| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Vaccines Research, Chiron Corporation, Emeryville, CA 94608; and
Genetronics, Inc., San Diego, CA 92121
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
One reason for the lack of efficacy in larger animals may be inefficient uptake of DNA by cells in situ. Hence, we sought to test whether cellular uptake of DNA was a significant limitation to efficient transfection in vivo and subsequent induction of immune responses. To this end, we used the technique of electroporation, which is widely used in vitro to effectively introduce DNA into eukaryotic cells and bacteria. Application of short electrical pulses to the target cells permeabilizes the cell membrane, thereby facilitating DNA uptake. Recently, it has been found that applying an electric field to tissues in vivo significantly increases DNA uptake and gene expression (for review, see Ref. 5). This has been shown for reporter genes and for genes of interest for therapeutic applications, such as erythropoietin (6) and HSV-TK (7). Among the tissues targeted for in vivo electroporation have been skin (8, 9), liver (10, 11), tumors (12, 13, 14), and muscle (15). Facilitation of gene expression in vivo by electroporation of plasmid DNA has implications for both vaccine and gene therapy applications. In this study, we show that increased Ag expression after DNA injection into muscle significantly increases the potency of DNA vaccines in mice, guinea pigs, and rabbits. Therefore, this technology may prove useful at increasing the effectiveness of DNA vaccines in larger animals, such as nonhuman primates and humans.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To generate the hepatitis B surface Ag
(HBsAg)2 expression
construct, the 1.4-kb BamHI fragment of pAM6 (American Type
Culture Collection (ATCC), Manassas, VA) was inserted into pEF-BOS, an
eukaryotic expression vector containing the human elongation factor
1
promoter and first intron and the polyadenylation signal from
human G-CSF cDNA in a pUC119 prokaryotic backbone (16).
pAM6 (ATCC 45020) is a genomic clone of hepatitis B virus (HBV)
serotype adw, and the 1.4-kb BamHI fragment was shown to
encode the "small" HBV surface Ag (HBsAg) (17). The
luciferase expression plasmid was obtained from Promega (Madison, WI).
Escherichia coli strain XL-1 Blue (Stratagene, La Jolla,
CA), carrying the expression plasmids, was grown in LB; antibiotic
selection used 50 µg/ml ampicillin. Plasmids were purified using
Qiagen Endo Free Plasmid Maxi Kits (Qiagen, Chatsworth, CA) according
to the manufacturer’s instructions.
The plasmid pCMV HIV gag (18) was grown in E. coli strain HB101, purified using a Qiagen Endofree Plasmid Giga kit (Qiagen), and resuspended in 0.9% sodium chloride (Abbott Laboratories, North Chicago, IL). The pCMV vector used contains the immediate early enhancer/promoter of cytomegalovirus and a bovine growth hormone terminator, and is described in detail (18). The HIV gag DNA vaccine (pCMV HIV gag) contains a synthetically constructed p55 gag gene, with codons reflecting mammalian usage, derived from the HIV-1 SF2 strain as previously described (19). The HIV env DNA vaccine (pCMV HIV env) contains a 2.1-kb EcoRI-XbaI fragment encoding a human tissue plasminogen activator (tPA) signal sequence and the reading frame for the ectodomain of the Env protein of the HIV-1 US4 strain codon optimized for expression in mammalian cells. The open reading frame in this construct is truncated before the transmembrane spanning region of the Env allowing high level expression of secreted gp140 protein in transfected cells (S. W. Barnett, L. Leung and H. Legg, unpublished observations).
Expression of the encoded Ags was verified by transient expression studies in B16 cells. For Lipofectin (Life Technologies/BRL) transfection, 1 µg of each plasmid DNA was used following the manufacturer’s protocol; 5 x 105 cells were used per 3-cm tissue culture dish; incubation time for DNA-Lipofectin on cells was 4 h. Supernatants were harvested 36 h after removal of the DNA-Lipofectin solution and cells were lysed in 500 µl PBS/0.5% TritonX100 (Mallinckrodt). Expression of HBsAg in cell lysates and supernatants was detected by the AUSZYME enzye immunoassay (Abbott). Luciferase activity in cell lysates was detected by commercial Luciferase Reporter Gene Assay (Roche, Indianapolis, IN). Expression of HIV gag and env protein was determined as described previously (18).
Animals
Female BALB/c, BALB/c x C57BL/6 F1 (CB6F1), and nude mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN), Charles River Breeding Laboratories (Wilmington, MA), Taconic Farms (Germantown, NY), or The Jackson Laboratory (Bar Harbor, ME) used at 6–10 wk of age and housed at Genetronics (San Diego, CA) and Chiron (Emeryville, CA). Rabbits were obtained and housed at Josman LLC (Napa, CA). Female guinea pigs were obtained from Elm Breeding Labs (Chelmsford, MA) and housed at Chiron in an American Association of Laboratory Animal Care-accredited facility.
DNA immunization and in vivo electroporation
Mice were anesthetized using 4 parts ketamine HCl, 100 mg/ml stock solution (Fort Dodge Animal Health, Fort Dodge, IA), to 1 part xylazine, 20 mg/ml (Lloyd Labs, Shenandoah, IA). The mice received 1 µl/g of body weight intramuscularly in the posterior thigh. The skin overlying the tibialis anterior (TA) muscle was shaved, and the animals were injected with amounts of plasmid DNA as described in a volume of 50 µl. To control needle depth, a 0.3-ml insulin syringe was covered with polyethylene tubing (inside diameter, 0.38) to expose only the bevel. Two-needle array electrodes (BTX, San Diego, CA) were inserted into the muscle immediately after DNA delivery for electroporation. The distance between the electrodes was 5 mm, and the array was inserted longitudinally relative to the muscle fibers. In vivo electroporation parameters were: 20 V/mm distance between the electrodes; 50-ms pulse length; 6 pulses with reversal of polarity after 3 pulses, at 1, given by a BTX 820 square wave generator.
In rabbits, a total of 0.5 mg HIV gag DNA and 1 mg HIV env DNA in 900 µl PBS was injected into the gracilis muscles of both hind limbs after shaving and anesthesia. In guinea pigs, a total of 50 or 500 µg each of HIV gag and HIV env DNA was given as above. Electroporation was performed with a six-needle electrode array forming a circle (Genetronics, San Diego, CA). The diameter of the electrode array was 1 cm, with a needle length of 1 cm. Six electroporation pulses of 20 V/mm, 50-ms pulse length, 1 pulse/s were given by a BTX 820 square wave generator, combined with an electronic switch (Genetronics) to rotate the electric field in 60-degree increments after each discharge (20).
Measurement of Ab responses
At various times following immunization, blood was collected from anesthetized animals and serum was recovered by centrifugation. Anti-hepatitis B surface Abs were measured using the AUSAB EIA Diagnostic Kit, and quantification in milli-International Units/milliliter was done in parallel with the AUSAB Quantification Panel following instructions provided by the manufacturer (Abbott).
Anti-HIV gag Abs in mice were measured by ELISA as follows. Wells of Immulon 2 HB U-bottom microtiter plates (Dynex Technologies, Chantilly, VA) were coated with HIV p55 protein at 5 µg/ml in PBS, 50 µl/well, and incubated at 4°C overnight. The plates were washed six times with wash buffer (PBS, 0.1% Tween 20 (Sigma, St. Louis, MO)) and blocked at 37°C for 1 h with 150 µl/well blocking buffer (PBS, 0.1% Tween 20 (Sigma), 1% goat serum). Test sera were diluted 1/25 followed by serial 3-fold dilutions in blocking buffer. The block solution was aspirated; then the plates were incubated at 37°C for 2 h with 50 µl/well of each serum dilution. After six washings, the plates were incubated for 1 h at 37°C with 50 µl/well goat anti-mouse IgG-HRP (Caltag, Burlingame, CA) diluted 1/40,000 in block buffer. After a final six washes, the plates were developed with OPD for 30 min. The OPD developer consists of 1 tablet (10 mg) o-phenylenediamine, 12 ml buffer (0.1 M citric acid, 0.1 M dibasic sodium phosphate), and 5 µl 30% H2O2. The reaction was stopped with 50 µl/well 4 N H2SO4, and optical density was measured at dual wavelengths 492–690. The reported titers correspond to the reciprocal of the serum dilution, producing an absorbance value of 1.0. For rabbits, the ELISA procedure was as for mice with the following changes. The blocking buffer was PBS, 0.5% casein, and 5% goat serum; the dilution buffer was blocking buffer plus 0.3% Tween 20; the secondary Ab was goat anti-rabbit IgG used at 1/20,000; and the OD cutoff used was 0.6. For guinea pigs, the ELISA procedure was as for mouse except that the secondary Ab was goat anti-pig IgG used at 1/20,000.
For measurement of anti-env Abs in rabbits and guinea pigs, Nunc Immunoplate U96 Maxisorp plates (Nalge Nunc International, Rochester, NY) were coated with 200 ng/well recombinant gp120SF2 protein and incubated for at least 14 h at 4°C. Between steps, the plates were washed in a buffer containing 137 mM NaCl and 0.05% Triton X-100. Serum samples were initially diluted 1/25 or 1/100 (in a buffer containing 100 mM sodium phosphate, 0.1% casein, 1 mM EDTA, 1% Triton X-100, 0.5 M NaCl, and 0.01% thimerosal, pH 7.5) and were serially diluted 3-fold. The plates were incubated for 50 min for rabbit sera or 1 h for guinea pig sera, at 37°C. After a washing in buffer containing 137 mM NaCl, 0.05% Triton X-100, the samples were then reacted with an HRP-conjugated Ab against the appropriate animal (50 min for rabbit sera; 30 min for guinea pig sera; at 37°C.) The plates were then developed using either a TMB substrate kit (Pierce, Rockford, IL) for rabbit sera (50 min at 37°C) or ABTS (Sigma), for the guinea pig sera (30 min at 37°C). The plates were stopped with either 2 N H2SO4 or 10% SDS, respectively, and read at wavelengths of 450 or 415 nm, respectively. Anti-env Ab responses were measured as the dilution at which an OD of 0.6 was achieved.
Measurement of T cell responses
A recombinant vaccinia virus encoding the
HIV-1SF2 gag-pol genes (rVVgag-pol) has been
described previously (21). Four weeks or more after gag
DNA immunization, mice were challenged with an i.p.injection of 1
x 107 PFU rVVgag-pol. Five days later, spleens
were harvested and stimulated with the
H-2d-restricted p7g gag peptide (21)
and then stained for intracellular IFN-
, as follows.
Erythrocyte-depleted single-cell suspensions were prepared by treatment
with Tris-buffered NH4Cl (Sigma), and 1–2
x 106 nucleated spleen cells were cultured in
duplicate at 37°C in the presence or absence of 10 µg/ml p7g
peptide. Monensin (PharMingen, San Diego, CA) was added to block
cytokine secretion. After 3–5 h cells were washed, incubated with
anti-CD16/32 (PharMingen) to block Fc receptors, stained with
FITC-conjugated CD8 mAb (PharMingen), and fixed overnight at 4°C in
2% (w/v) paraformaldehyde. The following day, cells were treated with
0.5% (w/v) saponin (Sigma) and then incubated with PE-conjugated mouse
IFN- mAb (PharMingen) in the presence of 0.1% (w/v) saponin,
washed, and analyzed using a FACSCalibur flow cytometer (Becton
Dickinson, San Jose, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has previously been shown that in vivo electroporation can
substantially increase gene expression in muscle up to 100-fold. To
test whether DNA vaccine potency could be improved by this method, we
used plasmid vectors encoding HBsAg, HIV env and HIV gag. A plasmid
expressing HBsAg driven by the human elongation factor 1 promoter
(E1-sAg) was injected into the tibialis anterior muscles of nude mice,
and in one cohort pulses of electric current were applied after DNA
injection. Because low voltage, long duration pulses have been found to
be most efficacious for increase of DNA uptake in tissues in vivo
(15, 22), we applied 6 square wave pulses of 100 V, 50-ms
pulse duration. For this purpose, a two-needle electrode array with a
gap of 5 mm between the needles was positioned so that the needles were
centered over the DNA injection site and was inserted directly into the
muscle without any surgical removal of skin. After 3 pulses, the
polarity of the electric field was reversed by switching the connectors
to the pulse generator. No signs of adverse reactions were observed in
any animal after muscle electroporation. Mice were sacrificed at
different time points after DNA administration, as indicated in Fig. 1
, and HBsAg expression was measured in
serum and muscle tissues, using the AUSZYME MONOCLONAL assay (Abbott),
which is an enzyme immunoassay for the detection of HBsAg in human
serum or plasma. No significant HBsAg levels were detected in the sera
or muscle tissues of immunodeficient nude mice receiving DNA only. In
contrast, in the in vivo electroporation-treated cohort, HBsAg was
detected within 1 day after DNA injection in muscle tissue. Peak levels
of HBsAg were reached by day 5 and remained practically unchanged at
least 20 days after DNA injection (Fig. 1A). No HBsAg was
detected in sera of any animal, unlike observations previously reported
by others (23), likely as a consequence of relatively low
level expression of HBsAg by the E1-sAg plasmid compared with vectors
containing the CMV promoter. In immunocompetent BALB/c mice, peak
expression of HBsAg was similarly found in electroporated muscle
tissues at day 5, but expression began to wane by day 13 (Fig. 1B), possibly as a consequence of CTL activity directed
toward transfected cells or clearance of Ag by the formation of Ag-Ab
complexes.
|
3000 relative light units vs 500,000 relative
light units). In the case of ß-galactosidase, staining of muscle
tissue sections revealed a substantial increase in the number of muscle
fibers detectably transfected, as well as an apparent increase in the
distribution of expression within the tissue (M. Dupuis, K. Denis-Mize,
C. Woo, C. Goldbeck, M. J. Selby, J. B. Ulmer, J. J. Donnelly, G. Ott,
and D. M. McDonald et al., unpublished observations). These results
correlated with an increase in the distribution and cellular uptake of
plasmid, as judged by fluorescence using a rhodamine-tagged plasmid.
Therefore, electroporation of DNA-injected muscle tissues resulted in
more efficient transfection of muscle cells in situ, leading to higher
levels of protein production. Enhancement of DNA vaccine potency by electroporation
To test whether in vivo electroporation could increase the
magnitude of immune responses induced by DNA vaccination, several lines
of experimentation were undertaken. First, BALB/c mice were immunized
with the low expressing HBsAg DNA vaccine at doses ranging from 0.5 to
50 µg. Anti-hepatitis B surface Ab titers were measured using the
ABBOTT AUSAB assay and expressed in standard milli-international units
per milliliter serum. A level of 10 mIU/ml is considered protective
against HBV infection in humans. In electroporation-treated cohorts
that received 3 µg DNA or more, strong and consistent Ab responses
were found 2 wk after the first immunization (Fig. 2). These responses were boosted to
titers of >10,000 mIU/ml 2 wk after the second immunization for the
high dose groups and remained stable for at least 3 mo. One microgram
or less of DNA was found not be sufficient to induce reliable immune
responses, whether or not animals were treated with electroporation
(not shown). In contrast, mice that received 5, 20, or 50 µg HBsAg
DNA without electroporation showed only weak to moderate anti-HBsAg
Ab titers (up to 30 mIU/ml), with not all animals responding even
after the boost. Therefore, electroporation substantially increased the
potency of a weakly immunogenic DNA vaccine.
|
and
4, readily primes Ab and T cell responses
in mice after a single immunization of 0.2 to 2 µg of DNA. Yet,
electroporation substantially enhanced these responses even further.
After a single immunization of 10 µg, anti-gag Ab titers were
increased up to 20-fold by electroporation, and this was maintained for
at least 12 wk (Fig. 3A). An even greater enhancement in Ab
responses was observed in BALB/c mice (>100-fold), possibly related to
an overall lower Ab response to HIV gag in this mouse strain. In
addition to increasing the magnitude of Ab responses, electroporation
significantly lowered the dose of DNA required to induce immune
responses. Strong Ab responses were seen in electroporation-treated
mice after a single dose of 20–200 ng of DNA, whereas similar titers
were achieved without electroporation only at a 100-fold higher DNA
dose (Fig. 3B). The variability of the titers from animal to
animal within a group appeared to be less in the
electroporation-treated mice. This observation is consistent with
previous reports on the consistency of expression levels after
electroporation (24). Anti-gag T cell responses were also
increased by electroporation. Immunized mice were challenged with a
recombinant vaccinia virus expressing gag, and 5 days later spleens
were removed and restimulated briefly in vitro with a known MHC class
I-restricted CTL peptide. IFN- production by
CD8+ T cells, as measured by flow cytometry,
indicated that up to 5% of total CD8+ T cells
were gag specific (Fig. 4). At doses of gag DNA ranging from 200 ng to
20 µg, electroporation increased the frequency of gag-specific CTL.
Therefore, electroporation increased the immunogenicity of DNA vaccines
in mice for both Ab and T cell responses.
|
|
A).
Thereafter, anti-env Ab titers were consistently 5- to 10-fold
higher in the treated group. In those same rabbits, significant
anti-gag Ab responses were seen only in animals that had received
electroporation treatment (Fig. 5B). In the untreated
DNA-vaccinated animals, little or no titers were detected at any time
during the study, even after three immunizations. In guinea pigs,
similar enhancement of Ab responses was seen in electroporation-treated
groups. Animals were immunized with a combination of 50 or 500 µg
each of HIV gag and HIV env DNA, and Ab responses were monitored after
one and two immunizations. No responses were seen in any animal,
treated or untreated, after a single immunization (data not shown). In
the untreated DNA-vaccinated animals, no measurable anti-gag or
anti-env Abs were detected even after two immunizations at the high
DNA dose (Fig. 6). However, Ab responses
against both Ags were seen in all guinea pigs immunized with the high
DNA dose and treated with electroporation. Therefore, electroporation
substantially enhanced the potency of DNA vaccines in mice and larger
animals.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this study, in vivo electroporation of muscle tissue was performed by six monopolar electric pulses using low voltage (nominal electric field of 200 V/cm), long pulse duration (50 ms) conditions. In addition to these conditions, trains of low voltage, high frequency bipolar pulses have been found to increase gene expression in muscle (6), demonstrating that the electrical stimuli can be delivered in different ways to the tissue. In vivo electroporation does differ, however, from conditions used in vitro, where high voltage, short pulse duration conditions are typically used. In vitro, electroporation pulses modify biological membranes and facilitate penetration of cells by DNA molecules, with only minor damage to these membranes (25). It is also conceivable that these conditions can affect the integrity of the nuclear membrane, thereby allowing freer passage of plasmid DNA into the nucleus. This may be particularly relevant for delivery of DNA into the nuclei of nondividing cells, such as muscle cells.
The means by which electroporation increases DNA vaccine potency is not yet known, but is likely to be related to increased expression of encoded Ag. This could simply provide more mass of Ag available for priming of immune responses. However, other factors may also be involved. For instance, whereas transfection of muscle cells is increased by electroporation, transfection of other cells, such as APCs, may also be facilitated. However, we have not detected any transfected APCs in muscle or draining lymph node after injection of DNA with or without electroporation (M. Dupuis, K. Denis-Mize, C. Woo, C. Goldbeck, M. J. Selby, J. B. Ulmer, J. J. Donnelly, G. Ott, and D. M. McDonald, unpublished observations). Another possibility is that the application of an electric field directly in the tissue could result in an inflammatory response that aids in the priming of immune responses against the DNA-encoded Ags. However, electroporation treatment after vaccination with protein-based vaccines did not result in an increase in immune responses (M. J. Selby, C. Goldbeck, and J. B. Ulmer, unpublished observations). Therefore, any inflammatory responses that may have been elicited by the conditions of electroporation treatment were not sufficient to alter immune priming. Hence, the observed increase in Ag expression in muscle cells likely plays a predominant role in the enhancement of DNA vaccine potency by electroporation. This hypothesis is consistent with previous observations that production of Ag by non-APCs, such as tumor cells (26), fibroblasts (27), and muscle cells (28, 29), is sufficient for priming immune responses.
DNA vaccines hold promise for use in humans. However, there are significant limitations with current technologies that have prevented the full effectiveness of DNA vaccines in larger animals. We have shown here that uptake of DNA and delivery to the cytoplasm is a barrier to efficient transfection of cells in vivo and that electroporation can circumvent this barrier. In vivo electroporation substantially increases DNA delivery and DNA vaccine potency, appears to be well tolerated by the animals, and is a simple technique that takes only a few seconds after inoculation. Electroporation has already been demonstrated to substantially increase the effectiveness of nonviral gene therapy in vivo, and our results extend the usefulness of this methodology to the field of DNA vaccination.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: HBsAg, hepatitis B surface Ag; TA, tibialis anterior; HBV, hepatitis B virus.
Received for publication December 10, 1999. Accepted for publication February 24, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  Y. Dai, Y. Zhu, D. A. Harn, X. Wang, J. Tang, S. Zhao, F. Lu, and X. Guan DNA Vaccination by Electroporation and Boosting with Recombinant Proteins Enhances the Efficacy of DNA Vaccines for Schistosomiasis Japonica Clin. Vaccine Immunol., December 1, 2009; 16(12): 1796 - 1803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Meoni, E. Faenzi, E. Frigimelica, L. Zedda, D. Skibinski, S. Giovinazzi, A. Bonci, R. Petracca, E. Bartolini, G. Galli, et al. CT043, a Protective Antigen That Induces a CD4+ Th1 Response during Chlamydia trachomatis Infection in Mice and Humans Infect. Immun., September 1, 2009; 77(9): 4168 - 4176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Chua, J. Gao, Y.-S. Ho, X. Xu, I-C. Kuo, K.-Y. Chua, H. Wang, R. C. Hamdy, J. C. Reed, and B. H.L. Chua Over-expression of a modified bifunctional apoptosis regulator protects against cardiac injury and doxorubicin-induced cardiotoxicity in transgenic mice Cardiovasc Res, January 1, 2009; 81(1): 20 - 27. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Mennuni, S. Ugel, F. Mori, B. Cipriani, M. Iezzi, T. Pannellini, D. Lazzaro, G. Ciliberto, N. La Monica, P. Zanovello, et al. Preventive Vaccination with Telomerase Controls Tumor Growth in Genetically Engineered and Carcinogen-Induced Mouse Models of Cancer Cancer Res., December 1, 2008; 68(23): 9865 - 9874. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Liu, R. Kjeken, I. Mathiesen, and D. H. Barouch Recruitment of Antigen-Presenting Cells to the Site of Inoculation and Augmentation of Human Immunodeficiency Virus Type 1 DNA Vaccine Immunogenicity by In Vivo Electroporation J. Virol., June 1, 2008; 82(11): 5643 - 5649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Schoenly and D. B. Weiner Human Immunodeficiency Virus Type 1 Vaccine Development: Recent Advances in the Cytotoxic T-Lymphocyte Platform "Spotty Business" J. Virol., April 1, 2008; 82(7): 3166 - 3180. [Full Text] [PDF]
|
|
|
|
 |
|
 K. C. Bagley Genetic adjuvant therapy for pancreatic cancer and other solid tumours Gut, March 1, 2008; 57(3): 289 - 291. [Full Text] [PDF]
|
|
|
|
|
|
D. Zhu, J. N. Williams, J. Rice, F. K. Stevenson, J. E. Heckels, and M. Christodoulides A DNA Fusion Vaccine Induces Bactericidal Antibodies to a Peptide Epitope from the PorA Porin of Neisseria meningitidis Infect. Immun., January 1, 2008; 76(1): 334 - 338. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Shirota, L. Petrenko, C. Hong, and D. M. Klinman Potential of Transfected Muscle Cells to Contribute to DNA Vaccine Immunogenicity J. Immunol., July 1, 2007; 179(1): 329 - 336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Luckay, M. K. Sidhu, R. Kjeken, S. Megati, S.-Y. Chong, V. Roopchand, D. Garcia-Hand, R. Abdullah, R. Braun, D. C. Montefiori, et al. Effect of Plasmid DNA Vaccine Design and In Vivo Electroporation on the Resulting Vaccine-Specific Immune Responses in Rhesus Macaques J. Virol., May 15, 2007; 81(10): 5257 - 5269. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-R. Huang, H.-L. Wu, P.-J. Chen, and D.-S. Chen An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection PNAS, November 21, 2006; 103(47): 17862 - 17867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Capone, I. Zampaglione, A. Vitelli, M. Pezzanera, L. Kierstead, J. Burns, L. Ruggeri, M. Arcuri, M. Cappelletti, A. Meola, et al. Modulation of the Immune Response Induced by Gene Electrotransfer of a Hepatitis C Virus DNA Vaccine in Nonhuman Primates J. Immunol., November 15, 2006; 177(10): 7462 - 7471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Hegde, Z. Liu, G. Mackay, M. Smith, Y. Chebloune, O. Narayan, and D. K. Singh Antigen Expression Kinetics and Immune Responses of Mice Immunized with Noninfectious Simian-Human Immunodeficiency Virus DNA J. Virol., December 1, 2005; 79(23): 14688 - 14697. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Lian, I. Srivastava, V. R. Gomez-Roman, J. zur Megede, Y. Sun, E. Kan, S. Hilt, S. Engelbrecht, S. Himathongkham, P. A. Luciw, et al. Evaluation of Envelope Vaccines Derived from the South African Subtype C Human Immunodeficiency Virus Type 1 TV1 Strain J. Virol., November 1, 2005; 79(21): 13338 - 13349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Beddows, N. Schulke, M. Kirschner, K. Barnes, M. Franti, E. Michael, T. Ketas, R. W. Sanders, P. J. Maddon, W. C. Olson, et al. Evaluating the Immunogenicity of a Disulfide-Stabilized, Cleaved, Trimeric Form of the Envelope Glycoprotein Complex of Human Immunodeficiency Virus Type 1 J. Virol., July 15, 2005; 79(14): 8812 - 8827. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Barouch, Z.-y. Yang, W.-p. Kong, B. Korioth-Schmitz, S. M. Sumida, D. M. Truitt, M. G. Kishko, J. C. Arthur, A. Miura, J. R. Mascola, et al. A Human T-Cell Leukemia Virus Type 1 Regulatory Element Enhances the Immunogenicity of Human Immunodeficiency Virus Type 1 DNA Vaccines in Mice and Nonhuman Primates J. Virol., July 15, 2005; 79(14): 8828 - 8834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Donnelly, B. Wahren, and M. A. Liu DNA Vaccines: Progress and Challenges J. Immunol., July 15, 2005; 175(2): 633 - 639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. R. Otten, M. Schaefer, B. Doe, H. Liu, I. Srivastava, J. zur Megede, J. Kazzaz, Y. Lian, M. Singh, M. Ugozzoli, et al. Enhanced Potency of Plasmid DNA Microparticle Human Immunodeficiency Virus Vaccines in Rhesus Macaques by Using a Priming-Boosting Regimen with Recombinant Proteins J. Virol., July 1, 2005; 79(13): 8189 - 8200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Buchan, E. Gronevik, I. Mathiesen, C. A. King, F. K. Stevenson, and J. Rice Electroporation as a "Prime/Boost" Strategy for Naked DNA Vaccination against a Tumor Antigen J. Immunol., May 15, 2005; 174(10): 6292 - 6298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Chen, L. Zhang, C. Qin, L. Ba, C. E. Yi, F. Zhang, Q. Wei, T. He, W. Yu, J. Yu, et al. Recombinant Modified Vaccinia Virus Ankara Expressing the Spike Glycoprotein of Severe Acute Respiratory Syndrome Coronavirus Induces Protective Neutralizing Antibodies Primarily Targeting the Receptor Binding Region J. Virol., March 1, 2005; 79(5): 2678 - 2688. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hechard, O. Grepinet, and A. Rodolakis Molecular cloning of the Chlamydophila abortus groEL gene and evaluation of its protective efficacy in a murine model by genetic vaccination J. Med. Microbiol., September 1, 2004; 53(9): 861 - 868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Quaglino, M. Iezzi, C. Mastini, A. Amici, F. Pericle, E. Di Carlo, S. M. Pupa, C. De Giovanni, M. Spadaro, C. Curcio, et al. Electroporated DNA Vaccine Clears Away Multifocal Mammary Carcinomas in Her-2/neu Transgenic Mice Cancer Res., April 15, 2004; 64(8): 2858 - 2864. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Giri, K. E. Ugen, and D. B. Weiner DNA Vaccines against Human Immunodeficiency Virus Type 1 in the Past Decade Clin. Microbiol. Rev., April 1, 2004; 17(2): 370 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Munoz-Montesino, E. Andrews, R. Rivers, A. Gonzalez-Smith, G. Moraga-Cid, H. Folch, S. Cespedes, and A. A. Onate Intraspleen Delivery of a DNA Vaccine Coding for Superoxide Dismutase (SOD) of Brucella abortus Induces SOD-Specific CD4+ and CD8+ T Cells Infect. Immun., April 1, 2004; 72(4): 2081 - 2087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. zur Megede, G. R. Otten, B. Doe, H. Liu, L. Leung, J. B. Ulmer, J. J. Donnelly, and S. W. Barnett Expression and Immunogenicity of Sequence-Modified Human Immunodeficiency Virus Type 1 Subtype B pol and gagpol DNA Vaccines J. Virol., June 1, 2003; 77(11): 6197 - 6207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Otten, M. Schaefer, C. Greer, M. Calderon-Cacia, D. Coit, J. Kazzaz, A. Medina-Selby, M. Selby, M. Singh, M. Ugozzoli, et al. Induction of Broad and Potent Anti-Human Immunodeficiency Virus Immune Responses in Rhesus Macaques by Priming with a DNA Vaccine and Boosting with Protein-Adsorbed Polylactide Coglycolide Microparticles J. Virol., May 15, 2003; 77(10): 6087 - 6092. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. K. Srivastava and M. A. Liu Gene Vaccines Ann Intern Med, April 1, 2003; 138(7): 550 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. E. Heslop, F. K. Stevenson, and J. J. Molldrem Immunotherapy of Hematologic Malignancy Hematology, January 1, 2003; 2003(1): 331 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Charo, M. Sundback, K. Wasserman, A.-M. T. Ciupitu, B. Mirzai, R. van der Zee, and R. Kiessling Marked Enhancement of the Antigen-Specific Immune Response by Combining Plasmid DNA-Based Immunization with a Schiff Base-Forming Drug Infect. Immun., December 1, 2002; 70(12): 6652 - 6657. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kalat, Z. Kupcu, S. Schuller, D. Zalusky, M. Zehetner, W. Paster, and T. Schweighoffer In Vivo Plasmid Electroporation Induces Tumor Antigen-specific CD8+ T-Cell Responses and Delays Tumor Growth in a Syngeneic Mouse Melanoma Model Cancer Res., October 1, 2002; 62(19): 5489 - 5494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Paster, M. Kalat, M. Zehetner, and T. Schweighoffer Structural Elements of a Protein Antigen Determine Immunogenicity of the Embedded MHC Class I-Restricted T Cell Epitope J. Immunol., September 15, 2002; 169(6): 2937 - 2946. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. O'Hagan, M. Singh, M. Ugozzoli, C. Wild, S. Barnett, M. Chen, M. Schaefer, B. Doe, G. R. Otten, and J. B. Ulmer Induction of Potent Immune Responses by Cationic Microparticles with Adsorbed Human Immunodeficiency Virus DNA Vaccines J. Virol., October 1, 2001; 75(19): 9037 - 9043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zucchelli, S. Capone, E. Fattori, A. Folgori, A. Di Marco, D. Casimiro, A. J. Simon, R. Laufer, N. La Monica, R. Cortese, et al. Enhancing B- and T-Cell Immune Response to a Hepatitis C Virus E2 DNA Vaccine by Intramuscular Electrical Gene Transfer J. Virol., December 15, 2000; 74(24): 11598 - 11607. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and electroporated
mice (
).
