Effect on antibody and T-cell responses of mixing five GMP-produced DNA plasmids and administration with plasmid expressing GM-CSF.

One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.

Enhancement of the immune response in rabbits to a malaria DNA vaccine by immunization with a needle-free jet device.

We compared the needle free jet device device Biojector with syringe/needle as a method to administer a DNA vaccine encoding the Plasmodium falciparum circumsporozoite protein (PfCSP) in albino rabbits. A group of three rabbits was injected by the intramuscular (IM) route using a syringe/needle combination, a second group IM with the Biojector device and a third group both IM and intradermal (ID) using the Biojector. When animals were immunized with the Biojector IM or IM/ID as compared to the syringe/needle IM, we observed 10- and 50-fold greater antibody titers, as measured by enzyme immunoassay (EIA) and indirect fluorescence antibody test (IFAT), respectively. We also observed that the Biojector conferred a greater ability to prime the immune system as compared with the needle. The subsequent boosting of all animals with a recombinant canary pox virus (ALVAC) expressing PfCSP induced significantly higher titers in both Biojector groups of rabbits as compared with the needle and naive animals. These results provided the foundation for a clinical trial using the same regime.

Induction of CD4(+) T cell-dependent CD8(+) type 1 responses in humans by a malaria DNA vaccine.

We assessed immunogenicity of a malaria DNA vaccine administered by needle i.m. or needleless jet injection [i.m. or i.m./intradermally (i.d.)] in 14 volunteers. Antigen-specific IFN-gamma responses were detected by enzyme-linked immunospot (ELISPOT) assays in all subjects to multiple 9- to 23-aa peptides containing class I and/or class II restricted epitopes, and were dependent on both CD8(+) and CD4(+) T cells. Overall, frequency of response was significantly greater after i.m. jet injection. CD8(+)-dependent cytotoxic T lymphocytes (CTL) were detected in 8/14 volunteers. Demonstration in humans of elicitation of the class I restricted IFN-gamma responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.

Safety of a GM-CSF adjuvant-plasmid DNA malaria vaccine.

MuStDO 5 is a multivalent plasmid DNA vaccine for malaria comprised of five plasmid DNAs encoding five proteins from Plasmodium falciparum and one plasmid DNA encoding human GM-CSF. To evaluate the safety of MuStDO 5, a series of pre-clinical studies were conducted in mice and rabbits. In pharmacology studies in mice, GM-CSF could not be detected in the serum following either intramuscular or a combined intramuscular/intradermal administration of the vaccine, but was readily detected in the muscle following intramuscular administration. In a tissue distribution study in mice, MuStDO 5 plasmid DNA was detected by PCR initially in highly vascularized tissues, while at later time-points the plasmid DNA was detected primarily at the site(s) of injection. In GLP safety studies in mice and rabbits, repeated intramuscular/intradermal administration of the MuStDO 5 vaccine was found to be safe and well tolerated without any evidence of autoimmune pathology.

Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus.

Intramuscular immunization with a naked DNA plasmid expressing the Plasmodium yoelii circumsporozoite protein (pPyCSP) protects mice against challenge with P. yoelii sporozoites. This protection can be improved either by coadministration of a plasmid expressing murine GM-CSF (pGMCSF) or by boosting with recombinant poxvirus expressing the PyCSP. We now report that combining these two strategies, by first mixing the priming dose of pPyCSP with pGMCSF and then boosting with recombinant virus, can substantially increase vaccine effectiveness. Not only were immune responses and protection improved but the pPyCSP dose could be lowered from 100 microg to 1 microg with little loss of immunogenicity after boost with recombinant poxvirus. Comparing mice primed by the 1-microg doses of pPyCSP plus 1 microg pGMCSF with mice primed by 1-microg doses of pPyCSP alone, the former were better protected (60% vs 0) and had higher concentrations of Abs (titers of 163, 840 vs 5, 120 by indirect fluorescent Ab test against sporozoites), more ex vivo CTL activity (25% vs 7% specific lysis), and more IFN-gamma-secreting cells by enzyme-linked immunospot assay (1460 vs 280 IFN-gamma spot-forming cells/106 cells). Priming with plasmid vaccine plus pGMCSF and boosting with recombinant poxviruses strongly improves the immunogenicity and protective efficacy of DNA vaccination and allows for significant reduction of dose.

Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers.

DNA-based vaccines are considered to be potentially revolutionary due to their ease of production, low cost, long shelf life, lack of requirement for a cold chain and ability to induce good T-cell responses. Twenty healthy adult volunteers were enrolled in a Phase I safety and tolerability clinical study of a DNA vaccine encoding a malaria antigen. Volunteers received 3 intramuscular injections of one of four different dosages (20, 100, 500 and 2500 microg) of the Plasmodium falciparum circumsporozoite protein (PfCSP) plasmid DNA at monthly intervals and were followed for up to twelve months. Local reactogenicity and systemic symptoms were few and mild. There were no severe or serious adverse events, clinically significant biochemical or hematologic changes, or detectable anti-dsDNA antibodies. Despite induction of excellent CTL responses, intramuscular DNA vaccination via needle injection failed to induce detectable antigen-specific antibodies in any of the volunteers.

Plasmid DNA malaria vaccine: tissue distribution and safety studies in mice and rabbits.

To evaluate the safety of a plasmid DNA vaccine, tissue distribution studies in mice and safety studies in mice and rabbits were conducted with VCL-2510, a plasmid DNA encoding the gene for the malaria circumsporozoite protein from Plasmodium falciparum (PfCSP). After intramuscular administration, VCL-2510 plasmid DNA was detected initially in all of the highly vascularized tissues, but at later time points was found primarily in the muscle at the site of injection, where it persisted for up to 8 weeks. After intravenous administration, plasmid DNA initially distributed at a relatively low frequency to all the tissues examined except the gonads and brain. However, plasmid DNA rapidly cleared, and by 4 weeks postadministration could be detected only in the lung of one of six animals evaluated. In a safety study in mice, eight repeated intramuscular injections of VCL-2510 at plasmid DNA doses of 1, 10, and 100 microg had no adverse effects on clinical chemistry or hematology, and did not result in any organ pathology or systemic toxicity. In a safety study in rabbits, six repeated intramuscular injections of VCL-2510 at plasmid DNA doses of 0.15 and 0.45 mg had no discernible effects on clinical chemistry, hematology, or histopathology. No evidence of autoimmune-mediated pathology, anti-nuclear antibodies (ANA), or antibodies to dsDNA were observed in the mouse or rabbit studies.

Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection.

Plasmid-based (naked DNA) genetic vaccines are now entering clinical trials to test their safety and efficacy in healthy human volunteers. A safety concern unique to this new class of vaccines is the potential risk of deleterious integration into host cell genomic DNA following direct intramuscular injection. To address this issue experimentally, a preclinical safety study was conducted in mice to determine the structural nature of plasmid DNA sequences persisting in total muscle DNA at both 30 and 60 days following a single intramuscular injection of a plasmid expressing the Plasmodium falciparum circumsporozoite protein. In a protocol described for the first time, total DNA was extracted from muscle tissue and was subsequently linearized with a restriction endonuclease to enable agarose gel size fractionation of all extrachromosomal plasmid DNAs from high molecular weight mouse genomic DNA. Using PCR assays to quantitate plasmid-specific sequences, it was found that the amount of plasmid DNA persisting in muscle tissue varied but averaged about 10 fg per microgram of genomic DNA (in the range of 1500 copies per 150,000 genomes). In two of four separate experimental injections of mouse muscle, PCR assays of genomic DNA fractions indicated that agarose gel purification removed plasmid DNA down to a level of < or =3 copies per 150,000 mouse genomes. In the two other experimental samples, 3-30 copies of plasmid DNA remained associated with purified genomic DNA. The time following injection (i.e., 30 or 60 days) was not a factor in the number of copies of plasmid associating with genomic DNA and it was not possible to conclude if such sequences were covalently linked to genomic DNA or simply adventitiously associated with the genomic DNA. However, if an assumption is made that the highest level plasmid DNA found associated with genomic DNA (i.e., 30 copies) represented covalently integrated plasmid inserts and that each insert resulted in a mutational event, the calculated rate of mutation would be 3000 times less than the spontaneous mutation rate for mammalian genomes. This level of integration, if it should occur, was not considered to pose a significant safety concern.

In vitro expression and in vivo immunogenicity of Plasmodium falciparum pre-erythrocytic stage DNA vaccines.

DNA vaccine plasmids were constructed that encoded four pre-erythrocytic antigens from the human malaria parasite Plasmodium falciparum: circumsporozoite protein (PfCSP); sporozoite surface protein 2 (PfSSP2); carboxyl terminus of liver stage antigen 1 (PfLSA-1 c-term); and, exported protein 1 (PfExp-1). Antigen expression was evaluated in vitro by immunoblot analysis of tissue culture cells following transient transfection with each plasmid. Clearly detectable levels of expression depended upon, or were markedly enhanced by, fusion of the antigen encoding sequences in-frame with the initiation complex and peptide leader sequence of human tissue plasminogen activator protein. Mice injected with these plasmids produced antigen specific antibody and cytotoxic T lymphocyte responses. However, the magnitudes of the responses were not always predicted by the in vitro expression assay. The results of this study provided the basis for further testing of these plasmids in primates and the formulation of multi-component pre-erythrocytic DNA vaccines for efficacy testing in human volunteers.

Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine.

CD8+ cytotoxic T lymphocytes (CTLs) are critical for protection against intracellular pathogens but often have been difficult to induce by subunit vaccines in animals. DNA vaccines elicit protective CD8+ T cell responses. Malaria-naïve volunteers who were vaccinated with plasmid DNA encoding a malaria protein developed antigen-specific, genetically restricted, CD8+ T cell-dependent CTLs. Responses were directed against all 10 peptides tested and were restricted by six human lymphocyte antigen (HLA) class I alleles. This first demonstration in healthy naïve humans of the induction of CD8+ CTLs by DNA vaccines, including CTLs that were restricted by multiple HLA alleles in the same individual, provides a foundation for further human testing of this potentially revolutionary vaccine technology.

A plasmid encoding murine granulocyte-macrophage colony-stimulating factor increases protection conferred by a malaria DNA vaccine.

Using the murine parasite Plasmodium yoelii (Py) as a model for malaria vaccine development, we have previously shown that a DNA plasmid encoding the Py circumsporozoite protein (PyCSP) can protect mice against sporozoite infection. We now report that mixing a new plasmid PyCSP1012 with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) increases protection against malaria, and we have characterized in detail the increased immune responses due to GM-CSF. PyCSP1012 plasmid alone protected 28% of mice, and protection increased to 58% when GM-CSF was added (p < 0.0001). GM-CSF plasmid alone did not protect, and control plasmid expressing inactive GM-CSF did not enhance protection. GM-CSF plasmid increased Abs to PyCSP of IgG1, IgG2a, and IgG2b isotypes, but not IgG3 or IgM. IFN-gamma responses of CD8+ T cells to the PyCSP 280-288 amino acid epitope increased but CTL activity did not change. The most dramatic changes after adding GM-CSF plasmid were increases in Ag-specific IL-2 production and CD4+ T cell proliferation. We hypothesize that GM-CSF may act on dendritic cells to enhance presentation of the PyCSP Ag, with enhanced IL-2 production and CD4+ T cell activation driving the increases in Abs and CD8+ T cell function. Recombinant GM-CSF is already used in humans for medical purposes, and GM-CSF protein or plasmids may be useful as enhancers of DNA vaccines.

Simultaneous induction of multiple antigen-specific cytotoxic T lymphocytes in nonhuman primates by immunization with a mixture of four Plasmodium falciparum DNA plasmids.

CD8(+) T cells have been implicated as critical effector cells in protective immunity against malaria parasites developing within hepatocytes. A vaccine that protects against malaria by inducing CD8(+) T cells will probably have to include multiple epitopes on the same protein or different proteins, because of parasite polymorphism and genetic restriction of T-cell responses. To determine if CD8(+) T-cell responses against multiple P. falciparum proteins can be induced in primates by immunization with plasmid DNA, rhesus monkeys were immunized intramuscularly with a mixture of DNA plasmids encoding four P. falciparum proteins or with individual plasmids. All six monkeys immunized with PfCSP DNA, seven of nine immunized with PfSSP2 DNA, and five of six immunized with PfExp-1 or PfLSA-1 DNA had detectable antigen-specific cytotoxic T lymphocytes (CTL) after in vitro restimulation of peripheral blood mononuclear cells. CTL activity was genetically restricted and dependent on CD8(+) T cells. By providing the first evidence for primates that immunization with a mixture of DNA plasmids induces CD8(+) T-cell responses against all the components of the mixture, these studies provide the foundation for multigene immunization of humans.

Protection of mice against Plasmodium yoelii sporozoite challenge with P. yoelii merozoite surface protein 1 DNA vaccines.

Immunization of mice with DNA vaccines encoding the full-length form and C and N termini of Plasmodium yoelii merozoite surface protein 1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titers after challenge. In C57BL/6 mice, two DNA vaccines provided protection comparable to that of recombinant protein consisting of the C terminus in Freund's adjuvant.

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine.

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-gamma-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0. 0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-gamma production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-gamma production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.

DNA vaccines for malaria: the past, the present, & the future.

The first clinical trial of a DNA vaccine designed to protect against malaria has just commenced. This vaccine has been designed to induce protective CD8+ T cell responses against Plasmodium falciparum infected hepatocytes. Herein, we review the rationale behind the development of vaccines that induce protective CD8+ T cells, the strategy for the development of a DNA vaccine designed to protect against falciparum malaria, and the experimental data in rodent models and nonhuman primates which has provided the foundation for trials of DNA vaccines against P. falciparum malaria in humans.

Toward clinical trials of DNA vaccines against malaria.

In mid 1997 the first malaria DNA vaccine will enter clinical trials. This single gene DNA vaccine encoding the Plasmodium falciparum circumsporozoite protein (PfCSP) will be studied for safety and immunogenicity. If these criteria are met, a multi-gene DNA vaccine designed to induce protective CD8+ T cell responses against P. falciparum infected hepatocytes will be subsequently assessed for safety, immunogenicity and capacity to protect immunized volunteers against experimental challenge with P. falciparum sporozoites. Our perspectives on malaria vaccine development in general, and on a multi-gene DNA vaccine in particular, have been recently reviewed. Herein, we review the rationale and experimental foundation for the anticipated P. falciparum DNA vaccine trials.

Strategy for development of a pre-erythrocytic Plasmodium falciparum DNA vaccine for human use.

Data generated in the Plasmodium yoelii rodent model indicated that plasmid DNA vaccines encoding the P.yoelii circumsporozoite protein (PyCSP) or 17 kDa hepatocyte erythrocyte protein (PyHEP17) were potent inducers of protective CD8+ T cell responses directed against infected hepatocytes. Immunization with a mixture of these plasmids circumvented the genetic restriction of protective immunity and induced additive protection. A third DNA vaccine encoding the P. yoelii sporozoite surface protein 2 (PySSP2) also induced protection. The P. falciparum genes encoding the homologues of these three protective P. yoelii antigens as well as another P. falciparum gene encoding a protein that is expressed in infected hepatocytes have been chosen for the development of a human vaccine. The optimal plasmid constructs for human use will be selected on the basis of immunogenicity data generated in mice and nonhuman primates. We anticipate that optimization of multi-gene P. falciparum DNA vaccines designed to protect against malaria by inducing CD8+ T cells that target infected hepatocytes will require extensive clinical trials during the coming years.

DNA vaccines against malaria: immunogenicity and protection in a rodent model.

Since the first demonstration of the technology a few years ago, DNA vaccines have emerged as a promising method of vaccination. In a variety of experimental systems, DNA vaccines have been shown not only to induce potent immune responses, but also to offer many advantages in terms of ease of construction, testing, and production. In this article we summarize the progress achieved in development of DNA vaccines that can protect mice from infection by the rodent malaria parasite Plasmodium yoelii, describe initial studies of immunogenicity of a malaria DNA vaccine in a primate model, and outline the strategies being employed to design the next generation of malaria DNA vaccines.