plasmid dna–recombinant opc protein complexes for nasal dna immunization

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Vaccine 19 (2001) 3692–3699 Plasmid DNA – recombinant Opc protein complexes for nasal DNA immunization Alexis Musacchio a, *, Diogenes Quintana a , Antonieta M. Herrera a , Belkis Sandez a , Jullio Cesar Alvarez a , Viviana Falco ´n b , Marı ´a Cristina la Rosa b , Fe ´lix Alvarez b , Dagmara Pichardo c a Diision of Vaccines, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, C. Habana, Cuba b Diision of Physical -Chemistry, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, C. Habana, Cuba c Diision of Animal Facilities, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, C. Habana, Cuba Received 6 September 2000; received in revised form 31 January 2001; accepted 27 February 2001 Abstract The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding genes that stimulate a specific immune response. Based on this, a new approach using pCMV-gal plasmid DNA complexed to the Opc meningococcal outer membrane protein was assayed for. Optimal conditions of interaction were established between recombinant Opc protein and pCMV-gal plasmid DNA. Complexes were fully characterized by electrophoresis analysis, DNAse resistance assay and transmission electron microscopy. DNA – protein complexes were also evaluated in in vitro transfection experiments. After the characterisation of complexes, Balb/c mice were intranasal (i.n.) and intramuscularly (i.m.) immunized. The humoral immune response against -galactosidase was measured by ELISA. The proliferative response in the spleen lymph nodes was also measured. Complexes administered by i.n. route induced both systemic and mucosal antibody responses. This behavior was not observed with the naked DNA. Finally, a lymphoproliferative response specific to -galactosidase induced by DNA – protein complexes was also detected. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: DNA immunization; Opc protein; Plasmid DNA www.elsevier.com/locate/vaccine 1. Introduction Nucleic acid vaccines represent a new approach to the control of infectious diseases, as novel means of expressing antigens in vivo for the generation of hu- moral and cellular immune responses [1]. This technology offers potential solutions to the de- velopment of vaccines: it allows presentation of anti- gens to the immune system in a native form, synthesized by the host in a similar way as antigens are synthesized during infection with the pathogen. This represents a great advantage over the use of purified recombinant-derived protein antigens, especially in the many cases in which it has proved difficult or impossi- ble to produce a complex antigen in a native form using traditional recombinant techniques in vitro. It should become possible to immunize with a complex mixture of DNA sequences in the case of pathogens that escape the immune system by modifying of their critical anti- gens. In addition, the antigens synthesized within host cells from directly injected genetic material, are directed to the MHC class I- and II-associated pathways in the same way as viral antigens during infection, and are therefore capable of eliciting similar cellular immune responses. An additional advantage of purified nucleic acid vaccination is the induction of a wide spectrum of a protective long lasting immune response. Finally, these vaccines are easy to obtain and produce. In practice most nucleic acid vaccines are in the form of plasmid DNA to take advantage of its safety and simplicity [2]. DNA vaccines have been administered by different routes, i.e. subcutaneous, intradermic, intraperitoneal, mucosal and intramuscular, obtaining the best results * Corresponding author. Tel.: +53-7-218008218164; fax: +53-7- 336008218070. E-mail address: [email protected] (A. Musacchio). 0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(01)00076-7

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Vaccine 19 (2001) 3692–3699

Plasmid DNA–recombinant Opc protein complexes for nasalDNA immunization

Alexis Musacchio a,*, Diogenes Quintana a, Antonieta M. Herrera a, Belkis Sandez a,Jullio Cesar Alvarez a, Viviana Falcon b, Marıa Cristina la Rosa b, Felix Alvarez b,

Dagmara Pichardo c

a Di�ision of Vaccines, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, C. Habana, Cubab Di�ision of Physical-Chemistry, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, C. Habana, Cuba

c Di�ision of Animal Facilities, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, C. Habana, Cuba

Received 6 September 2000; received in revised form 31 January 2001; accepted 27 February 2001

Abstract

The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding genes that stimulate a specific immuneresponse. Based on this, a new approach using pCMV�-gal plasmid DNA complexed to the Opc meningococcal outer membraneprotein was assayed for. Optimal conditions of interaction were established between recombinant Opc protein and pCMV�-galplasmid DNA. Complexes were fully characterized by electrophoresis analysis, DNAse resistance assay and transmission electronmicroscopy. DNA–protein complexes were also evaluated in in vitro transfection experiments. After the characterisation ofcomplexes, Balb/c mice were intranasal (i.n.) and intramuscularly (i.m.) immunized. The humoral immune response against�-galactosidase was measured by ELISA. The proliferative response in the spleen lymph nodes was also measured. Complexesadministered by i.n. route induced both systemic and mucosal antibody responses. This behavior was not observed with the nakedDNA. Finally, a lymphoproliferative response specific to �-galactosidase induced by DNA–protein complexes was also detected.© 2001 Elsevier Science Ltd. All rights reserved.

Keywords: DNA immunization; Opc protein; Plasmid DNA

www.elsevier.com/locate/vaccine

1. Introduction

Nucleic acid vaccines represent a new approach tothe control of infectious diseases, as novel means ofexpressing antigens in vivo for the generation of hu-moral and cellular immune responses [1].

This technology offers potential solutions to the de-velopment of vaccines: it allows presentation of anti-gens to the immune system in a native form,synthesized by the host in a similar way as antigens aresynthesized during infection with the pathogen. Thisrepresents a great advantage over the use of purifiedrecombinant-derived protein antigens, especially in themany cases in which it has proved difficult or impossi-ble to produce a complex antigen in a native form using

traditional recombinant techniques in vitro. It shouldbecome possible to immunize with a complex mixtureof DNA sequences in the case of pathogens that escapethe immune system by modifying of their critical anti-gens. In addition, the antigens synthesized within hostcells from directly injected genetic material, are directedto the MHC class I- and II-associated pathways in thesame way as viral antigens during infection, and aretherefore capable of eliciting similar cellular immuneresponses. An additional advantage of purified nucleicacid vaccination is the induction of a wide spectrum ofa protective long lasting immune response. Finally,these vaccines are easy to obtain and produce.

In practice most nucleic acid vaccines are in the formof plasmid DNA to take advantage of its safety andsimplicity [2].

DNA vaccines have been administered by differentroutes, i.e. subcutaneous, intradermic, intraperitoneal,mucosal and intramuscular, obtaining the best results

* Corresponding author. Tel.: +53-7-218008218164; fax: +53-7-336008218070.

E-mail address: [email protected] (A. Musacchio).

0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.PII: S0264-410X(01)00076-7

A. Musacchio et al. / Vaccine 19 (2001) 3692–3699 3693

using the latter [3–7]. However, special attention hasbeen given to the development of methods for adminis-tering DNA vaccines using the mucosal route, as theefficiency and safety of existing vaccines could be po-tentially increased. Hence, this route could increase theeffectiveness of vaccines in the elderly and young.

The ability of DNA expressing vectors, encoding forviral proteins, to elicit both humoral and cell mediatedimmune responses following intramuscular immuniza-tion has also suggested that this vaccination approachmay also elicit mucosal immunity if DNA is targeted tothe specialized inductive sites of the mucosal immunesystem. Based on the concept of a common mucosalsystem [8] and the experience of immunization withnaked DNA [1,9,10], we assayed an intranasal (i.n.)nucleic acid immunization strategy in mice to elicithumoral and cellular responses using DNA–proteincomplexes formed with the outer membrane protein(Opc) from Neisseria meningitidis [11]. This proteinfacilitates invasion of endothelial and epithelial cells bycapsule-deficient variants of N. meningitidis strains [12],so interaction of DNA–Opc protein complexes withmucosal cells may also be facilitated.

2. Material and methods

2.1. Reagents

All employed chemicals were from BDH, UK, whilemicrobiological culture media were from Oxoid, UK.Substrate p-nitrophenyl phosphate used in ELISA wasfrom Sigma, St. Louis, MO. Alkaline phosphatase la-beled anti-mouse IgG and IgA conjugate were fromSigma. Media and reagents for cell culture were ob-tained from Sigma.

2.2. Strains

Escherichia coli strain MC 1061 (F−, D(ara-leu)7696,araD139, DlacX74, galU, galK, hsdR, rpsL) [13] wasused as host for pCMV�-gal plasmid DNA transforma-tion. E. coli strain W3110 (thyA36 deoc2, IN(rrnD-rrnE)1 rph pyrE Lambda− F−) [14], bearing the opcgene [15], was used to purify the recombinant Opcantigen. COS-7 cells (Kidney, SV40 transformed,African green monkey, ATCC CRL 1651) [16] wereused in transfection experiments.

2.3. pCMV�-gal plasmid DNA and purification

pCMV�-gal plasmid DNA was obtained as previ-ously described [17]. Transformed E. coli MC 1061 wasgrown in Terrific Broth media and plasmid DNA waspurified by PEG6000 precipitation method [18]. Briefly,DNA was released from the cells by alkaline lysis; the

recovered lysate was precipitated sequentially in iso-propanol, lithium chloride and absolute ethanol. Thepellet was then incubated with RNAse and DNA wasprecipitated with 40% PEG6000/30 mM MgCl2 solu-tion. After phenol–chloroform treatment, DNA wasfinally precipitated in absolute ethanol and suspendedin Tris EDTA buffer. Purified DNA preparations werepooled and stored at −20°C until use. DNA wasquantitated by UV spectrophotometry and purity wasassessed by the ratio 260/280.

2.4. Purification of recombinant Opc protein

The recombinant Opc protein was purified from E.coli W3110 cells expressing this antigen, as previouslydescribed [19]. Briefly, after growth and cellular disrup-tion, the recombinant Opc protein was semi-purified bythe washed cell pellet procedure. Further purificationwas achieved in a high performance liquid chromatog-raphy (HPLC) system (Pharmacia, Sweden) using areverse phase C4 column (Vydac HPLC columns,USA), after extraction of insoluble material with 6 Mguanidinium hydrochloride (GuHCl) in 20 mM citrate-phosphate buffer pH 2.6. The eluted protein was freeze-dried, and kept at 4°C until used.

2.5. Plasmid DNA–protein complexes

Recombinant Opc protein was resuspended either in8 M Urea/20 mM citrate-phosphate buffer pH 2.6 anddesalting out to 20 mM citrate-phosphate buffer pH 2.6or in 8 M Urea/20 mM carbonate–bicarbonate bufferpH 10.6 and desalting out to 20 mM carbonate–bicar-bonate pH 10.6.

pCMV�-gal plasmid DNA was mixed with the re-combinant Opc protein in 1:1, 1:2, 1:10 (wt/wt) ratiosand incubated for 15 min at room temperature.

The formation of pCMV�-gal plasmid DNA–Opcprotein complexes was verified in 0.8% agarose gelsafter DNA visualization with ethidium bromide andCoomasie Blue for protein staining.

2.6. DNAse resistance assay

Formed pCMV�-gal plasmid DNA–Opc proteincomplexes (1:2 ratio) at pH 2.6 and pCMV�-gal plas-mid DNA were incubated with deoxyribonuclease I(0.25 �g ml−1) at 37°C, during 1 h. Samples wereapplied in a 0.8% agarose gel using 0.2 M NaOH in thesample buffer.

2.7. In �itro transfection and expression assay

In vitro transfection [18] was carried out using thepCMV�-gal plasmid DNA, and obtained plasmidDNA–protein complexes. The COS-7 cell line was

A. Musacchio et al. / Vaccine 19 (2001) 3692–36993694

grown at 37°C, 6% CO2 in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% heat-inacti-vated fetal calf serum, 4 mM L-glutamine, and 100 �gml−1 of penicillin and streptomycin. Cells were seededat 2×105 cells/60 mm plate. After 24 h, the pCMV�-gal plasmid DNA–Opc protein complexes, thepCMV�-gal plasmid DNA and the DEAE-Dextran-pCMV�-gal plasmid DNA were added, followed by adimethyl sulfoxide (DMSO) shock (10% DMSO inphosphate-buffered saline (PBS), pH 7.2 for 2 min).Cultures were washed 48 h after the DMSO shockswith PBS and fixed in 0.2% glutaraldehyde. Enzymaticactivity was visualized in an optical microscope byaddition of a 0.2% X-gal solution. Stained cells werecounted and the mean value of positive cells calculatedfrom four independent experiments.

2.8. Transmission electron microscopy

Purified pCMV�-gal plasmid DNA and DNA–protein complexes were processed for transmission elec-tron microscopy (TEM) using a negative stain/rotaryshadow technique. Fifteen microliter drops of freshlyprepared samples were placed on glow-discharged col-lodion/carbon-coated 400-mesh copper grids for 3 min.The solution was wicked off with filter paper andreplaced with 1% aqueous uranyl acetate for 30 s. Afterremoval of the solution, grids were rinsed in double-dis-tilled H2O and allowed to dry. Rotary shadowing wasperformed using 1 in. of 0.008-inch Pt/Pd 60/40 wire at7° angle. Grids were imaged in a Jeol JEM 2000EXelectron microscope operated at 80 kV.

2.9. Immunization schedule

To evaluate the immunogenicity of DNA–proteincomplexes, seven Balb/c mice per group were immu-nized either intra-nasal or intra-muscular. Four dosesof selected complexes were inoculated without adju-vant, at 2-week intervals. The immune serum was col-lected 2 and 10 weeks after the last injection. IgAantibody levels were determined in lungs after 10 weeksof the last inoculation. Lungs from individual animalswere macerated in 1 ml of PBS and after centrifugationthe supernatant (lung wash) collected. This suspensionwas used in ELISA assays.

Preparations used for immunization were:1. Intra-muscular immunization with 100 �g of

pCMV�-gal plasmid DNA.2. Intra-nasal immunization with 100 �g of pCMV�-

gal plasmid DNA–200 �g Opc protein complex.3. Intra-nasal immunization with 200 �g of recombi-

nant Opc protein.4. Intra-nasal immunization with 100 �g of pCMV�-

gal plasmid DNA.

2.10. Characterization of the immune response (ELISA)

Maxisorp plates (Nunc, Denmark) were coated with1 �g ml−1 of �-galactosidase, in 0.1 M Tris/HCl pH 8.5with 0.02% sodium azide, 3 h at 37°C. Plates werewashed 4 times in PBS with 0.05% Tween 20 (PBST)before serum dilutions were added. Sera were diluted inPBST and incubated 1 h at 37°C, in precoated plates.After washing, bound antibodies were detected by incu-bation for 1 h at 37°C with the alkaline phosphataselabeled anti-mouse IgG conjugate, or an alkaline phos-phatase labeled anti-mouse IgA conjugate. p-Nitro-phenyl phosphate (1 mg ml−1) in 0.5 M diethanolaminebuffer pH 9.8 was used to develop the antigen-antibodyreactions, and absorbance was read after 20–45 min at405 nm. Serum IgG or IgA anti-�-galactosidase anti-body levels were expressed as their absorbance (405nm) values in ELISA and used for statistical analysis.

2.11. Statistical methods

The Kruskal–Wallis method for the analysis of vari-ance and the Dunn’s multiple comparison test wereused to determined differences between data. A P valueof �0.05 was considered statistically significant. In thefigures, bars represent the mean of antibody levels�S.D. for each experimental group.

2.12. SDS-PAGE

SDS-PAGE was performed with a discontinuous gelsystem, using Tris/glicine buffer [20].

2.13. Lymphocyte proliferati�e assay

Induction of � galactosidase-specific lymphoprolifer-ative response was measured 11 weeks after of the lastimmunization dose by a conventional assay [21].Briefly, spleen cells from immunized BALB/c mice (n=3 each), were collected (viability higher than 95%),diluted in complete RPMI 1640 (Gibco, UK) contain-ing streptomycin (100 �g ml−1), penicillin (100 Uml−1) and 2-mercaptoethanol (50 �M), supplementedwith 10% serum fetal bovine (SFB). Spleen cells wereseeded at density of 2×105 cells per well, in presence of�-galactosidase at 0.5, 0.1 and 0.05 �g ml−1 per well.Murine phytohemagglutinin (1% v/v PHA-M, Gibco,UK) served as positive mitogenic control. Control wellsreceived only cells. Cells in all the wells were cultured in220 �l of medium. The plates were incubated in ahumidified atmosphere of 5% CO2 at 37°C, during 72 h.The cells were pulsed with [3H]Thymidine (1 �Ci perwell) (Amershan, UK), during the last 16 h of culture,and harvested onto glass microfiber filters (Pharmacia,Sweden). The incorporated radioactivity was deter-mined by using a LKB 1217 Rackbeta liquid scintilla-

A. Musacchio et al. / Vaccine 19 (2001) 3692–3699 3695

Fig. 1. (A) pCMV�-gal plasmid DNA used in DNA–protein com-plexes. (1) Molecular weight marker (� DNA-Hind III digest), (2)pCMV�-gal plasmid DNA. (B) SDS-PAGE of the recombinant Opcprotein used in DNA–protein complexes, (1) low molecular weightmarker, (2) purified recombinant Opc protein.

Fig. 3. Enzymatic treatment of pCMV�-gal plasmid DNA andpCMV�-gal plasmid DNA–Opc protein complex with deoxyribonu-clease I. Lanes: (1) molecular weight marker (� DNA-Hind IIIdigest), (2) pCMV�-gal plasmid DNA, (3) pCMV�-gal plasmidDNA–Opc protein complex, (4) pCMV�-gal plasmid DNA afterdeoxyribonuclease I treatment, (5) pCMV�-gal plasmid DNA–Opcprotein complex after deoxyribonuclease I treatment, (6) pCMV�-galplasmid DNA after deoxyribonuclease I treatment and applied with0.2 N NaOH in the sample buffer, (7) pCMV�-gal plasmid DNA–Opc protein complex after deoxyribonuclease I treatment and appliedwith 0.2 N NaOH in sample buffer.

tion counter to determine cellular growth (LKB/Wallac,Sweden). The stimulation indexes (SI) were calculatedas the mean counts per minute of the stimulated cellsdivided by the means counts per minute of the controlwells.

3. Results

3.1. Plasmid DNA-recombinant Opc protein complexes

To study the plasmid DNA–protein interaction,purified recombinant Opc protein and supercoiledpCMV�-gal plasmid DNA, were employed (Fig. 1).The formation of pCMV�-gal plasmid DNA–Opcprotein complexes are shown in Fig. 2. Different ratios(w/w) of plasmid DNA:Opc protein were assayed forcomplex formation (1:1, 1:2, 1:10) and applied intoagarose gel for electrophoresis analysis. A delay in

electrophoretic mobility of samples at DNA–proteinratios 1:2 and 1:10 was observed, when Opc protein atpH 2.6 was employed (lines 4 and 5). This effect wasnot observed when the Opc protein was used at pH 10.6(lanes 6–8).

3.2. DNAse resistance assay

Resistance to enzymatic degradation was tested in aDNAse assay, where formed complexes were exposedto the deoxyribonuclease I. Fig. 3 shows the results ofDNA-complex stability, submitted to this treatment.

Fig. 2. A total of 0.8% agarose gels of the plasmid DNA–Opc protein complexes visualized with ethidium bromide (A) and Comassie Blue (B).Lanes: (1) molecular weight marker (� DNA-Hind III digest), (2) pCMV�-gal plasmid DNA, (3–5) pCMV�-gal plasmid DNA-Opc proteincomplexes at ratios 1:1, 1:2 and 1:10 (pH 2.6), (6–8) pCMV�-gal plasmid DNA–Opc protein complexes at ratios 1:1, 1:2 and 1:10 (pH 10.6).

A. Musacchio et al. / Vaccine 19 (2001) 3692–36993696

pCMV�-gal plasmid DNA was protected from enzy-matic action when it was complexed to the Opcprotein (lanes 5 and 7) while a total degradation pat-tern was observed in case of naked DNA (lines 4 and6).

3.3. TEM

The shape of the DNA–protein complexes wasstudied in transmission electron microscopy using thenegative stain/rotary shadow technique. pCMV�-galplasmid DNA and pCMV�-gal plasmid DNA–Opcprotein complexes are shown in Fig. 4. DNA–proteincomplexes forming homogeneous packaged structureswere observed. The rounded shape size ranged be-tween 0.3 and 0.4 �m, although larger 0.6 �m sizecomplexes were also observed.

3.4. Transfection of COS-7 cells

As described above, the pCMV�-gal plasmid DNA(with and without DEAE-Dextran as a transfectingagent) and pCMV�-gal plasmid DNA–Opc proteincomplexes (at ratio 1:2 wt/wt) were used for in vitrotransfection experiments. �-galactosidase expressingcells are shown in Fig. 5. Transfected COS-7 cellsshowed a dense blue color, approximately 12 h fol-lowing the addition of X-gal solution. Fig. 5C shows

Fig. 5. Detection of �-galactosidase activity in transfected COS-7 cellswith (A) pCMV�-gal plasmid DNA-DEAE-Dextran, (B) pCMV�-galplasmid DNA without DEAE-Dextran and (C) pCMV�-gal plasmidDNA–Opc protein complex without DEAE-Dextran.

Fig. 4. Transmission electron microscopy images from (A) pCMV�-gal plasmid DNA–Opc protein complex and (B) pCMV�-gal plasmidDNA.

that pCMV�-gal plasmid DNA–Opc protein com-plexes were able to transfect COS-7 cells withoutDEAE-Dextran as transfecting agent. However, thepCMV�-gal plasmid DNA did not transfect suchcells, under the same conditions (Fig. 5B). TheDEAE-Dextran-pCMV�-gal plasmid DNA, used as apositive control in this experiment, transfected theCOS-7 cells, as expected (Fig. 5A). The frequency ofpositive stained cells using DEAE-Dextan-pCMV�-galplasmid DNA and pCMV�-gal DNA–protein com-plexes were 30% and 15–20%, respectively.

A. Musacchio et al. / Vaccine 19 (2001) 3692–3699 3697

3.5. Humoral immune response

After the characterization of complexes, Balb/cmice were immunized intranasally (i.n.) and intramus-cularly (i.m). IgG antibodies specific to �-galactosi-dase were detected in animals i.m. immunized withpCMV�-gal plasmid DNA, and also in mice whereOpc-pCMV-�gal plasmid DNA complex was i.n. ad-ministered (Fig. 6).

IgG antibody levels were higher (with statistical sig-nificance) in animals immunized i.m with pCMV�-galplasmid DNA, two weeks after the last inoculation.However, 10 weeks after immunization, differences inIgG antibody levels between groups immunized i.m.and i.n with plasmid DNA and plasmid DNA–protein complex respectively, were not observed.

IgA antibody levels in lungs, from groups that re-ceived complexes i.n. and pCMV�-gal plasmid DNAi.m., evaluated 10 weeks after immunization, had no

Fig. 7. IgA antibody levels against �-galactosidase in swap lungs ofmice after immunization. Samples (I) lung washes from mice immu-nized with pCMV�-gal plasmid DNA (administered i.m.), (II) lungwashes from mice immunized with pCMV�-gal plasmid DNA–Opcprotein complex (administered i.n.), (III) lung washes from miceimmunized with Opc protein (administered i.n.), (IV) swap lungsfrom mice immunized with pCMV�-gal plasmid DNA (administeredi.n.).

statistically significant differences, while no IgA re-sponse was detected either in mice immunized i.n.with pCMV�-gal plasmid DNA or in animals immu-nized by the same route with Opc protein (Fig. 7).

3.6. Induction of antigen-specific proliferati�e T cellresponse

Spleen cells from i.m. immunized mice with pCMV-�-gal plasmid DNA and those receiving plasmidDNA–Opc protein complexes proliferated in responseto pure �-galactosidase (Fig. 8). The stimulation in-dex was moderately higher in mice immunized i.n.with complexes, compared to mice inoculated i.m.with naked DNA. The response was antigen specific,since negligible proliferation was detected in the ab-sence of specific antigen.

Fig. 6. Serum antibody response (IgG) against �-galactosidase mea-sured in ELISA. (A) Antibody response 2 weeks after the lastimmunization. (B) Antibody response 10 weeks after the last immu-nization. Samples (I) sera from mice inmunized with pCMV�-galplasmid DNA (administered i.m.), (II) sera from mice inmunized withOpc protein (administered i.n.), (III) sera from mice inmunized withpCMV�-gal plasmid DNA–Opc protein complex (administered i.n.),(IV) sera from mice inmunized with pCMV�-gal plasmid DNA(administered i.n.), (V) Preimmune sera.

Fig. 8. Lymphoproliferative response against �-galactosidase. Spleencells of BALB/c immunized mice with (I) Opc protein (administeredi.n.), (II) pCMV�-gal plasmid DNA (administered i.m.), (III)pCMV�-gal plasmid DNA–Opc protein complex (administered i.n.).

A. Musacchio et al. / Vaccine 19 (2001) 3692–36993698

4. Discussion

Nasal delivery of vaccines is an alternative to injec-tion. Immunization results in mucosal immune re-sponses, which may be of particular importance inprotection of infection at mucosal surfaces, as well assystemic immune responses [22].

This route is also attractive for DNA immunization.However, naked DNA administered by the intranasalroute did not induce detectable antibody levels inserum, saliva or nasal washes, even when DNA hasbeen combined with protein adjuvants or specific typesof cationic liposomes [23,24]. To overcome these obsta-cles different alternatives have been assayed in combi-nation with naked DNA. Some of them are related topositive charged cationic lipids, polycationic liposomesand poly(DL-lactide-co-glycolide) particles [9,23,25], butthe elicited immune response appeared not to be soeffective as intramuscular delivery. Since the Opcprotein from N. meningitidis mediates attachment toboth endothelial and epithelial cells, we examined thefeasibility of an Opc–DNA complex for induction of aspecific immune response after nasal immunization.

The pCMV-�gal plasmid DNA, bearing the lacZgene, was used as reporter in our experiments [17]. Therecombinant Opc protein was purified from trans-formed E. coli strain W3110, as previously described[19]. For protein–DNA complex formation, differentpCMV-�gal plasmid DNA:Opc ratios, 1:1, 1:2 and 1:10(w/w), were assayed at two pH conditions (pH 2.6 andpH 10.6). Taking into account that Opc exhibits a highisoelectric point (9.6), a positive net charge should betheoretically expected at pH 2.6. Under this condition agood interaction between the Opc protein and pCMV-�gal plasmid DNA was observed. However, this behav-ior was not detected when Opc was used at pH 10.6,indicating that interactions between Opc and pCMV-�gal plasmid DNA in complex formation were mainlyelectrostatic. At pH 2.6 DNA was fully complexed at1:2 and 1:10 DNA:protein ratios.

The shape of selected pCMV-�gal plasmid DNA–Opc complexes at 1:2 ratio, was studied using a nega-tive stain/rotary shadow technique in TEM. Thestructure of observed Opc–DNA complexes was differ-ent to those obtained by others [26]. The roundedstructure observed contrasts to the rodlike structuresobtained when cationic lipids and poly-K were used asDNA condensing agents. Similar to the previous report,a number of DNA strands remained as unboundmolecules. However, DNA in formed structures wasprotected from enzymatic degradation when DNA–protein complexes were exposed to deoxyribonuclease Itreatment. The size of obtained DNA–protein com-plexes range mainly between 0.3 and 0.4 �m, althoughlarger size complexes were also observed. Since theoptimal size of particle processing for the mucosal

system seems to be less than 10 �m [27], the obtainedDNA–Opc complex size could facilitate the uptake bynasal route.

Prior to mice immunization, the ability of formedDNA–protein complexes to trasfect COS-7 cells invitro, was studied. Transfection experiments indicatedthat pCMV�-gal plasmid DNA–Opc complexes wereable to transfect COS-7 cells, without using any trans-fecting reagent. The transfection frequency was lower incomparison to the frequency obtained when the COS-7cells were trasfected with pCMV�-gal plasmid DNAusing DEAE-Dextran/DMSO procedures. Naked DNAwas unable to transfect COS-7 cells, as expected.

We investigated whether i.n. immunization with thepCMV�-gal plasmid DNA–Opc protein complexeswould elicit �-galactosidase specific IgG or IgA anti-bodies, either in serum or mucosal fluids (lung wash).Specific serum IgG was detected after the fourth admin-istration. In contrast, mice immunized i.m. with nakedDNA elicited specific IgG after the second inoculation.Both groups exhibited equal levels of specific serumIgG 10 weeks after the last immunization.

After the last inoculation, specific serum antibodyresponse was detected both in animals immunized i.m.with DNA or i.n. with protein–DNA complexes.Higher specific IgG antibody levels were detected inanimals stimulated by i.m. DNA injection, 15 days afterthe last immunization. However, at 10 weeks after thelast immunization the IgG levels increased in animalsi.n immunized, been similar to those i.m immunized. Aspecific IgA response was also detected in lungs of bothimmunized groups. The IgA antibody levels present inlung fluids were superior in mice received DNA com-plexed to the Opc protein. Several cellular and molecu-lar barriers has been shown to be limiting steps for geneexpression in the nucleus [23]. An explanation for theslower seroconvertion kinetics seen in mice immunizedi.n. could be associated with a process involving DNArelease from the DNA–proteins complexes after theiruptake by specialized cells.

In other mouse assays, low level of IgG antibodieshave also been observed after DNA nose drops admin-istration, in comparison with intramuscular inoculation[9]. Recently, it has been demonstrated that when plas-mid DNA in combination with CT was used for i.n.immunization, levels of specific antibodies in serum andmucosal fluids were undetectable. However, B cell re-sponses can be induced in the lung and spleen afterimmunization with specific protein encoding plasmidDNA in combination with CT [28].

Spleen cells from immunized mice were examined ina conventional [3H]thymidine uptake proliferation as-say. Taking into account the long-lasting specific anti-body response elicited against �-galactosidase, thecellular proliferative response against this antigen wasalso studied. Strong proliferative responses in groups

A. Musacchio et al. / Vaccine 19 (2001) 3692–3699 3699

either immunized i.m. within plasmid DNA or i.n. withplasmid DNA–protein complexes were observed. Thus,i.n. immunization with DNA–Opc protein complexeselicits a proliferative T cell response in the spleen.

Results presented in this work are conclusive in thatplasmid DNA–Opc protein complexes are more effi-cient to induce systemic and mucosal antibody re-sponses than naked DNA by the i.n. route. This couldbe a novel alternative for the improvement of futurenasal DNA vaccines.

Acknowledgements

We thank Dr J.V. Gavilondo and Professor E. Pen-ton for critical comments on the manuscript.

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