An Unstable Th Epitope of P. falciparum Fosters Central Memory T Cells and Anti-CS Antibody Responses

Malaria is transmitted by Plasmodium-infected anopheles mosquitoes. Widespread resistance of mosquitoes to insecticides and resistance of parasites to drugs highlight the urgent need for malaria vaccines. The most advanced malaria vaccines target sporozoites, the infective form of the parasite. A major target of the antibody response to sporozoites are the repeat epitopes of the circumsporozoite (CS) protein, which span almost one half of the protein. Antibodies to these repeats can neutralize sporozoite infectivity. Generation of protective antibody responses to the CS protein (anti-CS Ab) requires help by CD4 T cells. A CD4 T cell epitope from the CS protein designated T* was previously identified by screening T cells from volunteers immunized with irradiated P. falciparum sporozoites. The T* sequence spans twenty amino acids that contains multiple T cell epitopes restricted by various HLA alleles. Subunit malaria vaccines including T* are highly immunogenic in rodents, non-human primates and humans. In this study we characterized a highly conserved HLA-DRβ1*04:01 (DR4) restricted T cell epitope (QNT-5) located at the C-terminus of T*. We found that a peptide containing QNT-5 was able to elicit long-term anti-CS Ab responses and prime CD4 T cells in HLA-DR4 transgenic mice despite forming relatively unstable MHC-peptide complexes highly susceptible to HLA-DM editing. We attempted to improve the immunogenicity of QNT-5 by replacing the P1 anchor position with an optimal tyrosine residue. The modified peptide QNT-Y formed stable MHC-peptide complexes highly resistant to HLA-DM editing. Contrary to expectations, a linear peptide containing QNT-Y elicited almost 10-fold lower long-term antibody and IFN-γ responses compared to the linear peptide containing the wild type QNT-5 sequence. Some possibilities regarding why QNT-5 is more effective than QNT-Y in inducing long-term T cell and anti-CS Ab when used as vaccine are discussed.     

Naturally acquired CD8+ cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

In rodent malaria model systems, protective immunity induced by immunization with irradiated sporozoites is eliminated by in vivo depletion of CD8+ T cells, and adoptive transfer of CTL clones against the circumsporozoite protein protects against malaria. We recently demonstrated that volunteers immunized with irradiated Plasmodium falciparum sporozoites produce CTL against peptide 368-390 of the P. falciparum circumsporozoite protein. To determine whether natural exposure to malaria induced similar CTL, we studied 11 adult, male, life-long residents of a highly malarious area of Kenya, who were selected because their lymphocytes had been shown to proliferate after stimulation with peptides 361-380, 371-390, or 368-390 and because nine had been resistant to malaria in previous studies. In four of the 11 individuals there was peptide-specific, genetically restricted, CTL activity. In all four individuals, this activity was unaffected by depletion of CD4+ T cells. In three volunteers the activity was eliminated or reduced by depletion of CD8+ T cells; in the fourth volunteer the CD8+ T cell depletion was uninterpretable. This first demonstration of CD8+ T cell, genetically restricted, Ag-specific CTL against a malaria protein among individuals exposed to endemic malaria provides a foundation for studying the relationship between circulating CTL and resistance to malaria infection.     

A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT(*))(4)-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T(*), which was originally defined using CD4(+) T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T(*)-specific cellular responses with high anti-repeat Ab titers suggests that the T(*) epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.     

Pre-erythrocytic malaria vaccine: mechanisms of protective immunity and human vaccine trials

In order to provide a rational basis for the development of a pre-erythrocytic malaria vaccine we have aimed at: (a) elucidating the mechanisms of protection, and (b) identifying vaccine formulations that best elicit protection in experimental animals and humans. Based on earlier successful immunization of experimental animals with irradiated sporozoites, human volunteers were exposed to the bites of large numbers of Plasmodium falciparum or P. vivax infected irradiated mosquitoes. The result of this vaccine trial demonstrated for the first time that a pre-erythrocytic vaccine, administered to humans, can result in their complete resistance to malaria infection. However, since infected irradiated mosquitoes are unavailable for large scale vaccination, the alternative is to develop subunit vaccines. The human trials using irradiated sporozoites provided valuable information on the human immune responses to pre-erythrocytic stages and studies on mice an excellent experimental model to characterize protective immune mechanisms. The circumsporozoite protein, the first pre-erythrocytic antigen identified, is present in all malaria species, displaying a similar structure, with a central region of repeats, and two conserved regions, essential for parasite development. Most pre-erythrocytic vaccine candidates are based on the CS protein, expressed in various cell lines, microorganisms, and recently the corresponding DNA. We and others have identified CS-specific B and T cell epitopes, recognized by the rodent and human immune systems, and used them for the development of synthetic vaccines. We used synthetic peptide vaccines, multiple antigen peptides and polyoximes, for immunization, first in experimental animals, and recently in two human safety and immunogenicity trials. We also report here on our work on T cell mediated immunity, particularly the protection of mice immunized with viral vectors expressing CS-specific cytotoxic CD8+ T cell epitopes, and the striking booster effect of recombinant vaccinia virus. To what degree CD8+ T cells, and/or other T cells specific for sporozoites and/or liver stage epitopes, contribute to pre-erythrocytic protective immunity in humans, remains to be determined.     

Plasmodium falciparum: sporozoite boosting of immunity due to a T-cell epitope on a sporozoite vaccine

Plasmodium falciparum: Sporozoite boosting of immunity due to a T-cell epitope on a sporozoite vaccine. Experimental Parasitology 64, 64-70. The impact of a malaria sporozoite vaccine may be enhanced if protective immunity elicited by the vaccine is boosted by natural exposure to sporozoites. For this to occur, a helper T lymphocyte epitope present on the vaccine must be shared by sporozoites. These studies show that T cells from mice immunized with R32tet32, the Plasmodium falciparum sporozoite vaccine candidate, recognize an epitope of less than or equal to 7 amino acids derived from the circumsporozoite protein repeat region of R32tet32, as well as an epitope on the tet32 fusion protein tail of R32tet32. Exposure of R32tet32 immunized animals to P. falciparum sporozoites elicits a significant secondary antibody response which suggests that humans who are immunized and respond to this vaccine may be boosted by field exposure to sporozoite infected mosquitoes.     

Human lymphocyte proliferative response to a sporozoite T cell epitope correlates with resistance to falciparum malaria

To identify vaccine relevant T cell epitopes on the circumsporozoite (CS) protein of Plasmodium falciparum, the lymphocyte proliferative responses to 10 CS protein derived peptides were studied in 28 adult Kenyans, and correlated with resistance to malaria. Eight peptides, six of which were not overlapping, induced proliferation of lymphocytes from one to five volunteers, suggesting either genetic restriction of response to each of the T epitopes, or dominance of some T sites on the immunizing sporozoites. The 28 volunteers were radically cured of malaria and during the next 126 days 25 of the 28 were reinfected. Resistance to malaria was not correlated with antibodies to malaria Ag, but was significantly correlated with lymphocyte responses to CS protein residues 361-380 and 371-390. Among the 25 volunteers who became re-infected with malaria, lymphocytes from only two responded to a peptide including residues 361-380 of the P. falciparum CS protein, and only one to peptide 371-390. In contrast, lymphocytes from all three volunteers who did not become infected responded to peptide 361-380 (p = 0.003), and lymphocytes from two of the three responded to peptide 371-390 (p = 0.023). The significant correlation between proliferation to peptides 361-380 and 371-390 and resistance to malaria suggests that at least one epitope within these overlapping peptides is involved in a protective cellular immune response. The data support inclusion of these residues in future CS protein vaccines.     

HLA-DR-promiscuous T cell epitopes from Plasmodium falciparum pre-erythrocytic-stage antigens restricted by multiple HLA class II alleles

Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-gamma responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8. 7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.     

Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.     

Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development

CD8+ T cells against malaria liver stages represent a major protective immune mechanism against infection. Following induction in the peripheral lymph nodes by dendritic cells (DCs), these CD8+ T cells migrate to the liver and eliminate parasite infected hepatocytes. The processing and presentation of sporozoite antigen requires TAP mediated transport of major histocompatibility complex class I epitopes to the endoplasmic reticulum. Importantly, in DCs this process is also dependent on endosome-mediated cross presentation while this mechanism is not required for epitope presentation on hepatocytes. Protective CD8+ T cell responses are strongly dependent on the presence of CD4+ T cells and the capacity of sporozoite antigen to persist for a prolonged period of time. While human trials with subunit vaccines capable of inducing antibodies and CD4+ T cell responses have yielded encouraging results, an effective anti-malaria vaccine will likely require vaccine constructs designed to induce protective CD8+ T cells against malaria liver stages.     

Protective CD8+ T lymphocytes in primates immunized with malaria sporozoites

Live attenuated malaria vaccines are more potent than the recombinant protein, bacterial or viral platform vaccines that have been tested, and an attenuated sporozoite vaccine against falciparum malaria is being developed for humans. In mice, attenuated malaria sporozoite vaccines induce CD8(+) T cells that kill parasites developing in the liver. We were curious to know if CD8(+) T cells were also important in protecting primates against malaria. We immunized 9 rhesus monkeys with radiation attenuated Plasmodium knowlesi sporozoites, and found that 5 did not develop blood stage infections after challenge with live sporozoites. We then injected 4 of these protected monkeys with cM-T807, a monoclonal antibody to the CD8 molecule which depletes T cells. The fifth monkey received equivalent doses of normal IgG. In 3 of the 4 monkeys receiving cM-T807 circulating CD8(+) T cells were profoundly depleted. When re-challenged with live sporozoites all 3 of these depleted animals developed blood stage malaria. The fourth monkey receiving cM-T807 retained many circulating CD8(+) T cells. This monkey, and the vaccinated monkey receiving normal IgG, did not develop blood stage malaria at re-challenge with live sporozoites. Animals were treated with antimalarial drugs and rested for 4 months. During this interval CD8(+) T cells re-appeared in the circulation of the depleted monkeys. When all vaccinated animals received a third challenge with live sporozoites, all 5 monkeys were once again protected and did not develop blood stage malaria infections. These data indicate that CD8(+) T cells are important effector cells protecting monkeys against malaria sporozoite infection. We believe that malaria vaccines which induce effector CD8+ T cells in humans will have the best chance of protecting against malaria.     

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.     

Phase I trial of an alhydrogel adjuvanted hepatitis B core virus-like particle containing epitopes of Plasmodium falciparum circumsporozoite protein

The objectives of this non-randomized, non-blinded, dose-escalating Phase I clinical trial were to assess the safety, reactogenicity and immunogenicity of ICC-1132 formulated with Alhydrogel (aluminum hydroxide) in 51 healthy, malaria-naive adults aged 18 to 45 years. ICC-1132 (Malariavax) is a recombinant, virus-like particle malaria vaccine comprised of hepatitis core antigen engineered to express the central repeat regions from Plasmodium falciparum circumsporozoite protein containing an immunodominant B [(NANP)(3)] epitope, an HLA-restricted CD4 (NANPNVDPNANP) epitope and a universal T cell epitope (T*) (amino acids 326-345, NF54 isolate). We assessed an Alhydrogel (aluminum hydroxide)-adjuvanted vaccine formulation at three ICC-1132 dose levels, each injected intramuscularly (1.0 mL) on study days 0, 56 and 168. A saline vaccine formulation was found to be unstable after prolonged storage and this formulation was subsequently removed from the study. Thirty-two volunteers were followed for one year. Local and systemic adverse clinical events were measured and immune responses to P. falciparum and hepatitis B virus core antigens were determined utilizing the following assays: IgG and IgM ELISA, indirect immunofluorescence against P. falciparum sporozoites, circumsporozoite precipitin (CSP) and transgenic sporozoite neutralization assays. Cellular responses were measured by proliferation and IL-2 assays. Local and systemic reactions were similarly mild and well tolerated between dose cohorts. Depending on the ICC-1132 vaccine concentration, 95 to 100% of volunteers developed antibody responses to the ICC-1132 immunogen and HBc after two injections; however, only 29-75% and 29-63% of volunteers, respectively, developed malaria-specific responses measured by the malaria repeat synthetic peptide ELISA and IFA; 2 of 8 volunteers had positive reactions in the CSP assay. Maximal transgenic sporozoite neutralization assay inhibition was 54%. Forty-seven to seventy-five percent demonstrated T cell proliferation in response to ICC-1132 or to recombinant circumsporozoite protein (rCS) NF-54 isolate. This candidate malaria vaccine was well tolerated, but the vaccine formulation was poorly immunogenic. The vaccine may benefit from a more powerful adjuvant to improve immunogenicity. TRIAL REGISTRATION: ClinicalTrials.gov NCT00587249.     

Immune response gene regulation of immunity to Plasmodium berghei sporozoites and circumsporozoite protein vaccines. Overcoming genetic restriction with whole organism and subunit vaccines

We conducted a series of experiments to define Ir gene regulation of the immune response to Plasmodium berghei sporozoites and circumsporozoite (CS) protein-derived subunit vaccines. The studies demonstrated that there is no apparent genetic restriction of the capacity to develop protective immunity against a large sporozoite challenge after immunization with irradiation-attenuated P. berghei sporozoites; that the Th response to (Asp-Pro-Ala-Pro-Pro-Asn-Ala-Asn)n, the predominant protective B epitope on the P. berghei CS protein, is genetically restricted and regulated by Class II genes (I-Ab) and by genes in the Class I region (H-2Dk) or telomeric to this region; and that this restriction can be overcome by immunization with a r protein including the entire P. berghei CS protein. The results support the development of full length human CS protein vaccines to take advantage of all potential T epitopes on this protein.     

Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites

One of the primary strategies for malaria vaccine development has been to design subunit vaccines that induce protective levels of antibodies against the circumsporozoite (CS) protein of malaria sporozoites. In the Plasmodium yoelii mouse model system such vaccines have been uniformly unsuccessful in protecting against sporozoite-induced malaria. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of Navy yoelii sporozoite 1 (NYS1), an IgG3 mAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, a synthetic peptide derived from the repeat region of the P. yoelii CS protein, indicating that the epitope on sporozoites recognized by this mAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by indirect fluorescent antibody test and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing (QGPGAP)2 but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not absolute concentration could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective mAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. Although the results of the conventional antibody assays did not correlate with protection, sera from the protected animals inhibited sporozoite development in mouse hepatocyte cultures significantly more than did the sera from the unprotected, subunit vaccine-immunized animals, correlating with protection. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective mAb and nonprotective polyclonal antibodies will be important in the further development of malaria vaccines.     

P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections

In order to design P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x10⁶) and provided 80–100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.     

Chimeric epitopes delivered by polymeric synthetic linear peptides induce protective immunity to malaria

Polymeric linear peptide chimeras (LPCs) that incorporate Plasmodium vivax promiscuous T cell epitopes and the P. falciparum circumsporozoite protein B cell epitope have been shown to induce a high level of immunogenicity and overcome genetic restriction when tested as vaccine immunogens in BALB/c mice. The present study evaluates the biological relevance of several LPCs using a well characterized rodent malaria model. Polymeric peptide constructs based on P. berghei and P. yoelii sequences, and orthologous to the human malaria sequences included in the original LPCs, were designed and tested for immunogenicity in mice of different H-2 haplotypes. We demonstrate that robust immune responses are induced and that peptides containing the orthologous rodent Plasmodium sequences exhibited similar immunogenic capabilities. Unique to this report, we show that LPCs can also prime MHC class I-restricted cytotoxic T lymphocytes (CTLs) and, most relevantly, that a peptide construct prototype incorporating single B, T and CTL epitopes induced protection against an experimental challenge with P. berghei or P. yoelii sporozoites. Collectively, these results suggest that polymeric polypeptide chimeras can be used as a platform to deliver subunit vaccines.     

T cells as mediators of protective immunity against liver stages of Plasmodium

T cells from different subsets play a major role in protective immunity against pre-erythrocytic stages of malaria parasites. Exposure of humans and animals to malaria sporozoites induces (alphabeta CD8(+) and CD4(+) T cells specific for antigens expressed in pre-erythrocytic stages of Plasmodium. These T cells inhibit parasite development in the liver, and immunization with subunit vaccines expressing the respective antigenic moieties confers protection against sporozoite challenge. gammadelta and natural killer T cells can also play a role in protective immunity. Recent studies with mice transgenic for the alphabeta T-cell receptor have revealed the existence of complex mechanisms regulating the induction and development of these responses.     

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.     

Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

Background

Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection.

Methodology/Principal Findings

We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans.

Conclusions/Significance

We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.     

Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma

The Plasmodium falciparum circumsporozoite (CS) protein-based pre-erythrocytic stage vaccine, RTS,S, induces a high level of protection against experimental sporozoite challenge. The immune mechanisms that constitute protection are only partially understood, but are presumed to rely on Abs and T cell responses. In the present study we compared CS protein peptide-recalled IFN-gamma reactivity of pre- and RTS,S-immune lymphocytes from 20 subjects vaccinated with RTS,S. We observed elevated IFN-gamma in subjects protected by RTS,S; moreover, both CD4(+) and CD8(+) T cells produced IFN-gamma in response to CS protein peptides. Significantly, protracted protection, albeit observed only in two of seven subjects, was associated with sustained IFN-gamma response. This is the first study demonstrating correlation in a controlled Plasmodia sporozoite challenge study between protection induced by a recombinant malaria vaccine and Ag-specific T cell responses. Field-based malaria vaccine studies are in progress to validate the establishment of this cellular response as a possible in vitro correlate of protective immunity to exo-erythrocytic stage malaria vaccines.