virus-encoded redox sytem is required to form disulfide bonds . As part of our laboratory's effort to produce a candidate protein subunit smallpox vaccine, we demonstrated that proper folding and disulfide bond formation of the L1 protein occurs in insect cells if a cleavable signal peptide is appended to the N-terminus and the C-terminal transmembrane domain is removed [13, 30]. That study demonstrated that the chaperones and redox enzymes in the endoplasmic reticulum of insect cells could substitute for the cytoplasmic viral enzymes. The purpose of the present study was to determine whether a similar strategy would enhance the presentation of L1 expressed by a DNA vaccine in mammalian cells and enhance immunogenicity in mice. At the time this project was initiated, gene gun immunization with the natural L1 gene had been shown to provide partial protection against challenge with VACV and more complete protection when combined with genes encoding other MV and EV membrane proteins [11, 12, 39].
We started with a synthetic L1 gene for two reasons. First, our studies (unpublished) and others  have shown that codon optimization for mammalian cells can sometimes enhance expression of poxvirus genes. Second, the recombinant L1 protein expressed in insect cells was unnaturally glycosylated . Consequently, the L1 gene was codon optimized and had the three glycosylation sites mutated. Other modifications included addition of an N-terminal signal peptide sequence with or without removal of the C-terminal transmembrane domain. In addition, we used a cytomegalovirus promoter that had been modified for expression in mice , since the goal was to test immunogenicity. The transfected synthetic L1op genes were expressed to even higher levels than in VACV-infected cells and the signal peptide sequence produced increased protein on the cell surface. Removal of the transmembrane domain allowed secretion.
Gene gun immunization was used to deliver the recombinant L1 genes as described by Hooper . The highest binding and neutralizing antibody responses were achieved with the proteins containing a signal peptide with or without the transmembrane domain. The differences were more substantial when measured at two weeks after the second and third immunizations compared to three weeks after the fourth immunization, when protection studies were carried out. The protection against weight loss induced by any of the recombinant L1 constructs was statistically significant when compared to empty vector plasmid or untreated mice. However, differences between the individual L1 constructs were not significant. Although antibody responses are important in protection against VACV infection, it is possible that CD8+ T-cells also contributed.
It is useful to compare our study with recent reports of Golden and coworkers [31, 32]. This group took a similar approach in attaching a signal peptide to the N-terminus of L1, which retained the transmembrane domain, and also found that this increased induction of neutralizing antibody following gene gun immunization. When challenged with 3 times the LD50 of VACV strain IHD-J, all mice died regardless of whether they were vaccinated with modified or unmodified L1. However, the signal peptide modified L1 appeared superior to unmodified L1 when combined with the genes encoding additional MV and EV membrane proteins. Such combinations have been shown by several groups to be important for protection against lethal orthopoxvirus infections of mice and monkeys [11–14, 39, 41–43].
Modifications of the VACV L1 gene, including codon optimization, attachment of a signal peptide sequence, and removal of the transmembrane domain can enhance expression and immunogenicity for DNA vaccination.
Cell cultures and viruses
BS-C-1 cells (ATCC CCL-26) were grown in modified Eagle's minimal essential medium (Quality Biologicals, Inc, Gaithersburg, MD) that was supplemented with 10% heat inactivated fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine (Invitrogen, Carlsbad, CA), 100 U/ml of penicillin and 100 μg/ml streptomycin sulfate (Invitrogen). HeLa S3 (ATCC CCL-2.2) suspension cultures were grown in spinner cell Eagle's Minimal Essential Medium (Quality Biologicals, Inc) with the addition of 5% heat-inactivated equine serum (Hyclone). Propagation and purification of VACV strain WR (ATTC VR-1354) has been described [44, 45].
Plasmids and transfection
The natural L1 gene sequence from VACV strain WR was modified by removal of three potential glycosylation sites, mammalian codon optimized and inserted into PCR-Script by GENEART (Regenburg, Germany). A set of modified L1 sequences encoding the murine Ig κ-chain leader sequence  and/or truncated after codon 185 were assembled by PCR with terminal PstI and NotI sites and inserted into the corresponding sites of pVRC8400 . For in vitro expression of L1, BS-C-1 cells were transfected with 1.5 μg of plasmid in 10 μl of Lipofectamine™ 2000 (Invitrogen) per well of a 6-well plate.
Twenty-four hours after transfection, cells were washed with phosphate buffered saline and suspended with NuPAGE® LDS Sample Buffer (Invitrogen) and sonicated. The lysates were heated at 70°C for 10 min with or without NuPAGE® Sample Reducing Agent (Invitrogen) and the proteins were resolved by SDS-PAGE in NuPAGE® Bis-Tris gels (Invitrogen). Following transfer to a polyvinylidene difluoride membrane using iBlot PVDF Transfer Stack (Invitrogen), the membrane was incubated with rabbit polyclonal anti-L1 antibody (R180, provided by G. Cohen and R. Eisenberg, University of Pennsylvania) followed by anti-rabbit IgG conjugated to horseradish peroxidase. Bands were visualized with a chemiluminescence detection kit (Pierce, Rockford, IL).
Unfixed cells were incubated with anti L1 MAb 7D11  provided by Alan Schmaljohn followed by anti-mouse IgG conjugated to fluorescein isothiocyanate and analyzed by confocal microscopy as described .
Twenty-four hours after plasmid transfection, cells were washed with phosphate buffered saline and cell suspension made with versene EDTA chelating agent (Invitrogen). The non-permeabilized cells were incubated with MAb 7D11 followed by anti-mouse IgG antibody conjugated to fluorescein isothiocyanate, fixed with 2% paraformaldehyde and analyzed with a FACSCalibur flow cytometer using CellQuest (BD Biosciences) and FlowJo Software (Tree Star, Inc, Ashland, OR).
Gene gun immunization of mice
Seven weeks old female BALB/c mice were transfected with plasmids by Helios gene gun delivery (BIO-RAD, Hercules, CA). Individual cartridges were prepared with approximately 1 μg of plasmid and 0.5 mg gold particles. Briefly, plasmid DNA, spermidine, CaCl2 and 2 micron gold particles (DeGussa, Parsippany, NY) were mixed and washed with ethanol. The mixture were suspended in ethanol and dried onto Tefzel tubing (BIO-RAD). DNA-coated gold particles were delivered with a Helios Gene Gun at 400 pounds per square inch to three non-overlapping sites on the shaved abdomen.
Antibody binding assay
Antibody binding to purified L1 and VACV particles was carried out by ELISA  with some modifications. The 96-well plates (Immulon HB plate, Thermolab System, Hertfordshire, UK) were coated with 100 μl/well of affinity-purified L1 protein (600 ng/ml of phosphate buffered saline) and incubated ~24 h at 4°C. Following incubation with diluted sera followed by anti-mouse IgG-peroxidase (Roche, Branchburg, NJ), the plates were reacted with BM Blue substrate (Roche). The plates were read at wavelengths 370 nm and 492 nm using SpectraMax M5 Microplate Reader and SoftMaxPro Software System (Molecular Devices, Sunnyvale, CA). The endpoint was 0.1 absorbance unit after subtraction of the background absorbance of serum incubated in wells without protein.
Purified VACV expressing enhanced green fluorescent protein  was incubated with diluted serum in a 96-well plate for 1 h at 2.5 × 104 plaque forming units/well. HeLa S3 cells were treated with cytosine arabinoside for 10–15 min and then 1 × 105 cells were added to each well and the plates incubated for 16–18 h in a 37°C CO2 incubator. Incubated cells were fixed with 2% paraformaldehyde in phosphate buffered saline and analyzed on a FACSCalibur flow cytometer using CellQuest and FlowJo Software. IC50 values were calculated using PRISM software (GraphPad, La Jolla, CA)
p-value was determined by t-test using PRISM software (GraphPad).
The excellent assistance of Norman Cooper and Catherine Cotter in preparation of cells and purification of VACV is appreciated. Gary Nabel, Vaccine Research Center, NIAID, NIH generously provided the plasmid VRC8400 and Gary Cohen and Roselyn Eisenberg, University of Pennsylvania provided antiserum to the L1 protein. We thank Wolfgang Leitner, Dermatology Branch/NCI/NIH for supplying the gold particles used in the gene gun experiments. This study was supported by the Division of Intramural Research, NIAID, NIH.
- Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID: Smallpox and its eradication. first edition. Geneva: World Health Organization; 1988.Google Scholar
- Henderson DA, Inglesby TV, Bartlett JG, Ascher MS, Eitzen E, Jahrling PB, Hauer J, Layton M, McDade J, Osterholm MT, et al.: Smallpox as a biological weapon – Medical and public health management. JAMA-J Amer Med Assoc 1999, 281: 2127-2137. 10.1001/jama.281.22.2127View ArticleGoogle Scholar
- Lane JM, Goldstein J: Evaluation of 21st-century risks of smallpox vaccination and policy options. Ann Int Med 2003, 138: 488-493.View ArticlePubMedGoogle Scholar
- Fulginiti VA, Papier A, Lane JM, Neff JM, Henderson DA: Smallpox vaccination: A review, part I. Background, vaccination technique, normal vaccination and revaccination, and expected normal reactions. Clin Inf Dis 2003, 37: 241-250. 10.1086/375824View ArticleGoogle Scholar
- Fulginiti VA, Papier A, Lane JM, Neff JM, Henderson DA: Smallpox vaccination: A review, part II. Adverse events. Clin Inf Dis 2003, 37: 251-271. 10.1086/375825View ArticleGoogle Scholar
- Wiser I, Balicer RD, Cohen D: An update on smallpox vaccine candidates and their role in bioterrorism related vaccination strategies. Vaccine 2007, 25: 976-984. 10.1016/j.vaccine.2006.09.046View ArticlePubMedGoogle Scholar
- Moss B: Poxviridae: the viruses and their replication. In Fields Virology. Volume 2. Edited by: Knipe DM, Howley PM. Philadelphia: Lippincott Williams & Wilkins; 2007:2905-2946.Google Scholar
- Condit RC, Moussatche N, Traktman P: In a nutshell: structure and assembly of the vaccinia virion. Adv Virus Res 2006, 66: 31-124. 10.1016/S0065-3527(06)66002-8View ArticlePubMedGoogle Scholar
- Moss B: Poxvirus entry and membrane fusion. Virology 2006, 344: 48-54. 10.1016/j.virol.2005.09.037View ArticlePubMedGoogle Scholar
- Ward BM: The longest micron; transporting poxviruses out of the cell. Cellular Microbiology 2005, 7: 1531-1538. 10.1111/j.1462-5822.2005.00614.xView ArticlePubMedGoogle Scholar
- Hooper JW, Custer DM, Thompson E: Four-gene-combination DNA vaccine protects mice against a lethal vaccinia virus challenge and elicits appropriate antibody responses in nonhuman primates. Virology 2003, 306: 181-195. 10.1016/S0042-6822(02)00038-7View ArticlePubMedGoogle Scholar
- Hooper JW, Custer DM, Schmaljohn CS, Schmaljohn AL: DNA vaccination with vaccinia virus L1R and A33R genes protects mice against a lethal poxvirus challenge. Virology 2000, 266: 329-339. 10.1006/viro.1999.0096View ArticlePubMedGoogle Scholar
- Fogg C, Lustig S, Whitbeck JC, Eisenberg RJ, Cohen GH, Moss B: Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions. J Virol 2004, 78: 10230-10237. 10.1128/JVI.78.19.10230-10237.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Lustig S, Fogg C, Whitbeck JC, Eisenberg RJ, Cohen GH, Moss B: Combinations of polyclonal or monoclonal antibodies to proteins of the outer membranes of the two infectious forms of vaccinia virus protect mice against a lethal respiratory challenge. J Virol 2005, 79: 13454-13462. 10.1128/JVI.79.21.13454-13462.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Lai CF, Gong SC, Esteban M: The purified 14-kilodalton envelope protein of vaccinia virus produced in Escherichia coli induces virus immunity in animals. J Virol 1991, 65: 5631-5635.PubMed CentralPubMedGoogle Scholar
- Demkowicz WE, Maa JS, Esteban M: Identification and characterization of vaccinia virus genes encoding proteins that are highly antigenic in animals and are immunodominant in vaccinated humans. J Virol 1992, 66: 386-398.PubMed CentralPubMedGoogle Scholar
- Nelson GE, Sisler JR, Chandran D, Moss B: Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies. Virology 2008, 380: 394-401. 10.1016/j.virol.2008.08.009PubMed CentralView ArticlePubMedGoogle Scholar
- Sakhatskyy P, Wang SX, Chou THW, Lu S: Immunogenicity and protection efficacy of monovalent and polyvalent poxvirus vaccines that include the D8 antigen. Virology 2006, 355: 164-174. 10.1016/j.virol.2006.07.017View ArticlePubMedGoogle Scholar
- Lin CL, Chung CS, Heine HG, Chang W: Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo. J Virol 2000, 74: 3353-3365. 10.1128/JVI.74.7.3353-3365.2000PubMed CentralView ArticlePubMedGoogle Scholar
- Davies DH, McCausland MM, Valdez C, Huynh D, Hernandez JE, Mu YX, Hirst S, Villarreal L, Felgner PL, Crotty S: Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice. J Virol 2005, 79: 11724-11733. 10.1128/JVI.79.18.11724-11733.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Wolffe EJ, Vijaya S, Moss B: A myristylated membrane protein encoded by the vaccinia virus L1R open reading frame is the target of potent neutralizing monoclonal antibodies. Virology 1995, 211: 53-63. 10.1006/viro.1995.1378View ArticlePubMedGoogle Scholar
- Chung C-S, Hsiao J-C, Chang Y-S, Chang W: A27L protein mediates vaccinia virus interaction with cell surface heparin sulfate. J Virol 1998, 72: 1577-1585.PubMed CentralPubMedGoogle Scholar
- Hsiao JC, Chung CS, Chang W: Cell surface proteoglycans are necessary for A27L protein- mediated cell fusion: Identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain. J Virol 1998, 72: 8374-8379.PubMed CentralPubMedGoogle Scholar
- Vazquez MI, Esteban M: Identification of functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus. J Virol 1999, 73: 9098-9109.PubMed CentralPubMedGoogle Scholar
- Hsiao JC, Chung CS, Chang W: Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells. J Virol 1999, 73: 8750-8761.PubMed CentralPubMedGoogle Scholar
- Senkevich TG, Ward BM, Moss B: Vaccinia virus entry into cells is dependent on a virion surface protein encoded by the A28L gene. J Virol 2004, 78: 2357-2366. 10.1128/JVI.78.5.2357-2366.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Bisht H, Weisberg AS, Moss B: Vaccinia virus L1 protein is required for cell entry and membrane fusion. J Virol 2008, 82: 8687-8694. 10.1128/JVI.00852-08PubMed CentralView ArticlePubMedGoogle Scholar
- Franke CA, Wilson EM, Hruby MD: Use of a cell-free system to identify the vaccinia virus L1R gene product as the major late myristylated virion protein M25. J Virol 1990, 64: 5988-5996.PubMed CentralPubMedGoogle Scholar
- Senkevich TG, White CL, Koonin EV, Moss B: Complete pathway for protein disulfide bond formation encoded by poxviruses. Proc Natl Acad Sci USA 2002, 99: 6667-6672. 10.1073/pnas.062163799PubMed CentralView ArticlePubMedGoogle Scholar
- Aldaz-Carroll L, Whitbeck JC, Ponce de Leon M, Lou H, Pannell LK, Lebowitz J, Fogg C, White C, Moss B, Cohen GH, Eisenberg RJ: Physical and immunological characterization of a recombinant secreted form of the membrane protein encoded by the vaccinia virus L1R gene. Virology 2005, 341: 59-71. 10.1016/j.virol.2005.07.006View ArticlePubMedGoogle Scholar
- Hooper JW, Golden JW, Ferro AM, King AD: Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge. Vaccine 2007, 25: 1814-1823. 10.1016/j.vaccine.2006.11.017View ArticlePubMedGoogle Scholar
- Golden JW, Joselyn MD, Hooper JW: Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies. Vaccine 2008, 26: 3507-3515. 10.1016/j.vaccine.2008.04.017View ArticlePubMedGoogle Scholar
- Barouch DH, Yang ZY, Kong WP, Korioth-Schmitz B, Sumida SM, Truitt DM, Kishko MG, Arthur JC, Miura A, Mascola JR, et al.: A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates. J Virol 2005, 79: 8828-8834. 10.1128/JVI.79.14.8828-8834.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Su HP, Golden JW, Gittis AG, Hooper JW, Garboczi DN: Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein. Virology 2007, 368: 331-341. 10.1016/j.virol.2007.06.042PubMed CentralView ArticlePubMedGoogle Scholar
- Earl PL, Americo JL, Moss B: Development and use of a vaccinia virus neutralization assay based on flow cytometric detection of green fluorescent protein. J Virol 2003, 77: 10684-10688. 10.1128/JVI.77.19.10684-10688.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Turner GS: Respiratory infection of mice with vaccinia virus. J Gen Virol 1967, 1: 399-402. 10.1099/0022-1317-1-3-399View ArticlePubMedGoogle Scholar
- Law M, Putz MM, Smith GL: An investigation of the therapeutic value of vaccinia-immune IgG in a mouse pneumonia model. J Gen Virol 2005, 86: 991-1000. 10.1099/vir.0.80660-0View ArticlePubMedGoogle Scholar
- Hayasaka D, Ennis FA, Terajima M: Pathogeneses of respiratory infections with virulent and attenuated vaccinia viruses. Virol J 2007, 4: 22. 10.1186/1743-422X-4-22PubMed CentralView ArticlePubMedGoogle Scholar
- Hooper JW, Thompson E, Wilhelmsen C, Zimmerman M, Ichou MA, Steffen SE, Schmaljohn CS, Schmaljohn AL, Jahrling PB: Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox. J Virol 2004, 78: 4433-4443. 10.1128/JVI.78.9.4433-4443.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Barrett JW, Sun YM, Nazarian SH, Belsito TA, Brunetti CR, McFadden G: Optimization of codon usage of poxvirus genes allows for improved transient expression in mammalian cells. Virus Genes 2006, 33: 15-26. 10.1007/s11262-005-0035-7View ArticlePubMedGoogle Scholar
- Fogg CN, Americo JL, Lustig S, Huggins JW, Smith SK, Damon I, Resch W, Earl PL, Klinman DM, Moss B: Adjuvant-enhanced antibody responses to recombinant proteins correlates with protection of mice and monkeys to orthopoxvirus challenges. Vaccine 2007, 25: 2787-2799. 10.1016/j.vaccine.2006.12.037PubMed CentralView ArticlePubMedGoogle Scholar
- Xiao YH, Aldaz-Carroll L, Ortiz AM, Whitbeck JC, Alexander E, Lou H, Davis HL, Braciale TJ, Eisenberg RJ, Cohen GH, Isaacs SN: A protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost. Vaccine 2007, 25: 1214-1224. 10.1016/j.vaccine.2006.10.009PubMed CentralView ArticlePubMedGoogle Scholar
- Berhanu A, Wilson RL, Kirkwood-Watts DL, King DS, Warren TK, Lund SA, Brown LL, Krupkin AK, Vandermay E, Weimers W, et al.: Vaccination of BALB/c mice with Escherichia coli-expressed vaccinia virus proteins A27L, B5R, and D8L protects mice from lethal vaccinia virus challenge. J Virol 2008, 82: 3517-3529. 10.1128/JVI.01854-07PubMed CentralView ArticlePubMedGoogle Scholar
- Earl PL, Moss B: Characterization of recombinant vaccinia viruses and their products. In Current Protocols in Molecular Biology. Volume 2. Edited by: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. New York: Greene Publishing Associates & Wiley Interscience; 1998:16.18.11-16.18.11.Google Scholar
- Earl PL, Cooper N, Wyatt LS, Moss B, Carroll MW: Preparation of cell cultures and vaccinia virus stocks. In Current Protocols in Molecular Biology. Volume 2. Edited by: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. New York: John Wiley and Sons; 1998:16.16.11-16.16.13.Google Scholar
- Coloma MJ, Hastings A, Wims LA, Morrison SL: Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. J Immunol Meth 1992, 152: 89-104. 10.1016/0022-1759(92)90092-8View ArticleGoogle Scholar
- Husain M, Weisberg A, Moss B: Topology of epitope-tagged F13L protein, a major membrane component of extracellular vaccinia virions. Virology 2003, 308: 233-242. 10.1016/S0042-6822(03)00063-1View ArticlePubMedGoogle Scholar
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