Yupin Charoenvit

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.

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-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.

Synthetic oligodeoxynucleotides containing CpG motifs enhance immunogenicity of a peptide malaria vaccine in Aotus monkeys

Synthetic peptide and recombinant protein vaccines are optimally immunogenic when delivered with an effective adjuvant. Candidate vaccines currently insufficiently immunogenic may induce a protective immunity if they could be delivered with more effective adjuvants. For example, immunogens that induce promising responses when administered to mice with complete and incomplete Freunds adjuvants perform less well in primate animal models where complete Freunds adjuvant is not used. We report the use of synthetic oligodeoxynucleotides containing CpG motifs, the sequences of which are based on immunostimulatory bacterial DNA sequences, to enhance the immune response in Aotus monkeys to a synthetic peptide malaria vaccine. Monkeys were immunized with the synthetic peptide PADRE 45, a synthetic peptide containing amino acid sequences derived from the circumsporozoite protein (CSP) from Plasmodium falciparum, and delivered in an emulsion of saline and Montanide 720, a mannide oleate in oil solution, that also contained one of three oligodeoxynucleotides. The animals receiving oligodeoxynucleotides containing either three or four CpG motifs produced antibodies that bound a recombinant CSP as measured in ELISA, and reacted with P. falciparum sporozoites in a sporozoite immunofluorescent test. These responses were significantly greater than those seen in animals receiving the oligodeoxynucleotide without CpG motifs. These data indicate that oligodeoxynucleotides containing CpG motifs improve immunogenicity of peptide immunogens in non-human primates, and may be immunopotentiators useful in humans.

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 μg to 1 μg with little loss of immunogenicity after boost with recombinant poxvirus. Comparing mice primed by the 1-μg doses of pPyCSP plus 1 μg pGMCSF with mice primed by 1-μg 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-γ-secreting cells by enzyme-linked immunospot assay (1460 vs 280 IFN-γ 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.

Plasmid Vaccine Expressing Granulocyte-Macrophage Colony-Stimulating Factor Attracts Infiltrates Including Immature Dendritic Cells into Injected Muscles

Plasmid-encoded GM-CSF (pGM-CSF) is an adjuvant for genetic vaccines; however, little is known about how pGM-CSF enhances immunogenicity. We now report that pGM-CSF injected into mouse muscle leads to a local infiltration of potential APCs. Infiltrates reached maximal size on days 3 to 5 after injection and appeared in several large discrete clusters within the muscle. Immunohistological studies in muscle sections from mice injected with pGM-CSF showed staining of cells with the macrophage markers CD11b, Mac-3, IAd/Ed and to the granulocyte marker GR-1 from day 1 through day 14. Cells staining with the dendritic cell marker CD11c were detected only on days 3 to 5. Muscles injected with control plasmids did not stain for CD11c but did stain for CD11b, Mac-3, IAd/Ed, and GR-1. No staining was observed with the APC activation markers, B7.1 or CD40, or with markers for T or B cells. These findings are consistent with the infiltrating cells in the pGM-CSF-injected muscles being a mixture of neutrophils, macrophages, and immature dendritic cells and suggest that the i.m. APCs may be enhancing immune responses to coinjected plasmid Ags. This hypothesis is supported by data showing that 1) separation of injections with pGM-CSF and Ag-expressing plasmid into different sites did not enhance immune responses and 2) immune enhancement was associated with the presence of CD11c+ cells in the infiltrates. Thus, pGM-CSF enhancement may depend on APC recruitment to the i.m. site of injection.

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.

Safety, tolerability, and lack of antibody responses after administration of a PfCSP DNA malaria vaccine via needle or needle-free jet injection, and comparison of intramuscular and combination intramuscular/intradermal routes

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.

Induction in Humans of CD8 + and CD4 + T Cell and Antibody Responses by Sequential Immunization with Malaria DNA and Recombinant Protein

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-γ 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-γ responses, whereas, CD8+ T cells were involved only in the production phase. IFN-γ 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.

Identification and Characterization of the Protective Hepatocyte Erythrocyte Protein 17 kDa Gene of Plasmodium yoelii, homolog of Plasmodium falciparum Exported Protein 1

We recently reported the discovery of a 17-kDa Plasmodium yoelii protein expressed in infected hepatocytes and erythrocytes, P. yoelii hepatocyte erythrocyte protein 17 (PyHEP17), and have demonstrated that this protein is a target of protective antibodies and T cells. Here, we report the identification and characterization of the gene encoding this protein and reveal that it is composed of two exons. Immunization of mice with PyHEP17 plasmid DNA induces antibodies, cytotoxic T lymphocytes, and protective immunity directed against the infected hepatocyte. Based on extensive sequence homology, expression pattern, and antigenic cross-reactivity, the Plasmodium falciparum homolog of PyHEP17 is identified as the protein known as exported protein-1 (PfExp-1), also called antigen 5.1, circumsporozoite related antigen, or QF116. Identity between PyHEP17 and PfExp-1 is 37% at the amino acid level (60/161 residues), mapping primarily to two regions within the second exon of 73% (16/22 residues) and 71% (25/35 residues) identity. On this basis, PfExp-1 is proposed as an important component of pre-erythrocytic human malaria vaccines.

Boosting of DNA Vaccine-Elicited Gamma Interferon Responses in Humans by Exposure to Malaria Parasites

ABSTRACT 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-γ) 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-γ 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-γ 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-γ 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-γ 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.