Elsevier

Cellular Immunology

Volume 225, Issue 1, September 2003, Pages 12-20
Cellular Immunology

Mechanisms of increased immunogenicity for DNA-based vaccines adsorbed onto cationic microparticles

https://doi.org/10.1016/j.cellimm.2003.09.003Get rights and content

Abstract

Investigation into the mechanism of action of vaccine adjuvants provides opportunities to define basic immune principles underlying the induction of strong immune responses and insights useful for the rational development of subunit vaccines. A novel HIV vaccine composed of plasmid DNA-encoding p55 gag formulated with poly-lactide-co-glycolide microparticles (PLG) and cetyl trimethyl ammonium bromide (CTAB) elicits both serum antibody titers and cytotoxic lymphocyte activity in mice at doses two orders of magnitude lower than those required for comparable response to plasmid DNA in saline. Using this model, we demonstrated the increase in potency requires the DNA to be complexed to the PLG–CTAB microparticles. Furthermore, the PLG–CTAB–DNA formulation increased the persistence of DNA at the injection site, recruited mononuclear phagocytes to the site of injection, and activated a population of antigen presenting cells. Intramuscular immunization with the PLG–CTAB–DNA complex induced antigen expression at both the injection site and the draining lymph node. These findings demonstrate that the PLG–CTAB–DNA formulation exhibits multiple mechanisms of immunopotentiation.

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)

  • M. Dupuis et al.

    Dendritic cells internalize vaccine adjuvant after intramuscular injection

    Cell. Immunol.

    (1998)
  • J.J. Donnelly et al.

    DNA vaccines

    Annu. Rev. Immunol.

    (1997)
  • R.R. MacGregor et al.

    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.

    (1998)
  • J. zur Megede et al.

    Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene

    J. Virol.

    (2000)
  • G. Widera et al.

    Increased DNA vaccine delivery and immunogenicity by electroporation in vivo

    J. Immunol.

    (2000)
  • K. Roy et al.

    Oral gene delivery with chitosan–DNA nanoparticles generates immunologic protection in a murine model of peanut allergy [see comments]

    Nat. Med.

    (1999)
  • J.J. Kim et al.

    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.

    (2001)
  • M. Singh et al.

    Cationic microparticles: a potent delivery system for DNA vaccines

    Proc. Natl. Acad. Sci. USA

    (2000)
  • G.R. Otten, M. Chen, B. Doe, J. Kazzaz, Y. Lian, H.M. Liu, L. Leung, G. Ott, J.M. Polo, M. Shaefer, M. Selby, M. Singh,...
  • P. Matzinger

    Tolerance, danger, and the extended family

    Annu. Rev. Immunol.

    (1994)
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    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.

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