When introduced in the 1990s, immunization with DNA plasmids was considered potentially revolutionary for vaccine development, particularly for vaccines intended to induce protective CD8 T cell responses against multiple antigens. We conducted, in 1997-1998, the first clinical trial in healthy humans of a DNA vaccine, a single plasmid encoding Plasmodium falciparum circumsporozoite protein (PfCSP), as an initial step toward developing a multi-antigen malaria vaccine targeting the liver stages of the parasite. As the next step, we conducted in 2000-2001 a clinical trial of a five-plasmid mixture called MuStDO5 encoding pre-erythrocytic antigens PfCSP, PfSSP2/TRAP, PfEXP1, PfLSA1 and PfLSA3. Thirty-two, malaria-naïve, adult volunteers were enrolled sequentially into four cohorts receiving a mixture of 500 μg of each plasmid plus escalating doses (0, 20, 100 or 500 μg) of a sixth plasmid encoding human granulocyte macrophage-colony stimulating factor (hGM-CSF). Three doses of each formulation were administered intramuscularly by needle-less jet injection at 0, 4 and 8 weeks, and each cohort had controlled human malaria infection administered by five mosquito bites 18 d later. The vaccine was safe and well-tolerated, inducing moderate antigen-specific, MHC-restricted T cell interferon-γ responses but no antibodies. Although no volunteers were protected, T cell responses were boosted post malaria challenge. This trial demonstrated the MuStDO5 DNA and hGM-CSF plasmids to be safe and modestly immunogenic for T cell responses. It also laid the foundation for priming with DNA plasmids and boosting with recombinant viruses, an approach known for nearly 15 y to enhance the immunogenicity and protective efficacy of DNA vaccines.

Despite efforts to develop vaccines that protect against malaria by inducing CD8 + T cells that kill infected hepatocytes, no subunit vaccine has been shown to circumvent the genetic restriction inherent in this approach, and little is known about the interaction of subunit vaccineinduced immune effectors and infected hepatocytes. We now report that immunization with plasmid DNA encoding the Plasmodium yoelii circumsporozoite protein protected one of five strains of mice against malaria (H-2 a, 75%); a PyHEP17 DNA vaccine protected three of the five strains (H-2 a, 71%; H-2 k, 54%; H-2 a, 26%); and the combination protected 82% of H-2 a, 90% of H-2 k, and 88% of H-2 a mice. Protection was absolutely dependent on CD8 + T cells, IFN-~/, or nitric oxide. These data introduce a new target of protective preerythrocytic immune responses, PyHEP17 and its P. falciparum homologue, and provide a realistic perspective on the opportunities and challenges inherent in developing malaria vaccines that target the infected hepatocyte.

We have constructed a DNA plasmid vaccine encoding the C-terminal 42-kDa region of the merozoite surface protein1 (pMSP142) from the 3D7 strain of Plasmodium falciparum (Pf3D7). This plasmid expressed recombinant MSP142 after in vitro transfection in mouse VM92 cells. Rhesus monkeys immunized with pMSP142 produced antibodies reactive with Pf3D7 infected erythrocytes by IFAT, and by ELISA against yeast produced MSP119 (yMSP119). Immunization also induced antigen specific T cell responses as measured by interferon-γ production, and by classical CTL chromium release assays. In addition, immunization with pMSP142 primed animals for an enhanced antibody response to a subsequent boost with the recombinant yMSP119. We also evaluated Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as an adjuvant for pMSP142. We tested both rhesus GM-CSF expressed from a DNA plasmid, and E. coli produced recombinant human GM-CSF. Plasmids encoding rhesus GM-CSF (prhGM-CSF) and human GM-CSF (phuGM-CSF) were constructed; these plasmids expressed bio-active recombinant GMCSF. Co-immunization with a mixture of prhGM-CSF and pMSP142 induced higher specific antibody responses after the first dose of plasmid, but after three doses of DNA monkeys immunized with or without prhGM-CSF had the same final antibody titers and T cell responses. In comparison, rhuGM-CSF protein did not lead to accelerated antibody production after the first DNA dose. However, antibody titers were maintained at a slightly higher level in monkeys receiving GM-CSF protein, and they had a higher response to boosting with recombinant MSP119. The GM-CSF plasmid or protein appears to be less potent as an adjuvant in rhesus monkeys than each is in mice, and more work is needed to determine if GM-CSF can be a useful adjuvant in DNA vaccination of primates.

Introduction of a new vaccine requires choosing a delivery system that provides safe administration and the desired level of immunogenicity. The safety, tolerability, and immunogenicity of three monthly 2.5-mg doses of a PfCSP DNA vaccine were evaluated in healthy volunteers as administered intramuscularly (IM) by needle, IM by jet injection (Biojector or IM/intradermally (ID) by jet injection. Vaccine administration was well-tolerated. Adverse events were primarily mild and limited to the site of injection (98%). Jet injections (either IM or ID) were associated with approximately twice as many adverse events per immunization as needle IM, but nevertheless were strongly and consistently preferred in opinion polls taken during the study. No volunteers had clinically significant biochemical or hematologic changes or detectable anti-dsDNA antibodies. In conclusion, the injection of Plasmodium falciparum circumsporozoite (PfCSP) DNA vaccine appeared to be safe and well-tolerated when administered by any of the three modes of delivery. However, despite improved antibody responses following both jet injection and ID delivery in animal models, no antibodies could be detected in volunteers by immunofluorescence antibody test (IFAT) or enzyme-linked immunosorbent assay (ELISA) after DNA vaccination.

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.

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-γ 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-γ responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.

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.

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.

ABSTRACT 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.

We evaluated the capacity of the cationic lipid based formulation, Vaxfectin™, to enhance the immunogenicity and protective efficacy of DNA-based vaccine regimens in the Plasmodium yoelii murine malaria model. We immunized Balb/c mice with varying doses (0.4–50 μg) of plasmid DNA (pDNA) encoding the P. yoelii circumsporozoite protein (PyCSP), either in a homologous DNA/DNA regimen (D-D) or a heterologous prime-boost DNA-poxvirus regimen (D-V). At the lowest pDNA doses, Vaxfectin™ substantially enhanced IFA titers, ELISPOT frequencies, and protective efficacy. Clinical trials of pDNA vaccines have often used low pDNA doses based on a per kilogram weight basis. Formulation of pDNA vaccines in Vaxfectin™ may improve their potency in human clinical trials.

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.

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.

During the twentieth century, the primary approach to malaria prevention has been to interfere with transmission of the parasite between the infected mosquito and the human host using physical barriers, insecticides and prophylactic drugs. Despite these measures, approximately 40% of the world’s population is at risk and it is estimated that there are 300–500 million new Plasmodium infections and 1.5–2.7 million deaths annually due to malaria (WHO 1990). Accordingly, there have been major efforts to develop an efficacious malaria 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.

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 CDS8+ 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,1 and on a multi‐gene DNA vaccine in particular,2 have been recently reviewed. Herein, we review the rationale and experimental foundation for the anticipated P. falciparum DNA vaccine trials.

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.

According to the latest report from the World Health Organization (December 1996), malaria is still a public health problem in more than 90 countries, inhabited by a total of some 2.4 billion people or 40% of the world’s population. Worldwide incidence of the disease is estimated to be 300–500 million clinical cases each year. Mortality due to malaria is estimated to be in the range of 1.5–2.7 million deaths annually. The vast majority of deaths occur among young children in Africa, especially in remote rural areas with poor access to health services. Immunity to malaria seems to occur among residents of malarious areas with increasing age which prevents an even greater number of deaths. However, this type of protection is short-lived and requires frequent exposure to the malaria parasite.