Veterinary Immunology and Immunopathology 89 (2002) 67±81
A novel transcutaneous plasmid±dimethylsulfoxide delivery technique for avian nucleic acid immunization
R.A. Heckerta,*, S. Elankumarana, G.L. Oshopa, V.N. Vakhariab
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742-3711, USA Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, MD 20742-450, USA Received 12 April 2002; received in revised form 11 May 2002; accepted 6 June 2002
Abstract In this report, we show that dimethylsulfoxide (DMSO) enhances liposome-mediated transfection of nucleic acid in chicken macrophage cells and that this could be exploited for the transcutaneous delivery of naked DNA through the intact skin of chickens. We found that DMSO enhanced transfection ef®ciencies of lipofectamine and polyethyleneimine in HD-11 chicken macrophage cells. Based on this principle, we showed that transcutaneous delivery of a DNA plasmid±dimethylsulfoxide mixture (1:1) to untreated skin of chickens results in a wide distribution of the plasmid in the body. Distribution studies were done using plasmids encoding enhanced green ¯uorescent protein (EGFP) reporter gene and a bivalent DNA vaccine coding for infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) immunogenic protein genes. This bivalent vaccine induced mucosal and systemic immune responses, as evidenced by IgA and IgM production in the tears and serum of vaccinated chickens. Mucosal immune responses in the tears after topical vaccination were signi®cantly higher
P < 0:05 than after i.m. delivery of the same DNA vaccine and were characterized by the absence of an IgG response. The biodistribution of plasmid indicated that topical delivery with DMSO resulted in a wide distribution and persistence of the plasmid until 15 weeks postprimary vaccination. Both delivery methods resulted in insert-speci®c message being made in several body tissues, but after topical delivery the virus-speci®c mRNA could be detected in the bone marrow of one out of three chickens until 15 weeks postprimary vaccination. Furthermore, transcutaneous delivery of this DNA vaccine using DMSO conferred protection from challenge with virulent IBDV (86% survival) and NDV (86% survival). This novel transcutaneous method of delivery of a DNA vaccine shows promise as being an easy and effective way to deliver nucleic acids through intact skin for vaccination or therapeutic purposes. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: DNA vaccine; Skin; Transcutaneous immunization; Mucosal immunity; Chicken; Dimethylsulfoxide; Infectious bursal disease virus; Newcastle disease virus
Abbreviations: CMV, cytomegalovirus; DMSO, dimethylsulfoxide; EGFP, enhanced green ¯uorescent protein; HI, hemagglutination inhibition; HN, hemagglutinin-neuraminidase; IBDV, infectious bursal disease virus; NDV, Newcastle disease virus; PEI, polyethleneimine; PI, post-inoculation; SPF, speci®c-pathogen-free * Corresponding author. Tel.: 1-301-314-6830; fax: 1-301-314-6850. E-mail address: email@example.com (R.A. Heckert).
1. Introduction Nucleic acid (NA) vaccination or genetic immunization has been shown to elicit speci®c cellular and humoral immune responses to many different agents in many different species (Babiuk et al., 2000; Hasan et al., 1999; Kowalczyk and Ertl, 1999; Weiner and
0165-2427/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 1 8 6 - 1
we addressed whether genetic immunization by topical delivery of a DNA vaccine to the skin of chickens would elicit both systemic and secretory Ab responses. Shi È et al. Vectors Using standard molecular biology techniques.. To prevent virus transmission across the mucosal epithelium and to prevent virus dissemination to the target organs. It has been postulated that the strong and persistent humoral and cellular immune responses that follow intradermal DNA immunization are related to the important immune surveillance functions of the skin È and the skin-associated lymphoid tissues (Tuting et al.. The plasmid pCMVimhn was constructed by cloning a chimeric cDNA containing the hemagglutinin-neuraminidase (HN) gene from Newcastle disease virus (NDV) and the VP2. 1999. delivery of the same DNA vaccine and characterized by the absence of an IgG response. 1995. Glenn et al. This novel method shows promise as being an effective way to deliver nucleic acids through intact skin and establishes the proof-of-concept for transcutaneous immunization of chickens with DNA vaccines.. 1999). 1998). Such responses include secretory IgA and IgG antibodies that are reported to mediate virus neutralization and prevent virus adhesion and intraepithelial IgA. lack of reversion to virulence. 2. Heckert et al. Klavinskis et al. 1999.or NA-based vaccine was coupled to an adjuvant or liposome and the skin was either abraded or electroporation was used in an attempt to transfect cells.1 and used in transfection studies in HD-11 cells. Oshop et al. 2002). 3 and 4 genes from infectious bursal disease virus (IBDV) IM strain into pcDNA3. we show that topical delivery of a DNA vaccine combined with DMSO to untreated skin of chickens results in a wide distribution of the plasmid in the body and induces a very rapid mucosal and systemic immune responses. a safer and less cumbersome approach is needed. Tuting et al. Weisz-Carrington et al.. Immune responses were signi®cantly higher after topical administration than after i. injection or orally... Vaccines are usually delivered by i. Inc. 2000. Non-invasive vaccination by topical application has been shown with proteinbased vaccines (Misra et al.). In addition. 1999. 1997.A.m. 1992). Yu et al. 1998.) between the BamH I and Not I sites in pcDNA3. as evidenced by IgA and IgM production in the tears and serum. Systemic immunization generally fails to elicit mucosal protection.. Tang et al. Vaccinating animals or humans by delivering an immunogen onto the outer layer of the skin in a non-invasive mode is an appealing strategy. 1997) and mounts immune È responses to small amounts of Ag (Tuting et al.1 (Invitrogen.
. An important aspect of many effective vaccines is the production of mucosal immunity (McGhee et al. 1999..68
R.. Materials and methods 2. although a barrier to microbial invasion.. the protein. which may inhibit intracellular virus replication (Mazanec et al. In many of the previous studies. Fan et al. The cDNA was cloned between Nhe 1 and Kpn 1 sites in the vector under the control of cytomegalovirus (CMV) immediate early promoter and a bovine growth hormone polyadenylation signal. / Veterinary Immunology and Immunopathology 89 (2002) 67±81
Kennedy.. and the ability to address several diseases in a single vaccine construct.. For practicality. the plasmid pCMVegfp was made by inserting the EGFP gene derived from the plasmid pEGFP-1 (Clontech. this vaccination technique resulted in protection from challenge against the viruses for which the vaccine was intended.. 1998). 2000) and DNA-based vaccines in mice (Alexander and Akhurst. The skin. However. make DNA vaccines an attractive approach for vaccinating man and animals. 1979). 1979. Based on the concept of a common mucosal immune system (McDermott and Bienenstock. The low cost of production. is an organ of immunity (Bos. 1999). 1992).m.. Hammond et al. In this report. thermostability. Inc..1. 1998. We then evaluated the ef®ciency of gene transfer using a plasmid encoding for different viral proteins by the following: (1) the distribution of plasmid and duration of mRNA produced from the plasmid and (2) the systemic and mucosal immune responses to the viral proteins after topical DMSO-mediated genetic immunization. We used a plasmid expressing the enhanced green ¯uorescent
protein (EGFP) as a model to investigate: (1) the ef®ciency of dimethylsulfoxide (DMSO) to augment cationic lipid-mediated transfection of chicken macrophage cells in vitro and (2) the ef®ciency of DMSO to induce topical gene transfer through the skin of chickens. vaccines should ideally stimulate immune responses in mucosal tissues.. DNA vaccines have been shown to produce mucosal immunity when delivered to mucosal surfaces (Gautam et al.
USA) or a cationic polymer. Plasmid DNA was prepared using large-scale matrix-based puri®cation according to manufacturer's recommendations (Qiagen. 14 and 15 weeks post-vaccination by placing a pinch of salt on the conjunctiva (Elankumaran et al. 5 and 10% concentrations and the mixtures incubated for 5 min at room temperature. 3. These were housed and cared for in accordance with the animal care and use principles of the University of Maryland. Experiment 2 To evaluate the protective ef®cacy of the bivalent DNA vaccine.4. 14 and 15. Heckert et al. and two of the control birds at 8 and 15 weeks post-vaccination for DNA or mRNA determination. USA).m. 1 mg of the plasmid pCMVegfp was used per well of a 6-well cell culture dish which were seeded 1 day before transfection with 2:5 Â 106 HD-11 cells.1. MBI Fermentas. For each set of transfections. a challenge experiment was conducted. USA) were prepared as per manufacturer's recommendations. and 15 weeks post-vaccination. This concentration of DMSO (50%) was chosen arbitrarily..2. respectively. and topical routes. Storrs. 1996). The plasmids were given by i. and the large surface area of the chicken skin. 2. USA). muscle (at the site of inoculation) and bone marrow were collected from three of the vaccinated birds. injection (two sites in the breast muscle. Animals Four-week-old speci®c-pathogen-free (SPF) Leghorn chickens were obtained from a commercial supplier (SPAFAS. Groups A to C comprised of 14 birds per group.R. 2. Three groups of 4-week-old SPF chickens were employed. CT). the plasmids were mixed by pipetting 1:1 with DMSO and applied to the skin of the wing web (50 mg/bird. 8. / Veterinary Immunology and Immunopathology 89 (2002) 67±81
Expression of NDV and IBDV proteins was con®rmed by indirect immuno¯uorescence staining and or hemadsorption assays (for HN protein) of transiently transfected chicken embryo ®broblast cells. The plasmids were grown in Escherichia coli DH 10 B bacteria (GIBCO BRL. The birds in groups A and B received topically either pCMVimhn or pCMV-O plasmid DNA after mixing 1:1 with DMSO (100 mg/bird) at
. USA) were used. only three chickens from the vaccinated group and two from the control group were included. resuspended in endotoxin-free water and checked for endotoxin content by Limulus amoebocyte lysate test kit (E-Toxate. respectively.4.m. 5 and 10% concentrations was also tested to determine whether DMSO facilitates transfection of chicken macrophage cells in the absence of a cationic lipid. 2. 4. The liposome:DNA complexes in Opti-MEM (GIBCO BRL.1 without any insert. Sigma. USA). For the DNA vaccine study. The cell
culture dishes were spun at 800 Â g for 10 min and incubated for 3 h at 37 8C. based on the results of in vitro transfection. four groups of 4-week-old chickens were employed. For Ab analysis at weeks 12.4. For liposome-mediated transfection. Lipofectamine Plus (GIBCO BRL. 4. 25 mg plasmid/site) or by topical application (as above). a cationic lipid. two 4week-old chickens were administered the pCMVegfp plasmid and two chickens were maintained as controls that received plasmid pCMV-O.3. For topical application. The transfection ef®ciency of plasmid DNA:DMSO at 0. polyethleneimine (PEI) (Exgen 500..2. 8. 4. single site of 100 ml). The HD-11 cells were washed once with serum-free RPMI 1640 medium and the DNA:liposome:DMSO complexes added. DMSO was added at 0. 12. The pCMV-O control plasmid was pcDNA3. we initially tested the liposome-mediated transfection ef®ciencies in the presence of DMSO in HD-11 chicken macrophage cells.m. 6. Serum samples were collected at 2. 2. Tear samples were collected at 1.A. Spleen. Experiment 1 To evaluate the feasibility of topical delivery. To the plasmid:cationic lipid mixture. Transfection of chicken macrophage cells To test the proof of principle that DMSO can enhance transfection of DNA into mammalian cells and therefore be exploited for in vivo delivery of nucleic acid vaccines in chickens. Groups B and D (four birds/group) were administered pCMV-O control plasmid by the i. or topical routes. 2. The number of cells expressing EGFP was counted in at least ®ve microscopic ®elds and the mean reported. then further cultured for 24 h at 37 8C in RPMI 1640 with 7% FCS and evaluated for transfection ef®ciency in a ¯uorescent microscope. Groups A and C (six birds/group) received the pCMVimhn plasmid through i. 2. Inc. Vector administration 2. 4. 2.
2. 2. Two weeks after the revaccination. The downstream primer Tag-F (5H CTTATACGGATATCCTGGCAATTCGGACTT3H ) was exactly complimentary to the 30 base pair unique tag of the cDNA generated by the reverse transcription with the 47mer primer in the previous step. 1988) or NDV (15C4. Brie¯y. In addition to the commercial ELISA.6. Gaithersburg. polystyrene microtiter plates (Immulon 2. and 1 min at 68 8C. To avoid ampli®cation of DNA sequences in the plasmid. 1990). The percent survivability of inoculated chickens was determined over a period of 10 consecutive days. and 5X AMV/T¯ buffer (Promega). RNA-speci®c PCR (RS-PCR) Tissue samples (50±100 mg) were homogenized using a hand-held pellet pestle in a 1. 72 8C for 1 min). USA).. IgA or IgM antibodies (Bethyl Laboratories. an Ab capture (ABC) assay was developed to detect class-speci®c Ab responses to each virus. 7 U AMVreverse transcriptase. 200 mM dNTPs. The sense primer PCR-IM-R (5H ACAATAACCCTGATAAATGC3H ) and antisense primer PCR-IM-F (5H TACCAATGCTTAATCAGTC3H ) were complimentary to sequences in the vector backbone. 200 mM of each dNTP. the test samples at 1/50 dilution were applied to the plate and captured with sucrose gradient puri®ed viruses (IBDVor NDV).70
R. First strand products were then converted to double-stranded cDNA using primers of 30 bases in length. PCR ampli®cation was performed in 50 ml containing 5 ml DNA (processed tissue sample).
.. USA). After blocking the unbound sites with 1% bovine serum albumin in PBS. 100 ml in divided doses through the eye and cloacae). Thermal cycling was carried out for 40 rounds after initial denaturing at 94 8C for 5 min (94 8C for 1 min. eye and oral) or IBDV `IM' strain (103 mean embryo infective doses/ml. Gaithersburg. ampli®cation was carried out over 30 cycles of 1 min at 94 8C. 50 pmol of the 47mer primer
(5H CTTATACGGATATCCTGGCAATTCGGACTTAGACCTCGACCTACAAT3H ). After an initial denaturing step at 94 8C for 10 min. seven birds from each group were challenged with either NDV GB Texas strain (105 mean embryo infective doses/ml. Montgomery. USA). 0. 2. an RS-PCR was used to detect mRNA from the plasmid insert as described (Shuldiner et al. An oligonucleotide primer. PCR ampli®cation was then carried out essentially as outlined above. 40 U RNAsin. Heckert et al. 10D11 and B79) (Lana et al. 47 bases in length. was used to transcribe total RNA. GIBCO BRL. and 10 Â buffer (Panvera. USA).7. which contains a 30 base pair unique sequence or tag. Horseradish-peroxidase-conjugated goat anti-mouse IgG (Kirkegaard and Perry Laboratories. 1988) monoclonal Ab cocktails. 1. based on the assay described by van Zaane and Ijzerman (1984).25 U of Ex Taq DNA polymerase. Polymerase chain reaction Tissue samples (50±100 mg) were homogenized using a hand-held pellet pestle in a 1.and IBDV-speci®c antibodies as per manufacturer's recommendations. The bound Ab and Ag complexes were picked up with anti-IBDV (B69 and R63) (Snyder et al. A negative control reaction containing only water and no template RNA was run with each set of reactions to screen for reagent contamination. First strand cDNA was generated by incubation at 37 8C for 60 min in a 20 ml reaction mixture containing 5 mg total RNA. Dynatech) were coated with af®nity puri®ed goat anti-chicken IgG.5.A. The upstream primer sequence Tag-R (5H CTGGTTGGTACTGTGATGAGAATTGGTAAT3H ) was complimentary to a region of the insert RNA. 68 8C for 1 min. / Veterinary Immunology and Immunopathology 89 (2002) 67±81
4 weeks of age and revaccinated again at 8 weeks of age with the same dose. Analysis of systemic and mucosal immune responses Commercial ELISA kits (Kirkegaard and Perry Laboratories. whose sequence contains 17 bases at its 3H -end that are complimentary to a region in the target RNA and 30 bases at the 5H -end that are unique. MD) followed by TMB micro-well peroxidase substrate (Kirkegaard and Perry Laboratories. Group C remained as unvaccinated controls. 1 mM of each primer.. 1 min at 55 8C.8 ml Eppendorf tube and the RNA extracted as per the manufacturer's direction using a guanidine isothiocyanate-based reagent (Trizol. The product from this reverse transcription reaction is a single-stranded cDNA. followed by an ®nal extension step of 10 min at 68 8C.8 ml Eppendorf tube and the DNA extracted as per the manufacturer's direction using DNAzol (GIBCO BRL. The ®nal extension step was 70 8C for 10 min.5 ml/bird. MD) were used for the estimation of NDV.
There were signi®cantly higher
P < 0:05 IgA and IgM Ab responses in the tears early after immunization. After stopping the reaction with 0. The absorbance value of each sample was corrected by subtracting twice the mean of the absorbance from the birds. Statistical analyses All data were analyzed with the aid of a statistical software program (STATISTIX for Windows version 7. as evaluated by virus-speci®c IgA.1 M sulfuric acid. neutralizing antibodies by serum neutralization test. which received pCMV-O.2.8. we administered the pCMVegfp plasmid with DMSO to the untreated skin of two 4-week-old chickens and analyzed the expression of EGFP in various tissues 48 h later. lung and spleen (data not shown). 2. Tissues that were examined but showed no ¯uorescent protein expression were the intestines.
. heart. EGFP expression was detected at the site of delivery and the underlying muscle. If the overall test for a treatment effect in ANOVA was signi®cant.9. 8 and 12 weeks after primary immunization than those vaccinated intramuscularly. and HI antibodies to NDV (Table 1) over the course of the study in experiment 1. This value was selected as the cut-off. 1). i. No virus-speci®c IgG antibodies were detectable by commercial and ABC ELISAs for either virus in serum or tears.e. Comparison of topical and i. Analytical Software). The IgA antibodies against NDV in tears were signi®cantly higher in topical group only at 1 and 4 weeks post-primary vaccination. In the tears. However. Both routes of immunization resulted in IgM and IgA Ab responses in serum and tears. 3. 2.m. Transfection of cells in vitro and in vivo with a reporter gene We determined that liposome-mediated delivery of plasmid DNA to chicken macrophages in vitro could
be enhanced by the use of DMSO. using lipofectamine (Fig. the serum Ab responses were not signi®cantly different at any time point between the different routes of immunization
P > 0:05. The hemagglutination inhibition (HI) test was done as described by Allan and Gough (1974) employing four hemagglutination units of NDV B1 strain and fresh chicken erythrocytes. Serologic tests The neutralization titers in chicken serum and tears were determined by the constant virus and varying dilutions of serum method. 4.1.m. the absorbance of each well was read at 450 nm. NDV strain B1 or IBDV strain D78. To further test the hypothesis that DMSO could be used as delivery vehicle for plasmid DNA in vivo. / Veterinary Immunology and Immunopathology 89 (2002) 67±81
Gaithersburg. when the plasmid was delivered by the topical route. Results 3. 2). 2). and IgM ELISAs in serum and tears (Fig. Statistical signi®cance was set at P < 0:05 for all tests. or topical routes. The titers were expressed as log2 of the highest dilution of the Ab that caused a 50% reduction in cytopathic effects relative to virus controls. by either i. The transfection ef®ciencies were increased up to a ®nal concentration of 10% DMSO in the transfection mixture using PEI and up to 2±4% DMSO. Differences between the Ab responses in tears and serum of the vaccine groups were examined by one-way ANOVA. None of the tissues from the control birds showed EGFP expression. plasmid DNA (in the absence of cationic lipids) plus DMSO at varying concentrations failed to transfect HD-11 cells. at each time point. Heckert et al. The titer of serum or tears was expressed as log2 of the reciprocal of the highest dilution of the Ab that caused an inhibition of four hemagglutination units of the virus. are shown. IBDV IgA and IgM antibodies were signi®cantly higher
P < 0:05 in topically vaccinated chickens only at 2 weeks postvaccination. administration of an avian DNA vaccine The nature and location of the immune responses generated. The differences between treatment groups in the in vitro transfection experiments were also examined by one-way ANOVA. as it always exceeded the mean three times the standard deviation of the negative absorbance values.A. and the kidney (data not shown). MD) was used for detection. liver. but not intramuscularly (Fig. 3. NDV IgM antibodies were signi®cantly higher in the tears of topically vaccinated chickens at 2. However.R. then the means of the transformed variables were compared by Tukey's method.0.
Heckert et al. 1. / Veterinary Immunology and Immunopathology 89 (2002) 67±81 Fig. Means bearing the Ã indicate signi®cant differences between DNA:liposome mixtures at the same DMSO concentration. Transfection of HD-11 cells (chicken macrophage) with an EGFP encoding plasmid (pCMVegfp) and DMSO. The mean transfection ef®ciency for each type of transfection was estimated by counting the number of ¯uorescent cells at ®ve 40Â microscopic ®elds (A) and the mean transfection efficiency standard error for each concentration of DMSO was plotted (B).72 R.
.A. The means bearing the same superscript do not differ signi®cantly
P > 0:05 from each other within a group.
ÃP-values <0. / Veterinary Immunology and Immunopathology 89 (2002) 67±81 Fig. Birds were revaccinated 4 weeks later (arrows) with the same dose of the vaccine. The IgM and IgA Ab responses for IBDV (A±D) and NDV (E±H) were measured in the serum and tears of vaccinated birds at various time points by ELISA. Isotype-speci®c immune responses to NDV and IBDV in serum and tears of chickens vaccinated with 50 mg of DNA by the topical (&) or i. (^) routes or with a control plasmid DNA (with no viral genes) by the topical (*) or i.m.m.A. (~) routes.05 were considered signi®cant. Heckert et al. 2. 73
.A. (Continued ). / Veterinary Immunology and Immunopathology 89 (2002) 67±81
Fig. Heckert et al.74
as determined by HI. and ruf¯ed feathers commencing from day 2 post-challenge. however. The surviving birds in groups B and C had atrophied bursae at 10 days post-challenge. which was found dead by day 3 postchallenge. 3.3. 2. Neutralizing Ab titers of serum/tears from empty plasmid vaccinated chickens were below detectable levels (<16). 3. The protection ef®cacy against homologous challenge with the `IM' strain of IBDV in DNA vaccinated chickens was 86%. 12. Challenge with `GB' Texas strain of NDV induced clinical signs including temporal twitching and in coordination in groups B and C from day 4 and in one out of seven birds in group A on day 5. 6.
Serum neutralization (SN) titers of !16 to Newcastle disease and IBDVs were considered positive. Revaccination of the chickens. 8. Mortality commenced in these birds from day 2 and ended by day 4. 3. All the dead birds had hemorrhagic bursae. Neutralizing antibodies in serum to respective viruses were detectable only up to 4 weeks post-inoculation (PI) and the titers ranged from <16 to 64 (Table 1).R. HI titers of serum/tears from empty plasmid vaccinated chickens were below detectable levels. were detected in the serum of birds that received the vaccine by both routes and only in the tears of topically vaccinated chickens at two weeks post-vaccination. 4. are shown in Fig. Affected birds became paralyzed 1 day after the commencement of clinical signs. The antibodies to NDV. Birds immunized with the empty control plasmids by either route did not produce any systemic or mucosal immune response to the viruses. 14 and 15 weeks postvaccination. All the chickens were negative for neutralizing and HI antibodies to NDV and neutralizing antibodies to IBDV at 1.A. All the birds from group C were dead or euthanized between days 4 and 7. The range of titers was <8 to 1024. b The number of birds that tested positive/total number of birds vaccinated are shown for 2 and 4 weeks post-vaccination. / Veterinary Immunology and Immunopathology 89 (2002) 67±81 Table 1 Virus-speci®c Ab responses in serum or tears after intramuscular or topical delivery of a DNA vaccine to chickensa Route of vaccine delivery Weeks postvaccination NDV-specific antibodies HI Serum Intramuscular Topical Intramuscular Topical Two Four 6/6 4/6 0/6 0/6
IBDV-specific antibodies VN VN Tears 0/6 1/6 0/6 0/6 Serum 1/6 2/6 0/6 1/6 Tears 0/6 0/6 0/6 0/6
Tears 0/6 1/6 0/6 0/6
Serum 2/6 6/6 1/6 0/6
a The serum and/or tear samples were analyzed for neutralizing and HI antibodies to NDV and virus neutralizing (VN) antibodies to IBDV at 1. HI titers of !8 to NDV were considered positive. while those in group B died between days 4 and 6. The morbidity rate of chickens in groups B and C was 100%. Only one out of seven DNA vaccinated birds showed clinical signs on day 2. Heckert et al. 14 and 15 weeks post-vaccination. The mortality rate in group C birds was 29% and in group B was 33%. If birds were moribund they were humanely euthanized and scored as dead the next day. Neutralizing antibodies were detected in tears only in one of six topically vaccinated birds and only at 2 weeks PI. 4 weeks after the primary immunization elicited none to poor anamnestic responses by both routes. detectable only after 4 weeks post-boost in a few birds. 12. Challenge with the `GB' Texas
. The number of birds that responded to vaccination is shown in Table 2. Birds challenged with IBDV in groups B and C had clinical signs of IBDV including depression. 6. Protection ef®cacy of DNA vaccine against IBDV and NDV The survival rates of topically vaccinated and unvaccinated chickens after IBDV and NDV challenge
in experiment 2. 8. only the time points at which antibodies were detected are shown. 3. The other birds in the DNA vaccinated group remained healthy and survived the 10-day observation period with grossly normal bursae. The one sick bird from group A succumbed to challenge on day 6 while the rest of the vaccinated chickens survived challenge with the absence of clinical signs.
Detection of plasmid and mRNA in various tissues over time All PCR assays were found to be free of contamination as determined by the absence of any ampli®cation products in the `no DNA' control sample that were included in each set of PCRs. route. 3.m. 8. 4. / Veterinary Immunology and Immunopathology 89 (2002) 67±81
Table 2 Antibody responders by ELISA in DNA vaccinated chickens Weeks after primary vaccinationa IBDV (number responded/number tested) IgA Serum 1 2 3 4 6 8 12 14 15
NDV (number responded/number tested) IgA IgM Tears 6/6 5/6 5/6 1/6 3/6 0/6 3/6 0/6 2/6 0/6 3/6 3/6 0/3 0/3 0/3 0/3 0/3 0/3 Serum ND ND 6/6 4/6 ND ND 5/6 4/6 ND ND 5/6 5/6 ND ND ND ND 0/3 0/3 Tears 6/6 5/6 5/6 1/6 2/6 0/6 2/6 0/6 2/6 0/6 3/6 3/6 1/3 1/3 0/3 0/3 0/3 0/3
IgM Tears 0/6 0/6 5/6 1/6 1/6 0/6 4/6 1/6 1/6 1/6 3/6 3/6 1/3 0/3 0/3 0/3 0/3 0/3 Serum ND ND 1/6 4/6 5/6 ND 1/6 4/6 ND ND 1/6 0/6 ND ND ND ND 0/3 0/3 Tears 3/6 3/6 5/6 1/6 2/6 0/6 4/6 1/6 2/6 1/6 3/6 3/6 0/3 0/3 0/3 0/3 0/3 0/3
Serum ND ND 6/6 4/6 ND ND 5/6 4/6 ND ND 5/6 5/6 ND ND ND ND 0/3 0/3
Topical Intramuscular Topical Intramuscular Topical Intramuscular Topical Intramuscular Topical Intramuscular Topical Intramuscular Topical Intramuscular Topical Intramuscular Topical Intramuscular
ND ND 0/6 4/6 ND ND 5/6 0/6 ND ND 0/6 0/6 ND ND ND ND 0/3 0/3
Serum samples were collected at 2.76
R. we used a `no template' control in which water was added rather
than an RNA template in every set of reactions.A. To ascertain the speci®city of RS-PCR.
strain of NDV protected 86% of DNA vaccinated chickens and all the unvaccinated and pCMV-O administered chickens succumbed to challenge. 8. The chickens were vaccinated at 4 weeks of age and revaccinated at 8 weeks of age with 50 mg plasmid DNA either topically or intramuscularly. Tears were collected at 1. 14.4. Heckert et al. When double-stranded plasmid DNA or DNA contaminated RNA template was used as a starting template. and 15 weeks post-primary vaccination. Plasmid DNA was detected in most birds and in all tissues at 8 weeks PI. 12. No mRNA was found in any of the tissues from the birds given the empty control plasmid. 2. 6. while the RS-PCR method resulted in no signal. but only in one bird at 15 weeks PI in the bone marrow. 3. but in fewer birds at 15 weeks PI (Table 3). Insert-speci®c mRNA was found exclusively in topically vaccinated chickens at 8 weeks PI in the spleen and muscle. A greater number of birds had plasmid DNA in a wider variety of tissues when given the plasmid by the topical versus the i. one sample of total DNA from each control tissue was spiked with 1 mg of the respective plasmids. 4. and 15 weeks after primary vaccination. There appeared to be little correlation between the presence of the plasmid or mRNA and the Ab
. conventional RT-PCR resulted in a strong positive signal. as expected. As a positive control to verify the absence of PCR inhibitors in the DNA extracts. The PCR analysis employed in this study was intended to be qualitative.
Challenge protection against IBDV (A) and NDV (B). Irrespective of the presence of plasmid DNA or insert-speci®c mRNA. The birds were challenged 2 weeks after the booster inoculation with respective viruses.A. Groups of chickens were administered 100 mg of plasmid DNA vaccine or backbone or left unvaccinated at 4 weeks of age and boosted at 8 weeks of age. 3. While all of these birds were positive for IBDVspeci®c IgA and IgM in tears.
responses seen.R. The percent survivors over a 10-day observation period are plotted. Heckert et al. at the time points examined. only one topically vaccinated bird was positive for serum IgM and none
. all three birds tested at 8 weeks post-primary vaccination were positive for NDV-speci®c IgA and
IgM in tears and serum after both methods of delivery. / Veterinary Immunology and Immunopathology 89 (2002) 67±81
provided that there is a generalized and suf®cient expression of the antigens encoded by the vaccine to elicit immunity.A.. Although. we found that plasmid DNA could not be transfected into chicken macrophage cells in vitro using DMSO alone. At 8 weeks PI. Further. as indicated by their ability to neutralize virus and inhibit hemagglutination.
of them were positive for IgA in serum against IBDV.. All three birds tested at 15 weeks were negative for virus-speci®c antibodies after either route of delivery even though the plasmid was detected in the spleen and bone marrow (Table 3). as we could not demonstrate strong early (<4 weeks) IgA responses in the serum that would allow for transudation into the lachrymal compartment. Discussion Previously. In this study. 1996.. The most dramatic response was the early induction of IgA in the tears. The immune responses generated were better than the conventional i. possibly from the harderian gland.78
R. it has never been used in conjunction with DMSO. We could not detect neutralizing and HI antibodies after 4 weeks post-vaccination. Heckert et al.m. 1994). both mucosally and systemically and protect chickens against challenge with homologous viruses... / Veterinary Immunology and Immunopathology 89 (2002) 67±81
Table 3 Tissue distribution and longevity of plasmid DNA and messenger RNA in DNA vaccinated chickensa Tissues Number of birds tested positive/totalb Intramuscular Week 8 DNA Spleen Muscle Bone marrow
Topical Week 15 mRNA 0/3 0/3 0/3 DNA 1/3 0/3 0/3 mRNA 0/3 0/3 0/3 Week 8 DNA 2/3 2/3 2/3 mRNA 2/3 2/3 1/3 Week 15 DNA 2/3 0/3 1/3 mRNA 0/3 0/3 1/3
2/3 2/3 0/3
Six SPF chickens were vaccinated by the topical or i. liposome-mediated or ballistic penetration (Allioli et al. Watanabe et al. Vaccinating animals or humans by delivering nucleic acid-based vaccines topically is an appealing strategy. Li et al. Although. we then used this approach to deliver
DNA in vivo through the intact skin of chickens. We have shown that topical application of naked DNA in 50% DMSO to unaltered skin can induce a speci®c immune response. we showed that the addition of DMSO enhanced liposome-mediated transfection of chicken cells. 1995.. b The number of birds that tested positive/total number of birds tested for plasmid DNA or mRNA after respective routes of delivery of DNA vaccine to chickens. DMSO is considered to enhance uptake of adsorbed DNA by increasing the permeability of cell membranes (Kawai and Nishizawa. the antibodies produced were biologically active. we found that transfection of chicken macrophage cells with a DNA:PEI:DMSO mixture is better than lipofectamine-mediated transfection.m. We infer these lachrymal IgA antibodies are locally secreted. very rapid and consisted primarily of IgM and IgA immunoglobulins. 4. injection. 2000). 1994.. routes with 50 mg DNA vaccine (pCMVimhn) and four chickens with a control plasmid DNA (pCMV-O) at 4 weeks of age and revaccinated at 8 weeks of age with the same dose of the vaccine. Furthermore. Hong et al. Since we found that DMSO enhanced transfection of DNA in vitro. This study demonstrated the proof-of-concept that naked DNA could be delivered transcutaneously in the chicken using DMSO. 1998). methods for in vitro transfection of avian cells has been limited to retroviral infection. Others have shown that DMSO can increase electroporation ef®ciency in mammalian or in avian primordial germ cells and chicken embryo ®broblasts (Melkonyan et al. 1984). Gautam et al. it could ef®ciently mediate transcutaneous transfection in vivo. but detected virus-speci®c IgM and IgA antibodies by ELISA until 15 weeks
. 1995. polyethyleneimine (a cationic polymer) has been shown to be an effective transfection reagent both in tissue culture and in vivo (Boussif et al. three birds from each of the vaccine groups and two birds from the each of the control groups were sacri®ced and tissues harvested for analyzing tissue distribution of the plasmid DNA and insert speci®c messenger RNA (mRNA).
....R. 1996). priming for the systemic and mucosal immune responses reported herein. However. Alternatively. A recent review of transcutaneous immunization of domestic animals covered a wide range of species. 1987. Thayer et al. Chang et al. This may not be entirely surprising since lack of humoral immune responses after i. 2000. 1997.A. 1999. which is independent of CD40 ligation (Kawabe et al. DNA vaccines applied onto the skin in a noninvasive manner may penetrate into the body via hair follicles.. Fodor et al. early mucosal immune responses against virus-speci®c proteins can be achieved by this delivery method in chickens. 2001). 1992. but not the chicken (Hammond et al. from the highly vascularized skin.. 1996. 1994). Although several other papers have reported Ab responses after DNA immunization.. Sakaguchi et al. in the skin of the chicken (Carrillo-Farga et al. 1994).. 2000). The plasmid and mRNA time course and distribution studies showed that the plasmid was not only widely distributed and produced its message. Sephadex G-200 fractionated sera showed only SN activity in the IgM fraction. whereas other workers have used 100±800 mg of DNA per chicken in repeated doses (Fynan et al. Alternatively. The absence of IgG antibodies and the higher positive threshold (>24) used for scoring SN positive birds might also be reasons for the absence of HI and SN antibodies after 4 weeks post-vaccination in our study. showing that topical delivery of plasmid results in systemic distribution of the plasmid and by our results from tracking the plasmid and message. We immunized with two doses of 50 mg DNA per chicken. HI. Czifera et al.
. 2001). Detailed mechanistic studies on the uptake of plasmid DNA. 2001). This work is one of only two studies in which the humoral immune response to a DNA vaccine in the avian has been characterized into isotype-speci®c responses. The lack of IgG in our study and that of others may have been due to the low amount of DNA used for immunization. we were intrigued with the lack of IgG observed in this study. To our knowledge there are no other studies in the avian which have followed the plasmid and message to which we can compare our ®ndings. Besides transporting the plasmid to distant sites in the body. This wide dissemination of plasmid is supported by our observations using the reporter protein (EGFP). but also persisted for at least 15 weeks. plasmid DNA may directly enter the circulation and disseminate to the spleen and the harderian gland. 1983. and precipitating antibodies against NDV could be detected as IgM and IgG immunoglobulins. 1991) may have been transfected and transported the DNA to the various tissues. (1976) reported that after mercapto-ethanol treatment of chicken serum. To our knowledge.. This might be due to the increased sensitivity of the ELISA test over the HI (Snyder et al. Therefore. persistence of plasmid and expression of transgenes are imperative for a better understanding of this third generation of vaccine technology in the avian species.m. Chang et al. whereas the IgG fraction showed both HI and SN activity. Khare et al. In addition. Our studies demonstrated that DMSO... Russell and Mackie (2001) were unable to show a strong IgG response in chickens after topical ocular administration of a single dose of 65 mg plasmid DNA encoding the reporter protein betagalactosidase.. 1989. / Veterinary Immunology and Immunopathology 89 (2002) 67±81
post-primary vaccination.. and reduction or absence of this major contact-mediated signal may have diminished T-B cognate interaction in response to the viral antigens without affecting IgM secretion. as demonstrated in mice (Bouloc et al.. this is the ®rst report of transcutaneous immunization of the avian species with a DNA vaccine. enhanced liposome-mediated transfection in vitro and facilitated delivery of nucleic acids through intact skin. 1993.. It is possible that Langerhans cells. or be taken up by the underlying epidermal cells due to increased cell membrane permeability induced by DMSO. A wide variety of chemicals have been used as skin penetration enhancers (see review by Magnusson) including DMSO for transcutaneous delivery of drugs (Magnusson et al. These processes are dependent on the initial CD40-driven costimulatory signal delivered by activated helper T cells. which are bone marrow derived dendritic cells. Cvellic-Cabrillo et al. 1999). SN. its biodistribution. reduced isotype switching and memory cell responsiveness may indicate an inability of the DNA vaccine to generate germinal centers in lymphoid tissues (MacLennan. Adair et al.. Although the magnitude of immune responses
generated in this study were variable and unpredictable. detection of immune responses and protection from challenge suf®ced to show the proof of principle that we sought. DNA immunization in the chicken has been reported (Kodihalli et al. Heckert et al. most have not examined which isotype was involved.
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