Cytotoxic T cells recognize a peptide from the circumsporozoite protein on malaria-infected hepatocytes.

Irradiated malaria sporozoites can induce CD8+ T cells that are required for protection against infection. However, the parasite antigens targeted by this immune response are unknown. We have discovered a 16-amino acid epitope from the Plasmodium yoelii circumsporozoite (CS) protein that is recognized by cytotoxic T cells from immune mice. Lymphocytes stimulated with this peptide can kill P. yoelii liver stage parasites in vitro in an MHC-restricted, antigen-specific manner. Thus, epitopes from the CS protein are presented on the surface of infected hepatocytes and can be targets for T cells, even though intact CS protein has not been detected on the surface of the infected hepatocyte. A vaccine that induced CTL to parasite antigens might protect humans against malaria by eliminating liver stage parasites.

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.

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy.

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.

Host-parasite interactions and immunity to irradiated sporozoites.

We compare and contrast the results of immunizing mice with irradiated sporozoites of Plasmodium berghei and Plasmodium yoelii. Host genetic control of protective immunity is different in the two rodent malarias. Few mouse strains are strongly protected by P. yoelii sporozoites, while all are protected by P. berghei sporozoite immunization. The role of CD8+ T cells in the protective immune response to each of these malarias varies with the strain of mouse. Moreover, a single strain will use a CD8+ T cell-dependent mechanism against one malaria, and a CD8+ independent mechanism against the other. Thus, each host-parasite pairing in these rodent malarias engenders a unique set of immune responses. Such variety should be expected in the immune response to the human malarias, and may complicate the development of universally applicable vaccines.

Prevention of neonatal tolerance by a plasmid encoding granulocyte-macrophage colony stimulating factor.

A plasmid DNA vaccine encoding the circumsporozoite protein of malaria (pCSP) induces protective immunity in adult mice but persistent tolerance when administered to neonates. In an effort to improve antigen presenting cell (APC) function in newborns, we co-administered pCSP with a plasmid encoding granulocyte-macrophage colony stimulating factor (pGMCSF). This combination of plasmids prevented the development of neonatal tolerance, instead eliciting a primary IgG anti-CSP immune response. Mice primed as neonates and boosted as adults mounted anamnestic responses characterized by high serum antibody titers, cytotoxic T-cell activity and antigen-specific interferon gamma (IFNgamma) production. Neonatal administration of pGMCSF accelerated the maturation of local dendritic cells, suggesting that APC function plays a key role in determining whether tolerance or immunity results from neonatal exposure to antigen.

Daily primaquine is effective for prophylaxis against falciparum malaria in Kenya: comparison with mefloquine, doxycycline, and chloroquine plus proguanil.

Primaquine was tested as a prophylactic drug against Plasmodium falciparum in a region in western Kenya in which malaria is holoendemic. Children 9-14 years old were randomized to receive regimens of daily primaquine, daily doxycycline, daily proguanil plus weekly chloroquine, daily vitamin plus weekly mefloquine, or daily vitamin alone. Primaquine, doxycycline, and mefloquine were equally effective in preventing both symptomatic and asymptomatic malarial infections. Chloroquine plus proguanil was the least effective regimen. There was no toxicity from daily primaquine during the 11 weeks of the study. Findings show that primaquine can be successfully used as a causal prophylactic regimen against falciparum malaria in western Kenya; chloroquine plus proguanil was not as efficacious as the three other preventive regimens; most Kenyan children receiving standard doses of mefloquine and doxycycline had lower than expected serum trough drug levels; and some volunteers with adequate mefloquine or doxycycline levels at trough developed asymptomatic parasitemias and clinical malaria.

The role of CD4+ T cells in immunity to malaria sporozoites.

BALB/c mice are strongly protected against malaria after immunization with Plasmodium yoelii (PY) sporozoites, and this is an important model for malaria vaccine development in humans. This paper explores the role of CD4+ cells in the induction of the antimalarial immune response. The method used has been to treat animals with anti-CD4 mAb before immunization, resulting in a profound depletion of CD4+ T cells. The primary finding is that mice are not protected by sporozoite immunization after depletion of CD4+ cells. Such mice make little antibody to malaria sporozoite Ag, showing the strong T-cell dependence of these humoral responses. However, infusion of hyperimmune serum does not restore immunity to anti-CD4 treated animals. Neither does injection of exogenous IL-2 compensate for the absence of CD4+ cells. However, regrowth of CD4+ cells does allow successful immunization of animals, showing that long-term suppression against malaria Ag has not been induced by immunization in the absence of CD4+ cells. It is thought that infiltrating CD8+ T cells are critical effector cells against PY parasites in the pre-erythrocytic stages. Mice immunized while depleted of CD4+ cells have normal numbers of CD8+ T cells infiltrating their livers. In addition, they have normal numbers of splenic CD8+ CTL directed at the PY circumsporozoite protein. Thus, it appears that although CD8+ cells have been activated in the absence of CD4+ cells, they cannot protect mice against malaria. We conclude that a successful vaccine against the pre-erythrocytic stages of malaria must activate both CD4+ and CD8+ T cells.

CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites.

In recent malaria sporozoite vaccine trials in humans and mice, antibodies to the sporozoite coat protein have given only modest protection against sporozoite challenge. In contrast, irradiated sporozoites can protect mice against massive sporozoite infections. Evidence suggests that immunity in these mice is mediated by T cells. To identify the mechanism of immunity, we used monoclonal antibodies specific for either the CD4 or CD8 molecule to selectively deplete sporozoite-immunized mice of T-cell subsets. Though in vivo depletion of CD4+ T cells did not reduce immunity, depletion of CD8+ T cells abolished protection. Monoclonal antibody treatment did not affect anti-sporozoite antibody levels. Our data indicate that cytotoxic T cells are critical for immunity to large numbers of sporozoites and suggest that vaccine development should be reoriented toward stimulating cellular as well as humoral immunity.

Activity and safety of DNA plasmids encoding IL-4 and IFN gamma.

Cytokine-encoding DNA plasmids can act as 'genetic adjuvants', improving the immune response stimulated by co-administered DNA vaccines. We examined whether plasmids encoding the Th1 cytokine IFN gamma (pIFN gamma) or the Th2 cytokine IL-4 (pIL-4) have long-term effects on immune homeostasis when administered to adult mice, or alter immune maturation in neonates. Both plasmids boosted immunity against a co-administered vaccine, with pIFN gamma promoting the development of a Th1 response (characterized by the production of IgG2a antibodies), and pIL-4 preferentially stimulating a Th2 response (characterized by increased IgG1 antibody production). Both pIFN gamma and pIL-4 influenced the ratio of cells actively secreting Th1 versus Th2 cytokines, consistent with an effect on Th cell maturation. Interestingly, this effect persisted for only a few weeks and was not magnified by repeated plasmid administration. Cytokine-encoding plasmids had no long-term effect on the immune response of newborn or adult mice to subsequent antigenic stimulation, nor did they selectively induce the production of pathogenic anti-DNA autoantibodies. These results suggest cytokine-encoding plasmids may be safe as immune adjuvants.

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, IA(d)/E(d) 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, IA(d)/E(d), 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.

Genetic control of immunity to Plasmodium yoelii sporozoites.

Using a rodent malaria system, we have shown that protective immunity to the preerythrocytic stages of malaria is genetically controlled by MHC and non-MHC genes. Ten congenic strains of mice were immunized with irradiated sporozoites of Plasmodium yoelii. When challenged with viable sporozoites, only two strains had a high proportion of animals that did not develop blood stage infections. Immunity did not correlate with antisporozoite antibody levels. Two protective mechanisms exist determined by non-H-2 genes, and each mechanism is further controlled by H-2-linked Ir genes. On the BALB background only H-2d mice are protected, and protection is abolished by depleting CD8+ T cells. In contrast, on the B10 background only H-2q mice are strongly protected, and protection is not affected by CD8+ T cell depletion. If similar complex genetic regulation of immunity occurs in the human malarias, it will be a major hurdle for vaccine development.

A T cell clone directed at the circumsporozoite protein which protects mice against both Plasmodium yoelii and Plasmodium berghei.

Clone B is a cytotoxic T cell clone induced by immunization with Plasmodium yoelii sporozoites which recognizes an epitope on both the P. yoelii and Plasmodium berghei circumsporozoite proteins. It is CD8, uses the V beta 8.1 TCR, and is Kd restricted. When adoptively transferred, it protects mice against infection by both species of malaria sporozoites, and this protection is dependent on IFN-gamma. Clone B cells are more broadly reactive and protective than previously described murine T cell clones against malaria. Clone B may be an important model for immune protection against the spectrum of variant parasites in nature.

Factors associated with the development of neonatal tolerance after the administration of a plasmid DNA vaccine.

A plasmid DNA vaccine encoding the circumsporozoite protein of malaria (pCSP) induces tolerance rather than immunity when administered to newborn mice. We find that this tolerance persists for >1 yr after neonatal pCSP administration and interferes with the induction of protective immunity in animals challenged with live sporozoites. Susceptibility to tolerance induction wanes rapidly with age, disappearing within 1 wk of birth. Higher doses of plasmid are more tolerogenic, and susceptibility to tolerance is not MHC-restricted. CD8+ T cells from tolerant mice suppress the in vitro Ag-specific immune response of cells from adult mice immunized with pCSP. Similarly, CD8+ T cells from tolerant mice transfer nonresponsiveness to naive syngeneic recipients. These findings clarify the cellular basis and factors contributing to the development of DNA vaccine-induced neonatal tolerance.

Cloning, sequencing, and homology analysis of nonhuman primate Fas/Fas-ligand and co-stimulatory molecules.

The finding that a single administration of select recombinant human cytokines to nonhuman primates leads to potent cytokine-neutralizing antibody responses in the heterologous host despite >95% homology at the nucleotide and protein level prompted our laboratory to clone, sequence, and prepare recombinant nonhuman primate cytokines, chemokines, growth factors, and other immunoregulatory molecules. In the present report, we present findings on the gene sequences encoding the nonhuman primate homologues of human CD80, CD86, their ligands CD28 and CD152, CD154, CD95, and CD95-L from rhesus macaques and for phylogenetic analysis from pig-tailed macaques, African sooty mangabey monkeys, baboons, and vervets as well as select molecules from the New World aotus and marmoset monkeys. With the exception of CD95, the homology between nonhuman primate and human co-stimulatory molecules was above 95%. In contrast, CD95 was only 89.2% homologous to human CD95, but the differences were essentially found in the transmembrane and intracellular (death) domains. The extracellular portion of CD95 was more homologous which was in accordance with approximately 98% homology between Old World monkey and human CD95-L. In general, sequences from the New World monkey species appeared equidistant to sequences from Old World species and humans in terms of homology suggesting distinct evolutionary patterns. Of interest was the isolation of various splice variants of monkey CD86, CD152 (CTLA-4), CD154, and CD95 transcripts. This is also the first report documenting the occurrence of natural CD86 variants with deleted transmembrane domains, found both in sooty mangabeys and baboon RNA samples. Monkey CD95 showed various deletions and addition of residues in the transmembrane and intracytoplasmic domains compared with human CD95 and between Old and New World species. Subcloning of rhesus CD154 into an expression vector demonstrated expression of a functional protein in cell culture. The other genes are being cloned into expression vectors for the preparation and biological characterization of the nonhuman primate molecules. These investigations will provide novel reagents for in vivo use as immunomodulatory reagents in nonhuman primates in studies which may provide a rationale for their use in humans.

Generation of a cytotoxic T-lymphocyte response using a Salmonella antigen-delivery system.

We have constructed a general-use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium. Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelii in an aroA S. typhimurium strain. Mice immunized with CS-expressing Salmonella recombinants mount a CS-specific cytotoxic T-lymphocyte (CTL) response. This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice. The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Salmonella as an antigen-delivery system.

Genetic restriction of protective immunity to Plasmodium yoelii sporozoites.

Ten congenic strains of mice were immunized with irradiated sporozoites of Plasmodium yoelii. When challenged with viable sporozoites, only two strains had a high proportion of animals which did not develop blood-stage infections. Genes both within and outside the H-2 region affected the degree of protection. Immunity did not correlate with anti-sporozoite antibody levels. In vivo depletion of CD8+ T cells did not alter immunity in two of three congenic strains, implying the existence of a novel mechanism of cell-mediated immunity.

A CTL epitope on the circumsporozoite protein of P. yoelii.

Humans are infected with malaria by the bite of anophelene mosquitos carrying plasmodia sporozoites. These sporozoites pass quickly from the blood into hepatocytes, where they develop into mature liver-stage parasites over several days. The clinical stage of the illness begins only when the liver-stage parasites rupture into the bloodstream and erythrocytes are invaded. The pre-erythrocytic stages of malaria are inviting targets for vaccine development, because an effective immune response to these early stages would prevent symptomatic infections.

Recombinant pseudorabies virus carrying a plasmodium gene: herpesvirus as a new live viral vector for inducing T- and B-cell immunity.

In Balb/c mice, the sterile protective immunity induced by immunization with radiation-attenuated Plasmodium yoelii sporozoites is eliminated by in vivo depletion of CD8+ T lymphocytes, suggesting that cytotoxic T lymphocytes (CTL) against malaria antigens expressed on infected hepatocytes are required for mediating this protective immunity. To produce a vaccine that would induce CTL against the P. yoelii circumsporozoite protein (CS), we constructed an attenuated pseudorabies virus (PRV) containing a gene encoding this protein. Balb/c mice that received three doses of 10(7) plaque-forming units (p.f.u.) of this vaccine intravenously at 3 week intervals developed high levels of antibodies to sporozoites (indirect fluorescent antibody titre = 4096) and CTL against a 16 amino acid epitope (SYVPSAEQILEFVKQI, amino acids 281-296) from the P. yoelii CS protein designated PYCTL1. The cytotoxic activity of the CTL was antigen-specific, MHC-restricted, and dependent on CD8+ T cells. Furthermore, these CTL eliminated P. yoelii-infected hepatocytes from in vitro culture, indicating that they recognize this peptide on the surface of infected hepatocytes. However, all nine mice that were challenged with 200 sporozoites developed a blood-stage malaria infection. We attribute this lack of protection to the great difficulty of inducing sterile immunity against this highly infectious parasite P. yoelii. We conclude that recombinant pseudorabies virus (PRV) worked successfully as a live vaccine vector to induce both antibodies and CTL, albeit non-protective in vivo, and the herpesviruses should be considered as subunit vaccines where T- and B-cell immunity is required.

In vivo testing of subunit vaccines against malaria sporozoites using a rodent system.

To test the putative in vivo protective effects of antibodies to circumsporozoite (CS) protein repeats against malarial infection, different strains of mice were immunized against various repetitive regions of the Plasmodium yoelii CS protein in the form of synthetic peptides conjugated to keyhole limpet hemocyanin. Complete Freund's adjuvant or saponin was used as adjuvant. When vaccinated mice were challenged with 500 sporozoites almost all animals became infected. There were no significant protective effects in vaccinated versus unvaccinated mice. Furthermore, there was no correlation between the antibody titer to the CS repeats and infection. The parasites from infected animals were shown to encode a CS protein containing the same repeats as those used for immunization, indicating that the infections were not due to selection for variant parasites. These experiments demonstrate that antibodies to the CS repeats, as derived in vivo with peptides, despite being surface reactive, do not provide protection against sporozoite challenge in vivo. This conclusion is in contrast to previous conclusions based on studies showing protection by way of in vitro sporozoite neutralization procedures and passive transfer of monoclonal antibody.

Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques.

A candidate DNA vaccine expressing dengue virus type 1 pre-membrane and envelope proteins was used to immunize rhesus macaques. Monkeys were immunized intramuscularly (i.m.) or intradermally (i.d.) by three or four 1 mg doses of vaccine, respectively. Monkeys that were inoculated i.m. seroconverted more quickly and had higher antibody levels than those that were inoculated i.d. The sera exhibited virus-neutralizing activity, which declined over time. Four of the eight i.m.-inoculated monkeys were protected completely from developing viraemia when challenged 4 months after the last dose with homologous dengue virus. The other four monkeys had reduced viraemia compared with the control immunized monkeys. The i.d. -inoculated monkeys showed no reduction in viraemia when challenged with the virus. All vaccinated monkeys showed an anamnestic antibody response, indicating that they had established immunological memory. Vaccine-induced antibody had an avidity index similar to that of antibody induced by virus infection; however, no clear correlation was apparent between antibody avidity and virus neutralization titres.