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The Journal of Immunology, 1999, 163: 6834-6840.
Copyright © 1999 by The American Association of Immunologists

Development of Adenovirus Vectors Encoding Rat Complement Regulators for Use in Therapy in Rodent Models of Inflammatory Diseases1

Yvonne McGrath*, Gavin W. G. Wilkinson{dagger}, O. Brad Spiller* and B. Paul Morgan2,*

Departments of * Medical Biochemistry and {dagger} Medicine, University of Wales College of Medicine, Cardiff, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
C activation has been implicated in the pathogenesis of numerous inflammatory human diseases and disease models. A therapy based on C inhibition might therefore be of benefit to reduce inflammation and ameliorate disease. C inhibition in vivo can be accomplished by the delivery of soluble recombinant C regulators either systemically or directly to a target site, but effects are transitory. We have developed a strategy for the efficient delivery of the membrane-bound rat C inhibitors, CD59, Crry, and decay-accelerating factor (DAF), using replication-deficient adenovirus vectors with the intention of treating rat models of disease in which C is implicated. The adenovirus recombinants(RAd), RAdCD59, RAdCrry, and RAdDAF, respectively, have been tested for expression and function of the transgene in vitro. Infection of human fetal foreskin fibroblasts resulted in high levels of expression of each of the rat inhibitors. The constructs were also tested for inhibition of rat C-mediated cell lysis and C3b deposition. In a cell lysis assay, each inhibited to varying degrees of efficiency in the order RAdCD59 = RAdDAF > RAdCrry. In a C3b deposition assay, RAdDAF caused a greater reduction in C3b deposition than RAdCrry and RAdCD59 was ineffective. These agents, individually or in combination, provide the tools for testing the effects of prolonged inhibition of C at a target site on the progress of experimental models of disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The C system provides an efficient means of killing microorganisms and is involved in the clearance of immune complexes (1, 2). Where activation is excessive or occurs at an inappropriate site, however, C activation may cause injury to self-cells. Human diseases in which C activation has been shown to have a pathogenic role include, among others, adult respiratory distress syndrome, ischemic reperfusion injury, myocardial infarction, rheumatoid arthritis, and diseases of the nervous system such as multiple sclerosis, myasthenia gravis, and Alzheimer’s disease (3). Although C activation may not necessarily be the primary event in these diseases, it can exacerbate and perpetuate the inflammatory response both through direct cytotoxicity and through triggering the release of anaphylotoxins, cytokines, reactive oxygen metabolites, eicosanoids, and other inflammatory mediators (4). In healthy individuals, the activation of C on self-cells is tightly controlled by a number of naturally occurring endogenous C regulators. These regulators have been cloned and characterized and thus provide the potential for a therapy based on C inhibition (5). C activation in animal models of disease is similar to that in the human disease. Animal models can therefore provide a means to test the efficacy of C inhibition in inflammatory disease.

A soluble form of human C receptor 1 has been used to treat successfully numerous models of disease including ischemia reperfusion injury, demyelination, myasthenia gravis, autoimmune neuritis, and Ag-induced arthritis (6, 7, 8, 9, 10, 11, 12, 13). Human proteins, however, have several disadvantages when used in rodents; activity is reduced, half-life in plasma is shortened, and treatment is limited to ~1 week due to the production of neutralizing Abs (9). To better study C regulatory proteins as therapeutics in rat models, we have cloned and characterized the rat analogues of CD59, decay-accelerating factor (DAF),3 and membrane cofactor protein (MCP) (14, 15, 16). CD59 is a glycosylphosphatidylinositol (GPI)-anchored molecule, which acts late in the C cascade by inhibiting the assembly of the membrane attack complex (17). In contrast, DAF and MCP act during the activation stages to inhibit the C3 and C5 convertases, either by accelerating their decay (DAF) or by acting as a cofactor for the cleavage of C3b (MCP) (18). Another molecule, known as Crry, has been identified in rodents which expresses functions of both DAF and MCP (19, 20). Rat Crry is widely expressed and assumed to be the major regulator of C activation in this species (21, 22). Transgenic mice have been engineered to express a soluble recombinant form of mouse Crry to a high level, and this expression has been shown to confer protection from Ab-induced glomerular injury (23). Furthermore, the delivery of a soluble form of mouse Crry fused with Ig-1 to mice prevented the onset of Ab-induced glomerulonephritis (24).

Here we describe the development of recombinant adenovirus (RAd) constructs encoding the rat analogues of DAF, CD59, and Crry as a means of sustained delivery of C-regulatory proteins in rat models. These constructs have been designed to express the C regulators in their native GPI-anchored (DAF and CD59) or transmembrane (Crry) forms from the powerful CMV major immediate early promoter. High levels of surface expression have been obtained in infected fibroblasts with concomitant protection from rat C. Successful delivery of a transgene to rat synovium was demonstrated by intraarticular injection of an adenovirus recombinant encoding Escherichia coli ß-galactosidase (ß-gal).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and cDNAs

Mouse anti-rat CD59 mAb (6D1) and mouse anti-rat DAF mAb (RDIII-7) were raised in house and are described elsewhere (17, 25). 6D1 and RDIII7 Abs do not cross-react with their human counterparts. Crry polyclonal antiserum was raised in rabbits by standard techniques. Anti-rat Crry mAb TLD1C11 was a gift of Dr. W. F. Hickey (Dartmouth College, Hanover, NH). Mouse anti-human C3c mAb (C3/30; cross-reactive with rat C3c) was a gift of Dr. Peter Taylor (Norvartis, Horsham, U.K.). To sensitize human cells to C attack, antiserum to a subclone of the U937 cell line, which does not express CD59, was raised in rabbits (26). The serum was tested by immunoblotting and found not to cross-react with rat C inhibitors. Antiserum was incubated at 56°C for 30 min to inactivate C. Anti-human CD59 (Bric-229) was purchased from the International Blood Group Reference Laboratory (Bristol, U.K.). Anti-human DAF (1H4) was a gift from Dr. Wendell Rosse (Duke University, Durham, NC). The mAb to human factor H, OX23, was derived from a hybridoma obtained from the European Collection of Animal Cell Cultures (Porton Down, U.K.). Goat anti-mouse FITC conjugate and goat anti-rabbit PE conjugate were both obtained from Sigma (Poole, U.K.), goat anti-mouse PE was from Dako (Ely, Buckinghamshire, U.K.), and goat anti-mouse HRP conjugate was from Bio-Rad (Hemel Hempstead, Hertfordshire, U.K.).

Rat CD59 cDNA was obtained from Dr. N. Rushmere, rat DAF cDNA was from Dr. S. Hinchliffe (both at University of Wales College of Medicine, Cardiff, U.K.), and rat Crry cDNA was supplied by Dr. R. J. Quigg (University of Chicago, IL).

Reagents and animals

Cell lysates for Western blot analysis were prepared with Townsend lysis buffer (0.5% Nonidet P-40, 15 mM nananoyl-N-methylglucamide, 150 mM NaCl, 50 mM Tris (pH 7.5), and 0.05 mM EDTA) to which iodoacetamide and PMSF were added to final concentrations of 5 and 2 mM, respectively, immediately before use. Hanks’ medium was prepared with 10% HBSS, 10 mM HEPES, 0.15% NaHCO3, 10 U/ml penicillin, 10 mg/ml streptomycin, 100 ng/ml gentamicin, 20 ng/ml kanamycin, 5 mg/ml Fungizone (all Life Technologies, Paisley, U.K.). PBS and Veronal-buffered saline (VBS) were from Oxoid (Basingstoke, U.K.). All other reagents were from Sigma or Fisher Scientific (Loughborough, U.K.) unless otherwise stated. PVG-C rats were obtained from Banting and Kingman (Hull, U.K.). Rat blood was obtained from PVG-C rats by exsanguination under anesthesia, and serum was separated and stored immediately after preparation at -70°C. The control virus RAd35, which encodes the E. coli LacZ gene, was developed as described (27).

Cell lines and culture conditions

Viruses were propagated in monolayers of human embryonic kidney 293 cells transformed with the Ad5 early region E1. The 293 cells were originally developed and supplied by Dr. F. Graham (28) and are available from the European Collection of Animal Cell Cultures. The human fetal foreskin fibroblast primary cell line (HFFFs) was used in passages 8 to 20 and was from Dr. G. Farrar at the Centre for Applied Microbiology and Research (Salisbury, U.K.). Both cell lines were grown in MEM supplemented with streptomycin (0.1 mg/ml), penicillin (100 U/ml), L-glutamine (2 mM), and 10% FCS (all supplied by Life Technologies with the exception of MEM which was supplied by Sigma).

Generation of RAd vectors

The RAd vectors were generated by the method of McGrory et al. (29). Briefly, the gene of interest was inserted into a transfer vector downstream of the CMV major immediate early promoter and upstream of a polyadenylation signal. The transfer vector was then cotransfected with pJM17 by calcium phosphate transfection into 293 cells (30). The plasmid pJM17 contains the complete Ad5 dl 309 genome with a prokaryotic vector pBRX DNA insertion, rendering it too large to be packaged into Ad nucleocapsids. After cotransfection, the transfer vector replaces the prokaryotic vector DNA and the E1 portion of the genome by homologous recombination and thereby renders it replication deficient but sufficiently small to be packaged into viable viral particles. Viral replication is made possible by the provision of the E1 function in trans by the 293 cells.

RAd were plaque purified, grown to high titer in 293 cells as described previously (31), and extracted using an equal volume of Arklone P (Basic Chemical Company, High Wycombe, U.K.). The virus was further purified by cesium chloride gradient ultracentrifugation and dialysis against 1 mM MgCl2, 135 mM NaCl, 10 mM Tris-HCl (pH 7.8), 10% glycerol (31). Titers were determined by standard plaque assay in 293 cells (32). Three viruses were generated: RAdCD59 which encodes GPI anchored rat CD59; RAdDAF which encodes GPI-anchored rat DAF; and RAdCrry encoding the seven SCR (short consensus repeat) form of transmembrane rat Crry. The fidelity of the C regulator sequences in each of the viruses was confirmed by sequencing.

Testing expression in HFFFs by immunofluorescence

HFFFs were grown to 80% confluence on coverslips, and infections were set up in a minimum amount of medium. A crude Arklone P extract of the third plaque purification was used for immunofluorescence microscopy to test for expression of the C regulator before large scale production of the virus. Cells were infected for 4 h at 37°C at which time the medium was replaced. After a further incubation for 16 h at 37°C, the cells were washed in PBS, dried, and fixed in methanol-acetone (1:1) for 30 s. Cells were incubated with 10 µg/ml CD59 mAb 6D1, Crry mAb TLD1C11, or DAF mAb RDIII7 for 30 min at 37°C, washed in PBS, and detected by incubating with 10 µg/ml goat anti-mouse FITC conjugate at 37°C for 30 min. Cells were mounted in Dabco (Sigma, 2% in PBS-glycerol, 1:10) and viewed with a Leica fluorescence microscope for evidence of expression.

Testing for expression in HFFFs by Western blot analysis

HFFFs were infected in 35-mm dishes at a multiplicity of infection (MOI or number of PFU per cell) of 100 as described above. Four hours postinfection (p.i.) the cells were washed in PBS and overlaid with serum-free medium. Cell lysates were prepared day 3 p.i. by resuspending cells in 100 µl Townsend lysis buffer, vortexing, and incubating on ice for 15 min. Cell lysates were stored at -70°C in nonreducing SDS-PAGE buffer (Bio-Rad) until use. Uninfected cells were used to detect nonspecific binding of either the primary or secondary Abs. RAdCD59-infected cell lysates were run on a 15% SDS-PAGE nonreducing gel and RAdDAF- and RAdCrry-infected cell lysates on a 10% nonreducing gel before blotting onto nitrocellulose membranes (Sartorius, Göttingen, Germany). After transfer, blots were blocked in 5% nonfat milk powder in PBS for 30 min at room temperature and then probed with CD59 mAb 6D1, Crry mAb TLD1C11, or DAF mAb RDIII7 at 1 µg/ml for 1 h at 4°C. Blots were washed in PBS, 0.1% Tween 20 and incubated for 1 h at 4°C with 1 µg/ml goat anti-mouse HRP conjugate. Development was with the ECL detection kit (Amersham, Little Chalfont, U.K.) according to the manufacturer’s instructions.

Flow cytometric analysis and PIPLC cleavage of the GPI anchor

Cells were infected in six-well trays at 80% confluency at MOIs of 50 and 500 in a minimum amount of medium. Medium was changed after 4 h, and cells were incubated for a further 3 days. Cells were detached with flow cytometry solution (15 mM EDTA, 30 mM NaN3, 1% BSA in PBS), and protein expression was detected by incubating 105 cells with 5 µg/ml primary Ab for 30 min at 4°C. CD59 mAb 6D1, Crry polyclonal antiserum and DAF mAb RDIII7 were used to detect CD59, Crry, and DAF, respectively. For all three viruses, a control Ab, OX23, was used to quantify nonspecific Ab binding due to adenovirus infection. The cells were washed in flow cytometry solution, and the primary Ab was detected with a goat anti-mouse or anti-rabbit PE conjugate at 4°C for 30 min. The cells were washed, the signal detected on a Becton Dickinson FACScalibur flow cytometer (Heidelberg, Germany) and analyzed using Cell Quest software (Becton Dickinson). To assess the integrity of the CD59 and DAF GPI anchor, the ability of phosphatidylinositol-specific phospholipase C (PIPLC, Roche, Lewes, U.K.) to release the protein from the membrane was assessed. Cells (105) infected at an MOI of 100 with RAdCD59, RAdCrry, or RAdDAF were either treated with PIPLC at 1/100 for 45 min at 37°C or incubated without PIPLC. Cells were then washed and stained for FACS analysis as described above.

C3 deposition assays

Pilot studies were used to determine optimum serum and sensitizing Ab concentrations for maximum C3 deposition with minimum cell lysis on uninfected cells. HFFFs were infected with RAd either singly or in combination at MOIs of 100, 200, and 300 or left uninfected. On day 3 p.i., cells were left unsensitized and were incubated with a 1/5 dilution of rat serum in VBS for 30 min at 37°C. Negative controls were performed using VBS, heat-inactivated serum, or serum containing 10 mM EDTA. The cells were washed in PBS and detached in flow cytometry solution. C3 deposition was detected with anti-C3c mAb (C3/30 ascites at 1/500) by flow cytometry as described.

Results were analyzed by one-way ANOVA. Posttests were done on relevant groups of data and Bonferroni corrected for multiple comparisons.

Cell lysis assays

HFFFs in 24-well plates were infected at MOIs of 100, 200, and 300 with RAdCD59, RAdCrry, and RAdDAF either singly or in combination or left uninfected. On Day 3 p.i., the cells were loaded with calcein-AM (Molecular Probes, Eugene, OR) at 2 µg/ml in MEM medium (including supplements) at 37°C for 1 h. The medium was aspirated off, and the cells were sensitized with 100 µl/ml of rabbit anti-U937 serum in Hanks’ medium for 15 min at 37°C. During sensitization, human CD59 and DAF were blocked on the HFFFs using 10 µg/ml of Bric-229 and 1H4. Blocking and sensitization had previously been shown to increase rat serum-induced lysis. Cells were washed and exposed to C attack with rat serum diluted to 1/5 in VBS. Supernatant was removed completely after 30 min at 37°C, and the remaining unlysed cells were lysed by addition of 0.1% Triton X-100 in water. The lysate was saved, and calcein was measured in the supernatant and lysate on a Wellfluor fluorometer (excitation filter wavelength, 485 nm; emission filter wavelength, 530 nm). Percentage of cell lysis was calculated as [calcein release by serum/(calcein release by serum + calcein release by detergent)] x 100.

Results were analyzed by one-way ANOVA. Posttests were done on relevant groups of data and Bonferroni corrected for multiple comparisons.

In vivo delivery of RAd to rat synovium

PVG-C rats were used for in vivo delivery experiments. Rats were anesthetized, and 3.5 x 108 PFU of RAd35 were injected intraarticularily into the right hind knee joint. Control animals were mock-injected. On day 3 postinjection, the rats were sacrificed, and the patella and surrounding tissue were dissected off both the experimental and control knees. The tissue was frozen in CRYO-M-BED (Bright Instrument, Huntingdon, Cambridgeshire, U.K.), and 12-µm sections were cut on a cryostat (Bright Instrument). Sections were fixed in 0.5% glutaraldehyde and stained in 3 mM potassium ferrocyanide, 3 mM potassium ferricyanide, 1.3 mM MgCl2, and 130 µg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactoside for 2 h at room temperature before viewing.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of rat C inhibitors on human fibroblasts

Expression of rat CD59, Crry, and DAF on HFFFs infected with the relevant recombinant virus was first demonstrated by immunofluorescence microscopy (Fig. 1Go). As is typical for adenovirus expression in human fibroblasts, the expression was heterogeneous, with some cells showing much higher expression than others. This heterogeneity can be accounted for by the dependence of the CMV promoter strength on the cell cycle stage of the host cell. Background binding on uninfected cells for all three Abs was negligible; a typical example is shown in Fig. 1GoD. Western blot analysis was used to confirm that the recombinant proteins were of the correct size as compared with the native protein expressed on rat erythrocytes (Fig. 2Go). Rat CD59 in the RAdCD59-infected cells comigrated with the major erythrocyte CD59 band at ~18–20 kDa as expected (Fig. 2GoA). Rat DAF in the RAdDAF-infected cells ran as a band of ~65 kDa, which correlates well with that seen in the rat erythrocyte lane (Fig. 2GoC). A higher m.w. species of ~120 kDa was also seen in the lanes positive for DAF, which represents DAF dimers. Rat Crry in the RAdCrry-infected cells ran as a single band of 65–68 kDa, whereas rat erythrocytes contained bands of 55 and 65 kDa, corresponding to the 6 SCR and 7 SCR forms of Crry found on rat erythrocytes (Fig. 2GoB) (20, 22). All uninfected control lysates were consistently negative. By flow cytometry, it was apparent that each of the regulators was expressed at a high level on infected cells (Fig. 3Go). The level of expression was proportional to an increasing MOI up to an MOI of 500. Fig. 3Go shows representative traces at a MOI of 50 and 500 to illustrate this point. At a MOI of 30, almost 100% of the cells expressed the transgene, and the dose-dependent increase in expression above this MOI is likely due to multiple infection of individual cells. No further increase in expression could be demonstrated above MOIs of 500 for any of the recombinants. Because the Ab was not limiting, this ceiling on expression was probably due to the saturation of the expression machinery of the cells. Indeed at the highest MOIs, we observed virus-induced cytotoxicity, manifesting as holes in the cell monolayer. In vitro expression levels were followed for 7 days by flow cytometry. Expression levels peaked at day 3 but remained high at day 7 p.i. (data not shown). No increase in binding of the control mAb OX23 was seen in any infection at any MOI (results not shown).



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  		FIGURE 1. Immunofluorescence microscopy demonstrating the expression of rat C inhibitors. HFFFs were infected with RAdCD59 (A), RAdCrry (B), and RAd DAF (C), and transgene expression was detected with the primary mAbs 6D1, TLD1C11, and RDIII7, respectively. Secondary detection was with goat anti-mouse FITC conjugate. Negative controls were performed by staining uninfected cells. The uninfected cells shown in D are stained with 6D1 and represent a typical negative result.

 


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  		FIGURE 2. Size comparison of recombinant rat C inhibitors to endogenous by Western blot analysis. HFFFs were infected with RAdCD59 (A), RAdCrry (B), and RAdDAF (C). Cell lysates were prepared 3 days p.i. and run alongside rat erythrocyte ghosts as positive controls. Cell lysates of uninfected cells were run as negative controls. Detection of CD59, Crry, and DAF was with the primary mAbs 6D1, TLD1C11, and RDIII7, respectively. Goat anti-mouse HRP conjugate was used as the secondary Ab.

 


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  		FIGURE 3. Flow cytometric analyses of expression of C inhibitors on infected human fibroblasts. Cells were infected with RAdCD59 (A), RAdDAF (B), and RAdCrry (C) at MOIs of 50 (hatched line) and 500 (solid line) or left uninfected (filled) and stained for the relevant Ag. Detection of CD59 and DAF was with the primary mAbs 6D1 and RDIII7, respectively. Crry was detected with monospecific polyclonal antiserum. Secondary detection was with a goat anti-mouse or anti-rabbit PE conjugate. FL, fluorescence.

 
Release of CD59 and DAF by PIPLC demonstrates the integrity of the anchor

A 69% reduction in mean cell fluorescence was demonstrated on treatment of RAdCD59-infected cells with PIPLC (Fig. 4GoA). Similarly, DAF showed a 57% reduction in mean cell fluorescence after PIPLC treatment (Fig. 4GoB). In contrast, the mean cell fluorescence for Crry actually increased by 71% after PIPLC treatment.



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  		FIGURE 4. Treatment of infected HFFF with PIPLC. Cells infected with a MOI of 100 of RAdCD59 (A), RAdDAF (B), or RAdCrry (C) were either treated with PIPLC (solid line) or left untreated (filled). Cells were stained for the relevant Ag and analyzed as described in legend to Fig. 3Go. FL, fluorescence.

 
Expression of rat C inhibitors by RAd infection inhibits C3 deposition on human fibroblasts

RAdCrry significantly inhibited C3 deposition on human fibroblasts in a dose-dependent manner at MOIs 100, 200, and 300 over uninfected controls (p < 0.01, p < 0.01, and p < 0.001, respectively) (Table IGo). Similarly for RAdDAF, significant and dose-dependent inhibition of C3 deposition was seen at all three MOIs tested (p < 0.001; Table IGo). At each of the MOIs, RAdDAF was consistently better at inhibiting C3 deposition than RAdCrry. C3 deposition on cells infected with a combination of both RAdDAF and RAdCrry at an MOI of 100 each was less than on cells infected with either RAdCrry or RAdDAF alone at a MOI of 100. Although this was significant (p < 0.01) only for comparison with RAdCrry, these data indicate that the viruses can act additively. Protection conferred by infection by the combination of two viruses was marginally greater than that conferred by double the dose of either virus alone, but the differences were not statistically significant. RAdCD59, as expected, did not inhibit C3 deposition on human fibroblasts at any MOI. Background C3 deposition obtained with either heat-treated serum or serum containing 10 mM EDTA was comparable with that of VBS only controls.


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  		Table I. Reduction in C3 deposition on human fibroblasts by expression of rat C inhibitors using recombinant adenovirus vectors1

 
Expression of rat C inhibitors by RAd infection protects human fibroblasts from rat C-mediated lysis

Over a range of MOIs, each of the adenoviruses conferred protection on human fibroblasts from rat C lysis (Fig. 5Go). RAdDAF and RAdCD59 at MOIs of 100 and 300 conferred significant protection on infected cells when compared with control virus (p < 0.001 for each comparison). RAdCrry, although less effective, also offered a significant degree of protection over control virus (p < 0.05 and p < 0.01 for MOI of 100 and 300). RAdCD59 and RAdDAF gave significantly better protection than RAdCrry (p < 0.01 for RAdCD59 vs RAdCrry and p < 0.001 for RAdDAF vs RAdCrry at a MOI of 100). There was no significant difference between RAdDAF and RAdCD59 at any of the MOIs tested. RAd35, at an MOI of 30, conferred a small but significant degree of protection from lysis over uninfected controls (p = 0.02).



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  		FIGURE 5. Protection of HFFF from rat C-mediated lysis with RAd. Fibroblasts were infected in triplicate with RAdCD59, RAdCrry, RAdDAF, or RAd35 at MOIs of 100, 200, and 300. On day 3 p.i., the cells were Ab sensitized and exposed to rat C. Experiments were done in triplicate, and results are shown as mean ± SD of triplicate measurements from a typical experiment.

 
Double infection of human fibroblasts with combinations of virus confers increased protection

We wished to examine whether pairs of regulators inhibiting C at the same or different stages expressed on the same cell could act synergistically to protect from lysis. Double infection of a HFFF cell population was confirmed by infecting cells singly with virus at a MOI of 200, or doubly, with each virus at a MOI of 200 and comparing expression levels by flow cytometry. At this MOI, all cells expressed and expression levels of each individual protein in dual-infected cell populations were equal to those obtained in single infected populations (data not shown). These data demonstrate dual infection of single cells with each adenovirus. Comparison of single infections with each virus at a MOI of 200 with double infections each at a MOI of 200 demonstrated that, in all cases, the combination of two inhibitors protected better than either alone (Fig. 6Go). To better assess whether any combination of two inhibitors acted synergistically, cells infected with each single recombinant at a MOI of 400 were compared with cells infected with pairs of recombinants, each at a MOI of 200 (Fig. 6Go). In each combination, protection was better than that conferred by double the dose of either virus alone. Even combinations including the least effective virus, RAdCrry, conferred better protection than twice the dose of RAdCD59 or RAdDAF. The differences were small and impossible to assess statistically because of the nature of the comparisons. Nevertheless, the results do suggest that combining viruses to express multiple C regulators will further enhance protection of cells.



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  		FIGURE 6. Effect of expression of combinations of rat C regulators. Fibroblasts were infected in triplicate either with a single virus, RAdCD59, RAdCrry, or RAdDAF each at a MOI of 400, or with a combination of two viruses each at an MOI of 200. At day 3 p.i., the cells were Ab sensitized and exposed to rat C. Experiments were done in triplicate, and results are shown as mean ± SD of triplicate measurements from a typical experiment.

 
RAd delivery of ß-gal to rat synovium via RAd35

Sections of synovium from rat knees injected with RAd35 showed abundant areas of blue stain, corresponding to infected cells (data not shown). Sections from the concomitant control knee were negative.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The potential for utilizing C regulators in therapy is now well recognized, in large part because of the successes obtained in animal models with agents such as soluble human C receptor 1. Animal studies to date have been limited to acute situations, because of the short half-life and immunogenic nature of human C regulators in rodents (12). To develop therapies for chronic conditions such as rheumatoid arthritis, C inhibition will need to be sustained over long periods in vivo. Using the rodent C regulators should overcome the problem of immunogenicity in models, enabling the testing in vivo of long term therapy with C regulators. A second problem is that the repeated systemic administration of C-regulatory proteins will be technically difficult and prohibitively expensive and might render the individual more susceptible to infection. A possible solution to these problems is to utilize gene therapy to deliver the cDNA encoding the C inhibitor directly to the relevant tissue with the use of a viral vector. This means of delivery should result in the expression at the site of sustained levels of protein over weeks or months (33, 34, 35). We have chosen RAd as a vehicle for delivery because of its capacity to infect both replicating and quiescent cells to a high level in vivo (36). Expression of the rat C regulators in this system should permit sustained, local C inhibition in relevant rat models of disease. Roessler et al. (37) have demonstrated the expression of ß-gal in rabbit knee joints after intraarticular injection of a RAd encoding ß-gal for >8 wk. There is to our knowledge only one previous study in which adenovirus vectors have been used to express C regulators. Charreau et al. (38) generated an adenovirus recombinant encoding human CD59 and demonstrated expression of human CD59 on rat endothelial cells infected with the virus.

Here we describe the generation of adenovirus recombinants expressing rat CD59, rat DAF, and rat Crry. Infection of human fetal fibroblasts with each of these viruses caused high levels of membrane expression of the corresponding C regulator. Expression was dependent on the dose of adenovirus used. The expressed proteins were similar in size to the endogenous regulators on rat erythrocytes. The GPI-anchored proteins, CD59 and DAF, were efficiently cleaved from expressing cells by treatment with PIPLC, indicating that the anchor signal was correctly processed. Transmembrane Crry was not released by PIPLC but instead showed an apparent increase in expression, likely due to increased accessibility of the protein to Ab after PIPLC release of other membrane proteins.

Each of the expressed proteins conferred protection against attack by rat C. In C lysis assays, RAdCD59 and RAdDAF were equally effective, and each provided human fibroblasts with better protection than did RAdCrry. This was surprising, given the known importance of Crry in protection against C attack in vivo (39, 40, 41). This result may be attributable to the expression system in that RAdDAF and RAdCD59 might promote higher levels of expression of their transgene than RAdCrry. Indeed, at very high MOIs of RAdCrry, inhibition of lysis did reach the levels achieved with RadCD59 and RAdDAF (data not shown). The differences may also relate to the lytic assay used, in which C activation was achieved through the classical pathway. It is possible that rat DAF functions better in the classical pathway whereas rat Crry favors the alternative pathway. Although this possibility has not been formally tested in rats, in humans, MCP, which has many features in common with Crry, functions only in the alternative pathway (42, 43). Infection with either RAdDAF or RAdCrry inhibited C3b deposition, the former again being more active in the assay used. RAdCD59, acting on the terminal pathway had no effect on C3b deposition, as expected. Of interest, the control ß-gal virus (RAd35) caused a small but significant inhibition of lysis in infected cells. It has previously been shown that infection with adenovirus recombinants encoding either ß-gal or human CD59 caused activation of rat endothelial cells with 2-fold up-regulation of MHC class I and endogenous CD59 (38). We examined the effects of infection with each virus on expression of endogenous C regulators on HFFFs. Infection with RAd35 caused a 50% increase in expression of CD59 and DAF and a small increase in expression of MCP (data not included), providing an explanation for the observed increased resistance to lysis. Infection with the other viruses used in this study resulted in a small increase in expression of endogenous DAF but did not alter expression of CD59 or MCP. These increases were seen across the cell population and not as a shift in a particular subset of cells. None of the viruses tested had an effect on the levels of MHC class I expression.

HFFFs cotransfected with two recombinant viruses encoding different regulators expressed each regulator at levels similar to those obtained following transfection with a single virus. Cotransfection demonstrated a modest degree of synergy in that some combinations provided better protection from lysis than twice the dose of either virus alone. Statistical analysis was not possible in these studies, but trends toward synergy were apparent for all recombinant virus combinations. These data suggest that combination therapies with regulators blocking the C cascade at different stages or through different mechanisms can offer better protection than any single inhibitor. The implication for vector use in vivo is that coadministration of two or three recombinant viruses encoding different C regulators may be much more effective than the administration of even high doses of one. Indeed, multiple C regulators could be delivered via a single polycistronic vector, which would reduce the virus dose required and potentially minimize virus immunogenicity.

Adenovirus vectors are able to infect and express at high level in many different cell types, including cells recalcitrant to transfection with other expression vectors. The recombinant viruses described here thus provide useful tools for examining the effects of C inhibitors on these difficult cells. For example, we have preliminary evidence that these adenovirus vectors infect rodent brain cells and induce abundant expression of C regulators on neurones and glia. For use in therapy, either cells can be infected ex vivo and injected into the target site or the adenovirus can be injected directly into the target site to infect cells in situ. We have chosen initially to express the C regulators as membrane-anchored molecules. Only direct injection of virus will thus be effective in obtaining protection of cells in the target site. Adenovirus vectors encoding soluble forms of the C regulators offer an alternative approach to inhibiting C activation at a target site; these could be administered either directly or after cell infection ex vivo.

Our primary target for therapy with the adenovirus vectors is inflammatory joint disease. A role for C has been clearly demonstrated in human arthritis and in animal models (44, 45, 46, 47, 48). Both systemic and local therapy with C regulators has been shown to inhibit disease in several rodent models of arthritis (13, 49, 50). Adenovirus recombinants are particularly suited to therapy of arthritis because of the ease of access to the joint space. In a recent study, adenovirus vector encoding a modified form of the TNF-{alpha} receptor was delivered directly into the rat joint and was effective in suppressing collagen-induced arthritis (51). Our preliminary studies utilizing the RAd35 vector expressing ß-gal demonstrate that sustained, high level expression of the gene can be attained in synovium by this approach without inducing inflammation. Studies of the efficacy of the C regulator-encoding adenoviruses administered intraarticularly in Ag arthritis are in progress.


   Footnotes
 
1 This work was supported by the Arthritis Research Council and The Wellcome Trust. Y.M. is the holder of an Arthritis Research Council Studentship. Back

2 Address correspondence and reprint requests to Dr. B. Paul Morgan, Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XX, U.K. E-mail address: Back

3 Abbreviations used in this paper: DAF, decay-accelerating factor; GPI, glycosylphosphatidylinositol; PIPLC, phosphatidylinositol-specific phospholipase C; MCF, mean cell fluorescence; MCP, membrane cofactor protein; p.i., postinfection; MOI, multiplicity of infection; SCR, short consensus repeat; VBS, Veronal-buffered saline; RAd, recombinant adenovirus; HFFF, human fetal foreskin fibroblast; ß-gal, ß-galactosidase. Back

Received for publication July 22, 1999. Accepted for publication October 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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