ABSTRACT
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.
Subject(s)
Antigens, Protozoan/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Malaria Vaccines/immunology , Malaria Vaccines/therapeutic use , Plasmodium falciparum/immunology , Plasmodium falciparum/pathogenicity , Sporozoites/immunology , Vaccines, DNA/immunology , Vaccines, DNA/therapeutic use , Adult , Female , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Humans , Malaria Vaccines/administration & dosage , Male , Middle Aged , Plasmids/genetics , Vaccines, DNA/adverse effects , Young AdultABSTRACT
A mixture of DNA plasmids expressing five Plasmodium falciparum pre-erythrocyte-stage antigens was administered with or without a DNA plasmid encoding human granulocyte-macrophage colony-stimulating factor (hGM-CSF) as an immune enhancer. After DNA immunization, antigen-specific gamma interferon (IFN-gamma) responses were detected by ELISPOT in 15/31 volunteers to multiple class I- and/or class II-restricted T-cell epitopes derived from all five antigens. Responses to multiple epitopes (=7) were detected simultaneously in some volunteers. By 4 weeks after challenge with P. falciparum parasites, 23/31 volunteers had positive IFN-gamma responses and the magnitude of responses was increased from 2- to 143-fold. Nonetheless, none was protected against malaria. Volunteers who received hGM-CSF had a reduced frequency of IFN-gamma responses to class I peptides compared to those who only received plasmids expressing P. falciparum proteins before challenge (3/23 versus 3/8; P = 0.15) or after parasite challenge (4/23 versus 5/8; P = 0.015) but not to class II peptides before or after challenge. The responses to one antigen (P. falciparum circumsporozoite protein [PfCSP]) were similar among volunteers who received the five-gene mixture compared to volunteers who only received the PfCSP DNA plasmid in a previous study. In summary, DNA-primed IFN-gamma responses were boosted in humans by exposure to native antigen on parasites, coadministration of a plasmid expressing hGM-CSF had a negative effect on boosting of class I-restricted IFN-gamma responses, and there was no evidence that immunization with PfCSP DNA in the mixture reduced T-cell responses to PfCSP compared to when it was administered alone.
Subject(s)
Antigens, Protozoan/immunology , Interferon-gamma/blood , Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Vaccines, DNA/immunology , Animals , Antigens, Protozoan/administration & dosage , Antigens, Protozoan/genetics , Bites and Stings , Culicidae , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Humans , Immunization , Malaria Vaccines/administration & dosage , Plasmids , T-Lymphocytes/immunology , Treatment Outcome , Vaccines, DNA/administration & dosageABSTRACT
Optimal protection against malaria may require induction of high levels of protective antibody and CD8(+) and CD4(+) T cell responses. In humans, malaria DNA vaccines elicit CD8(+) cytotoxic T cells (CTL) and IFNgamma responses as measured by short-term (ex vivo) ELISPOT assays, and recombinant proteins elicit antibodies and excellent T cell responses, but no CD8(+) CTL or CD8(+) IFNgamma-producing cells as measured by ex vivo ELISPOT. Priming with DNA and boosting with recombinant pox virus elicits much better T cell responses than DNA alone, but not antibody responses. In an attempt to elicit antibodies and enhanced T cell responses, we administered RTS,S/AS02A, a partially protective Plasmodium falciparum recombinant circumsporozoite protein (CSP) vaccine in adjuvant, to volunteers previously immunized with a P. falciparum CSP DNA vaccine (VCL-2510) and to naïve volunteers. This vaccine regimen was well tolerated and safe. The volunteers who received RTS,S/AS02A alone had, as expected, antibody and CD4(+) T cell responses, but no CD8(+) T cell responses. Volunteers who received PfCSP DNA followed by RTS,S/AS02A had antibody and CD8(+) and CD4(+) T cell responses (Wang et al., submitted). Sequential immunization with DNA and recombinant protein, also called heterologous prime-boost, led to enhanced immune responses as compared to DNA or recombinant protein alone, suggesting that it might provide enhanced protective immunity.
Subject(s)
Antibodies, Protozoan/biosynthesis , Malaria Vaccines/immunology , Plasmodium falciparum/immunology , Vaccines, DNA/immunology , Adolescent , Adult , Animals , Antibodies, Protozoan/analysis , B-Lymphocytes/immunology , Female , Hepatitis B Antibodies/biosynthesis , Hepatitis B Surface Antigens/immunology , Humans , Immunization Schedule , Malaria Vaccines/adverse effects , Male , Spores, Protozoan/immunology , T-Lymphocytes/immunology , Vaccines, DNA/adverse effects , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/immunologyABSTRACT
Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8(+) and CD4(+) T cell and Ab responses in the same individual. In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8(+) T cells by CTL or short-term (ex vivo) IFN-gamma ELISPOT assays, and partial short-term protection. P. falciparum DNA vaccines elicit CD8(+) T cells by these assays, but no protection. We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4(+) T cells, and CD8(+) cytotoxic and Tc1 T cells. Depending upon the immunization regime, CD4(+) T cells were involved in both the induction and production phases of PfCSP-specific IFN-gamma responses, whereas, CD8(+) T cells were involved only in the production phase. IFN-gamma mRNA up-regulation was detected in both CD45RA(-) (CD45RO(+)) and CD45RA(+)CD4(+) and CD8(+) T cell populations after stimulation with PfCSP peptides. This finding suggests CD45RA(+) cells function as effector T cells. The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines.
Subject(s)
Antibodies, Protozoan/biosynthesis , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunization Schedule , Malaria Vaccines/administration & dosage , Plasmodium falciparum/immunology , Protozoan Proteins/administration & dosage , Vaccines, DNA/administration & dosage , Amino Acid Sequence , Animals , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cytotoxicity Tests, Immunologic , Epitopes, T-Lymphocyte/administration & dosage , Epitopes, T-Lymphocyte/immunology , Hepatitis B Antibodies/biosynthesis , Hepatitis B Surface Antigens/immunology , Humans , Immunization, Secondary/methods , Interferon-gamma/biosynthesis , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Malaria, Falciparum/prevention & control , Molecular Sequence Data , Protozoan Proteins/genetics , Protozoan Proteins/immunology , T-Lymphocyte Subsets , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Vaccines, DNA/genetics , Vaccines, DNA/immunologyABSTRACT
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.