Viral Oncology Unit, Division of Medicine, ICSM at St Marys Hospital1, BS-ICSM at St Marys Hospital2, Norfolk Place, London W2 1PG, UK
Division of Life Sciences, FranklinWilkins Building, Kings College London, 150 Stamford Street, London SE1 8WA, UK3
Author for correspondence: Wilson Caparrós-Wanderley. Fax +44 20 7402 1037. e-mail w.caparros-wanderley{at}ic.ac.uk
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Abstract |
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Introduction |
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There is a large body of literature on potential DNA delivery vehicles, most of which are based on live virus vectors, and their use in gene therapy (for reviews see Alemany et al., 2000 ; Clayman, 2000
; Lesch, 1999
; Verma & Somia, 1997
). However, less work appears to have been carried out to assess the immune response induced by these vehicles and any resulting inherent potential drawbacks for their use, particularly in elderly and/or immunocompromised individuals. In order to assess the immunological properties of VP1 VLPs with regard to their use as a gene therapy vehicle, we have analysed both the transfection efficiency of VLPDNA complexes in vitro and the immune response induced to both vehicle (VP1 VLPs) and transgene product (
-gal) following intranasal delivery of these complexes to mice.
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Methods |
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The pCMV- vector (7·2 kbp) (Clontech) is a mammalian expression vector which contains the
-gal gene cloned downstream from the CMV immediate early promoter. The pc-EGFP vector (6·2 kbp) (kindly provided by R. K. Afghan) is a mammalian expression vector derived from pcDNA3.1(+) (Invitrogen) which contains the enhanced green fluorescent protein (EGFP) gene cloned downstream from the CMV immediate early promoter. All plasmid DNAs were purified from recombinant E. coli cultures using QIAGEN plasmid midi kits.
Production and purification of polyomavirus VP1 VLPs.
A 500 ml Sf9 culture growing in a 2 l stirrer flask, at a cell density of 1·21·6x106 cells/ml, was infected with a VP1 recombinant baculovirus (Forstová et al., 1995 ) at an m.o.i. of 10. After 3 h incubation at 25 °C, 500 ml of fresh complete medium was added and incubation continued for 72 h at 25 °C. At the end of this period, cells were harvested by centrifugation (1500 g, 4 °C, 10 min) and the cell pellet was resuspended in 10 ml of PBS and frozen at -70 °C for later processing.
For purification of VP1 VLPs, frozen Sf9 cell pellets were thawed and lysed by sonicating in ice seven times for 10 s with 10 s cooling intervals, also in ice. The resulting lysate was cleared by centrifugation in a microcentrifuge (9000 r.p.m., 10 min, 4 °C) and the supernatant loaded onto a two step 2060% sucrose gradient in PBS. Following centrifugation in a SW41Ti rotor (Beckman) (217000 g, 90 min, 4 °C), the 2060% interphase was harvested, diluted 1:5 with PBS and CsCl added to the solution (0·41739 g CsCl/g of solution). After centrifugation to equilibrium in a SW41Ti rotor (217000 g, 40 h, 25 °C), fractions (0·650 ml) were harvested from the top and analysed for the presence of VP1 by SDSPAGE. Fractions containing empty VP1 VLPs, as determined by VLP density (1·29 g/ml), were pooled and dialysed overnight against PBS at 4 °C.
Preparation of VLPDNA complexes and in vitro transfection.
COS-7 cells were seeded in 6-well plates (Nunc) at a density of 2x105 cells per well and incubated overnight. Transfection mixtures containing VLP/pCMV- DNA were prepared by adding 58, 29, 14·5, 2·9, 0·58, 0·29 and 0·145 µg of pCMV-
DNA to 10 µg of VP1 VLPs (DNA/VLP molar ratios of 20:1, 10:1, 5:1, 1:1, 1:5, 1:10 and 1:20 respectively) in a final volume of 105 µl. After 10 min incubation at room temperature, 0·4 ml of serum-free DMEM was added to the mixture. VLP/pc-EGFP DNA mixtures were prepared as described above, but using only DNA/VLP molar ratios of 20:1, 1:1 and 1:20. DNA-only solutions were prepared by diluting the same amounts of pCMV-
or pc-EGFP DNA with PBS to 105 µl. Liposomal transfection controls were prepared with 2·9 µg of either pCMV-
or pc-EGFP DNA and DOTAP (Boehringer Mannheim) according to the manufacturers instructions.
For transfection, the COS-7 cell monolayers were washed once with serum-free medium and overlaid with the VLP/DNA mixtures. After 90 min incubation at 37 °C with occasional rocking, DMEM with FCS was added to a final FCS concentration of 10%. Transfections with DOTAP were carried out according to the manufacturers instructions. Following 48 h incubation at 37 °C, cells were washed twice with PBS, fixed with 0·1% glutaraldehyde in PBS, washed twice with PBS and finally overlaid with the chromogenic substrate [5 µM K-ferricyanide (Sigma), 5 µM K-ferrocyanide (Sigma), 2 µM MgCl2 and 400 µg/ml X-Gal (Promega)]. The total number of blue cells in each well, after 24 h incubation in the dark at room temperature, was established by visual examination under the microscope. All transfection experiments were carried out in triplicate and statistical analysis (t test) was carried out on the data sets.
Immunizations.
Six-week-old female barrier-reared BALB/c mice (Harlan Laboratories) were bled prior to intranasal dosing by tail snipping under local anaesthetic and tested by ELISA for previous exposure to polyomavirus VP1. ELISA-negative animals were divided into four groups, with each animal being immunized by intranasal delivery under anaesthesia with either PBS, 50 µg of VP1 VLPs, 50 µg VP1 VLPs+3 µg pCMV- or 3 µg pCMV-
, all in 30 µl volumes. On day 14, all animals were boosted with the same antigens and on day 28 they were killed by cardiac puncture and their spleens removed. Genital lavages were obtained from all animals before culling by rinsing the vaginal cavity twice with 50 µl PBS. Each experimental group, with the exception of the PBS group, for which n=2, contained four animals. Experiments were carried out in duplicate.
Cell proliferation analysis.
Mouse spleens were gently pressed through cell strainers and red blood cells removed by treatment with red cell lysis buffer (9 parts 0·16 M NH4Cl and 1 part 0·17 M Tris, pH 7·2). Splenocyte suspensions from each experimental group were prepared at a density of 2x106 cells/ml containing either no antigen, phytohaemagglutinin (PHA) (1 and 10 µg/ml), lysozyme (3 and 20 µg/ml), VP1 VLP (1, 3 and 20 µg/ml), recombinant -gal type VIII (Sigma) (3 and 20 µg/ml), pCMV-
DNA (1 and 3 µg/ml) or pc-EGFP DNA (1 and 3 µg/ml). These splenocyte suspensions were seeded in quadruplicate in 96-well plates (Nunc) at a density of 2x105 cells per well. After 4 days incubation at 37 °C, 0·25 µCi of [3H]thymidine (Amersham-Pharmacia) was added to each well and plates were incubated for 6 h at 37 °C. Proliferation levels were measured for each experimental group in a 3H counter. The results obtained for each group from the duplicated experiments were pooled and statistical analysis (t test) was carried out on the data sets.
ELISA.
Maxisorp 96-well plates (Nunc) were coated overnight at 4 °C with 100 µl per well of either 5 µg/ml VP1 VLPs or 1 µg/ml recombinant -gal type VIII (Sigma), both in PBS. After removing the antigen solution, wells were coated with 200 µl of 2% milk powder in PBS. After 2 h at room temperature, the blocking solution was removed and triplicate wells were overlaid with 100 µl of decreasing tripling dilutions of the different experimental sera (starting at dilution 1/60) and vaginal (starting at dilution 1/15) samples. Following 1 h incubation at room temperature, plates were washed four times with PBSTween 20 (0·1%) and overlaid with 100 µl of either rabbit anti-mouse HRP-conjugated sera (Dako) or goat anti-mouse IgA HRP-conjugated ascites fluid (Sigma). After 1 h incubation at room temperature, plates were washed four times with PBS/Tween 20 (0·1%) and overlaid with 100 µl of substrate solution [0·1 mg/ml 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) in 0·05 M phosphatecitrate buffer pH 5·0 containing a 1/4000 dilution of 30% H2O2]. The reaction was terminated by adding 50 µl of stopping solution (50% dimethylformamide, 20% SDS) and the absorbance of each well at 405 nm was determined.
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Results |
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Splenocyte suspensions from each experimental group were stimulated with either PHA (1 and 10 µg/ml), lysozyme, -gal (3 and 20 µg/ml), VP1 VLP (1, 3 and 20 µg/ml), pCMV-
DNA or pc-EGFP DNA (1 and 3 µg/ml). Stimulation indices achieved by each experimental group against the tested antigens are shown in Fig. 2
. In order to increase the stringency of these assays the positive response threshold was set at a stimulation index (SI) of 3. The average non-stimulated counts (background proliferation) of all groups in the two independent experiments carried out was 272±26 c.p.m.
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For stimulation with -gal, only animals immunized with either VLPDNA complexes or DNA alone achieved SI values above the activation threshold of 3. These SI values were found to titrate, with both groups achieving their maximum SI at the highest concentration of
-gal used. A major difference between these groups was that animals immunized with VLPDNA reached a statistically significant (P<0·05, t test) higher maximum SI (4·36±0·30) than those immunized with DNA only (3·24±0·13). This corresponds to a 35% increase in the
-gal-specific response.
For stimulation with pCMV- or pc-EGFP DNA, no experimental group achieved SI values above the activation threshold of 3. The data indicate that our choice of antigen concentration was low enough so as not to induce non-specific proliferation of B-cells caused by unmethylated CpG motifs in the bacterial plasmid DNA (Holt, 1995
). In addition, they also strongly suggest that DNA concentrations flanking that at which maximum COS-7 transfection is achieved in vitro (1·45 µg/ml, which corresponds to 0·58 µg of DNA) are not necessarily those at which maximum transfection of splenocytes, and by deduction other cell types, is achieved.
Regarding the humoral immune response to VP1 and -gal, pre-dosing sera from all animals were found to be negative to these antigens by ELISA (data not shown). Following intranasal delivery, ELISA assays for total serum antibody and mucosal IgA to these antigens gave the data presented in Fig. 3
. All animals immunized with either VLPDNA complexes or VLP alone developed serum antibodies against VP1. The titre of these antibodies, despite the differences observed in the proliferative responses to VP1 among the experimental groups, was similar. Our data do not discriminate between antibody isotype, hence we cannot specify whether a differential degree of response at the IgM and IgG levels is masked by the similarity in the antibody titre. Anti-VP1 IgA antibodies were also found in the vaginal lavages, but again no differences in titre were observed between the VLPDNA complex or VLP alone groups. This IgA response clearly shows that intranasal delivery of polyomavirus VP1 VLPs can, as shown for human papillomavirus (HPV) VLPs (Balmelli et al., 1998
), induce mucosal antibody responses. No antibodies to
-gal were detected in the sera of animals immunized with VLPDNA complexes or DNA alone.
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Discussion |
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Short-term (48 h) in vitro transfection efficiency analysis of VP1 VLPDNA complexes and DNA alone showed that both systems were significantly less efficient at transfecting COS-7 monolayers than commercially available liposomal systems (DOTAP). Overall, the transfection efficiency of VLPDNA complexes appeared to be lower than that of DNA alone, but these differences, with the exception of those observed for DNA/VLP ratios of 1:20 and 20:1, were not statistically significant. Our data indicate that murine polyomavirus VP1 VLPDNA complexes are poor short-term in vitro transfection reagents. This observation contrasts with other reports indicating that VLPs of human polyomavirus JC (Goldmann et al., 1999 ) and HPV16 (Touze & Coursaget, 1998
) can efficiently deliver DNA to cells in vitro. In these reports, however, DNA was encapsidated into the VLPs by a process of assembly and disassembly, whilst in our case this route is not very efficient and hence DNA was simply mixed with the VLPs. This is apparently an important difference since one report (Touze & Coursaget, 1998
) clearly indicates, whilst another suggests (Ou et al., 1999
), that mixing DNA and VLPs results in poor short-term in vitro transfection. Despite this, our earlier findings (Soeda et al., 1998
; Krauzewicz et al., 2000a
) suggest that DNA introduced by VP1 VLPs remains in the cell for longer than DNA delivered via a liposomal route. As established by electron microscopy (our unpublished observations), not all supercoiled plasmid DNA added to the VLPs appears associated with the particles; a significant proportion remains free in the preparations. Therefore, it is possible that the observed level of short-term in vitro transfection may reflect primarily transfection by free DNA.
The peak of transfection efficiency observed at a DNA/VLP ratio of 1:5 is based on a short-term in vitro experiment using COS-7 cells. This observation, whilst not evidence for such a ratio being the most appropriate for the efficient transfection of mouse tissues in vivo, does provide a guide. Consequently, this ratio was used to dose mice intranasally with the aim of studying the immune response to both components of the VP1 VLPDNA complexes. Our data show that murine polyomavirus VP1 VLPs are very immunogenic, inducing high cellular proliferation and antibody titres both in serum and mucosal surfaces (IgA). This observation differs with those regarding human JC polyomavirus VLPs, which fail to activate dendritic cells (Lenz et al., 2001 ) and to induce an immune response (Goldmann et al., 1999
). We show that intranasal immunization with VLPDNA complexes induces a lower proliferative response to VP1 than immunization with VP1 alone, but these differences are not observed in the antibody response. Differences were observed also in the immune response to
-gal. Animals dosed with VLPDNA had higher proliferative responses to
-gal than animals dosed with DNA alone. Despite this difference in the proliferative response, no serum or mucosal antibody responses were detected against
-gal. Our data offer no explanation for the differential degree of proliferation to VP1 in the VLPDNA and VLP-only groups. However, the increased proliferation against
-gal of the VLPDNA group compared to the DNA-only group clearly suggests that the VP1 VLPs are acting as physically attached adjuvants in the induction of immunity. The lack of a detectable antibody response to
-gal may reflect the lag period existing between delivery of the plasmid and transgene expression and the short time between first immunization and terminal bleeding (28 days). Alternatively, it may also reflect lower levels of transgene expression and ultimately low amounts of the protein being released from the cells, an event found to be required for the development of transgene-specific immunity following DNA immunization (Ulmer et al., 1996
).
Despite a higher in vitro transfection efficiency, we were unable to compare the immune response to -gal induced following immunization with pCMV-
combined with DOTAP, since an effective preparation of the liposomal solution for the amount of DNA required could not be prepared in the 30 µl volume allowed for intranasal immunization.
Considered together, our earlier and current data suggest a possible explanation for our observations. After intranasal delivery, the plasmid DNA, either delivered on its own or present free in the VLPDNA preparation, as well as some VLPDNA complexes, would be taken up by the cells of the nasal mucosa, lung epithelium or by immune cells present in these tissues. Following transgene expression and antigen processing and presentation, an immune response to -gal would be induced. In the case of the animals dosed with VLPDNA complexes, the anti-
-gal response would benefit from the high immunogenicity and likely adjuvant properties of the VP1 VLPs, resulting in the higher SI observed. It is proposed that a proportion of the VLPDNA complexes not processed immediately may, in contrast to DNA delivered on its own, reach the bloodstream, where they would travel for a period of time to other organs, infecting other cell types or being processed by immune cells. This would result in a continuous stimulation of the immune system through sustained low level presentation of antigen at different sites delivered by a highly immunogenic VLP, probably also acting as an adjuvant. This first initial boost, followed by the continuous low level presentation of the antigen, may be responsible for the increased proliferative response we observe against
-gal. As suggested earlier, the lack of detectable antibodies to
-gal may be due to low in vivo transfection efficiency, the lag period existing before transgene expression and/or low level of transgene expression and extracellular release.
If this postulate is not correct and instead all VLPDNA complexes are taken up and processed at the same time, VP1 may simply act as an adjuvant in the development of the immune response to -gal. Indeed VLPs of other human viruses have been reported to induce strong immune responses (Kirnbauer et al., 1992
; Rose et al., 1994
). This option, however, does not explain the reported longer-term expression of the transgene product observed when the DNA is delivered associated with VP1 VLPs, rather than on its own (Soeda et al., 1998
; Krauzewicz et al., 2000a
). Furthermore, although VP1 VLPs may well act as an adjuvant, it is difficult to reconcile an adjuvant-only idea with the lack of a detectable antibody response to
-gal, since in this case a much greater number of cells would be expected to be expressing the antigen.
Neither of the above hypotheses can explain why the proliferative response to VP1 is lower in animals immunized with VLPDNA complexes than in those that received VLPs alone. Similarly, our present data cannot account for the lack of an anti--gal antibody response. Further long-term experiments are required to address these questions.
In summary, our data show that murine polyomavirus VP1 VLPDNA complexes are poor short-term in vitro transfection reagents and, as currently used with a wild-type VP1 sequence, too immunogenic for repeated use as gene therapy vehicles. A potential solution to this problem would be to use less immunogenic mutants of VP1, an alternative currently being explored. Finally, the high immunogenicity of these VLPs and VLPDNA complexes suggests that they could be of value in vaccination, either on their own or complexed with DNA. A modified approach would involve the incorporation of epitopes encoded by the transgene in the sequence of the VLP so that, post-delivery, VLPs would provide the initial challenge and the DNA, aided by the adjuvant properties of the VLPs, the continuous low level stimulation required for long-term immunity.
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Acknowledgments |
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This work was supported by grants from the Wellcome Trust (grant 048711/2/96) and the European Community, as well as by a studentship to B.C. from the BBSRC (contract BI04 CT 972147).
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Received 26 March 2001;
accepted 2 August 2001.