Mechanisms of increased immunogenicity for DNA-based vaccines adsorbed onto cationic microparticles☆
Introduction
DNA vaccines induce both humoral and cellular immunity in a variety of preclinical models for infectious disease and provide a promising option for the development of subunit vaccines [1]. Naked DNA vaccines induce significant CTL activity in humans [2], but are unable to generate significant humoral response in humans after i.m. administration [2], [3], and require milligram doses of DNA to be effective. These limitations have led to investigation of strategies designed to increase the potency of DNA vaccines. Significant increases in immunogenicity have been demonstrated with enhanced gene expression [4], novel methods of physical delivery [5], [6], and adjuvant approaches including both co-expression of cytokines [7] and use of particulate adjuvant/delivery systems [8], [9]. Recently, we have described the enhanced immunogenicity obtained after i.m. administration of a pCMV plasmid expressing codon-optimized HIV p55 gag antigen formulated with novel cetyl trimethylammonium bromide-stabilized PLG3 particles [10]. The microparticle/DNA formulation induced significantly greater antibody responses than naked DNA and induced potent CTL responses at doses two orders of magnitude less than those required for naked DNA. Results obtained with rhesus macaques confirmed the efficacy of the formulation for antibody induction in primates [11].
Developing DNA vaccines potent enough to be clinically useful will likely require an understanding of the mechanism of action both of naked DNA vaccines themselves, and of adjuvant formulations that may be combined with DNA. Various adjuvants have been defined as activators of innate immunity [12], [13], delivery vehicles/depots, and agents which alter cell trafficking [14]. Mechanisms of action attributed to naked DNA vaccines described thus far include transfection of keratinocytes [15], muscle cells [16], and dendritic cells [17], as well as activation of innate immunity by unmethylated CpG-containing immunostimulatory sequences [18].
Recently, we have described the effects of tissue distribution and levels of expression for the gene of interest on the immunogenicity of naked DNA vaccines after i.m. injection in mice [19]. In the present study we administered PLG–CTAB–DNA to mice by i.m. injection and determined the distribution of the fluorescence-labeled vaccine formulations by microscopy. Persistence of DNA at the site of injection was measured by PCR, and expression of the encoded antigen by RT-PCR. Cellular influx to the injection site and activation of cells taking up fluorescence-labeled DNA have been characterized by flow cytometry. Improved delivery of DNA-encoding HIV p55 gag was correlated with the enhanced potency of the PLG–CTAB–DNA formulation.
Section snippets
Plasmids and formulations
The pCMVKm2.GagMod.SF2 plasmid encoding the HIV-1 p55 gag protein under the control of the cytomegalovirus early promoter [4] was purified by ion exchange chromatography using a Qiagen Endo Free Giga Kit and determined to be endotoxin free (<2.5 EU/ml). For distribution experiments, plasmid DNA-encoding β-galactosidase was labeled with a rhodamine-peptide nucleic acid (PNA)-clamp (Gene Therapy Systems, San Diego, CA). Plasmid DNA complexed with PNA-clamp does not alter the conformation or
Results
Several strategies were used to determine the fate of plasmid DNA adsorbed onto cationic microparticles in vivo. First, we used fluorescence-labeled DNA and cationic microparticles to delimit the macroscopic and microscopic distribution of the formulation following i.m. injection. Second, we characterized the stability of DNA and cellular influx at the site of injection. Third, we analyzed transgene expression at the injection site and in the draining lymph node by RT-PCR. Finally, we evaluated
Discussion
The enhancement of both humoral and cellular responses achieved by formulating an HIV-p55gag expressing DNA vaccine with PLG–CTAB microparticles has been clearly demonstrated in both mouse and guinea pig models [10], [26]. Antibody titers 1000-fold greater and CD8+ T cell responses 100-fold greater than those induced with threshold doses of naked HIV-p55gag DNA have been obtained with PLG–CTAB–DNA in mice. Titers equivalent to those obtained with gp120 protein in the presence of the potent
Acknowledgements
The authors thank Tim Brown for assistance with FACS analysis and Chris Kirk for assistance with cell isolation protocols. The authors acknowledge Jeffrey Ulmer, Marty Giedlin, and Gillis Otten for helpful discussions, and Noelle Cronen and Suzanne Stevenson for assistance in manuscript preparation.
References (41)
- et al.
Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers
Vaccine
(2000) - et al.
Immune responses and protection obtained by oral immunization with rotavirus VP4 and VP7 DNA vaccines encapsulated in microparticles
Virology
(1999) - et al.
Enhanced type I immune response to a hepatitis B DNA vaccine by formulation with calcium- or aluminum phosphate [In Process Citation]
Vaccine
(2000) The immune system evolved to discriminate infectious nonself from noninfectious self
Immunol. Today
(1992)Immunological concepts of vaccine adjuvant activity
Curr. Opin. Immunol.
(2000)Selective extraction of polyoma DNA from infected mouse cell cultures
J. Mol. Biol.
(1967)- et al.
Distribution of adjuvant MF59 and antigen gD2 after intramuscular injection in mice
Vaccine
(1999) - et al.
Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8(+) T-cell responses in vivo
Blood
(2000) - et al.
Immunomodulation by iscoms, immune stimulating complexes
Methods
(1999) - et al.
Revisiting PLA/PLGA microspheres: an analysis of their potential in parenteral vaccination
Eur. J. Pharm. Biopharm.
(2000)
Dendritic cells internalize vaccine adjuvant after intramuscular injection
Cell. Immunol.
DNA vaccines
Annu. Rev. Immunol.
First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response [see comments]
J. Infect. Dis.
Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene
J. Virol.
Increased DNA vaccine delivery and immunogenicity by electroporation in vivo
J. Immunol.
Oral gene delivery with chitosan–DNA nanoparticles generates immunologic protection in a murine model of peanut allergy [see comments]
Nat. Med.
Engineering enhancement of immune responses to DNA-based vaccines in a prostate cancer model in rhesus macaques through the use of cytokine gene adjuvants
Clin. Cancer Res.
Cationic microparticles: a potent delivery system for DNA vaccines
Proc. Natl. Acad. Sci. USA
Tolerance, danger, and the extended family
Annu. Rev. Immunol.
Cited by (77)
Characterization of rabies pDNA nanoparticulate vaccine in poloxamer 407 gel
2018, International Journal of PharmaceuticsCalcium phosphate embedded PLGA nanoparticles: A promising gene delivery vector with high gene loading and transfection efficiency
2012, International Journal of PharmaceuticsEffect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines
2011, VaccineCitation Excerpt :Besides, differences in the vaccination protocol with regard to pDNA dose, administration frequency, number of injections, site of injection and read-out may explain the discrepancy. As tissue localization may play a role in the efficacy of DNA vaccines [20], we studied the biodistribution at different time points after subcutaneous administration of dual radiolabeled pDNA-liposomes. Our results indicate that encapsulation of pDNA into cationic liposomes results in its retention at the site of injection after subcutaneous administration, related to the formation of a lipid depot.
Effect of nanoparticle coating on the immunogenicity of plasmid DNA vaccine encoding P. yoelii MSP-1 C-terminal
2011, VaccineCitation Excerpt :These requirements and low potency of DNA vaccines have attracted the development of several complexes and immunostimulatory molecules for their use in DNA-based vaccination [9–11] and how their application could be optimized for better immune response in malaria and other diseases [12–17]. High levels of gene expression in vitro were observed in different kinds of cell lines transfected with nanoparticle-formulated plasmid DNA [18–20], and immunization with nanoparticle- or cationic lipid-formulated plasmid DNA has improved antibody response and protection in malaria and other diseases [21–26]. However, in malaria DNA vaccination, levels of antibody induced and protection observed in immunized mice against challenge with Plasmodium are variable; from lower antibody response and no protection to higher antibody response and partial protection [21,27–29].
In vivo electroporation enhances the potency of poly-lactide co-glycolide (PLG) plasmid DNA immunization
2010, VaccineCitation Excerpt :The efficacy of PLG microparticle formulations such as ZYC300 relies on efficient recruitment of antigen presenting cells (APCs), and a particle size distribution that allows for uptake by APC [9–11]. Our lab and others [12] have previously shown that for polymer-based systems to be effective, the DNA needs to be physically associated with, or formulated into, the PLG particles. Reports do exist, however, in which intradermal coinjection of naked plasmid DNA and gold particles augments humoral responses without the nucleic acids being adsorbed onto or bound by particles [13].
Immunologic basis of vaccine vectors
2010, Immunity
- ☆
This work was supported in part by Grant S97-25 from the University of California BioSTAR Project.
- 1
Present address: Apoxis SA, 20 Avenue de Sevelin, Lausanne CH-1004, Switzerland.
- 2
Present address: Dynavax Technologies, 717 Potter Street, Suite 100, Berkeley, CA 94710, USA.