Cellular immunity induced by the recombinant Plasmodium falciparum malaria vaccine, RTS,S/AS02, in semi-immune adults in The Gambia.

Vaccination of malaria-naive humans with recombinant RTS,S/AS02, which includes the C-terminus of the circumsporozoite protein (CS), has been shown to induce strong T cell responses to both the whole protein antigen and to peptides from CS. Here we show that strong T cell responses were also observed in a semi-immune population in The Gambia, West Africa. In a Phase I study, 20 adult male volunteers, lifelong residents in a malaria-endemic region, were given three doses of RTS,S/AS02 at 0, 1 and 6 months. Responses to RTS,S, hepatitis B surface antigen and peptides from CS were tested using lymphocyte proliferation, interferon (IFN)-gamma production in microcultures, and IFN-gamma ex vivo and cultured ELISPOT, before and after vaccination. Cytotoxic responses were tested only after vaccination and none were detected. Before vaccination, the majority of the volunteers (15/20) had detectable responses in at least one of the tests. After vaccination, responses increased in all assays except cytotoxicity. The increase was most marked for proliferation; all donors responded to RTS,S after the third dose and all except one donor responded to at least one peptide after the second or third dose. There was a lack of close association of peptide responses detected by the different assays, although in microcultures IFN-gamma responses were found only when proliferative responses were high, and responses by cultured ELISPOT and proliferation were found together more frequently after vaccination. We have therefore identified several peptide-specific T cell responses induced by RTS,S/AS02 which provides a mechanism to investigate potentially protective immune responses in the field.

Prospective randomised phase II study of gemcitabine at standard or fixed dose rate schedule in unresectable hepatocellular carcinoma.

The present randomised phase II study was an effort to evaluate single-agent gemcitabine as a first-line systemic treatment of Asian patients with unresectable hepatocellular carcinoma (HCC). Gemcitabine was given via intravenous infusion at 1250 mg m(-2) on days 1 and 8 of 3-week cycles. Patients were randomised to receive gemcitabine as a 30-min intravenous infusion (standard schedule) or at a fixed dose rate (FDR) of 10 mg m(-2) min(-1). A total of 50 patients were enrolled in the study, of whom 48 received study therapy. One patient on standard schedule had a partial response, for an overall response rate of 2.1% (95% CI: 0.05-11.1%). The median time to progression and survival time were 46 and 97 days, respectively. The overall rates of Grade 3 or 4 haematological and nonhaematological toxicities were 39.6 and 64.6%, respectively, with no significant difference between the two treatment arms. There were no drug-related deaths and severe clinical toxicities were rare. Both schedules of gemcitabine were safe and toxicity was well manageable in this patient population. However, gemcitabine seems no more active than other cytotoxic agents when used alone for systemic treatment of advanced HCC.

Naturally exposed populations differ in their T1 and T2 responses to the circumsporozoite protein of Plasmodium falciparum.

T-cell responses directed against the circumsporozoite protein (CS) of Plasmodium falciparum can mediate protection against malaria. We determined the frequency of T cells reactive to different regions of the CS in the blood of donors naturally exposed to P. falciparum by examining T1 (gamma interferon [IFN-gamma] ELISPOT assay), T2 (interleukin 4 [IL-4] ELISPOT assay), and proliferative T-cell responses. The proliferative responses were weak, which confirmed previous observations. The responses to the CS in the IL-4 and IFN-gamma ELISPOT assays were also weak (<40 responding cells per 10(6) cells), much weaker than the response to the purified protein derivative of Mycobacterium tuberculosis in the same donors. Moreover, a response in one assay could not be used to predict a response in either of the other assays, suggesting that although these assays may measure different responding cells, all of the responses are weakly induced by natural exposure. Interestingly, the two different study populations used had significantly different T1 and T2 biases in their responses in the C terminus of the protein, suggesting that the extent of P. falciparum exposure can affect regulation of the immune system.

Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial.

BACKGROUND: RTS,S/AS02 is a pre-erythrocytic malaria vaccine based on the circumsporozoite surface protein of Plasmodium falciparum fused to HBsAg, incorporating a new adjuvant (AS02). We did a randomised trial of the efficacy of RTS,S/AS02 against natural P. falciparum infection in semi-immune adult men in The Gambia. METHODS: 306 men aged 18-45 years were randomly assigned three doses of either RTS,S/AS02 or rabies vaccine (control). Volunteers were given sulfadoxine/pyrimethamine 2 weeks before dose 3, and kept under surveillance throughout the malaria transmission season. Blood smears were collected once a week and whenever a volunteer developed symptoms compatible with malaria. The primary endpoint was time to first infection with P. falciparum. Analysis was per protocol. FINDINGS: 250 men (131 in the RTS,S/AS02 group and 119 in the control group) received three doses of vaccine and were followed up for 15 weeks. RTS,S/AS02 was safe and well tolerated. P. falciparum infections occurred significantly earlier in the control group than the RTS,S/AS02 group (Wilcoxon's test p=0.018). Vaccine efficacy, adjusted for confounders, was 34% (95% CI 8.0-53, p=0.014). Protection seemed to wane: estimated efficacy during the first 9 weeks of follow-up was 71% (46-85), but decreased to 0% (-52 to 34) in the last 6 weeks. Vaccination induced strong antibody responses to circumsporozoite protein and strong T-cell responses. Protection was not limited to the NF54 parasite genotype from which the vaccine was derived. 158 men received a fourth dose the next year and were followed up for 9 weeks; during this time, vaccine efficacy was 47% (4-71, p=0.037). INTERPRETATION: RTS,S/AS02 is safe, immunogenic, and is the first pre-erythrocytic vaccine to show significant protection against natural P. falciparum infection.

Unique T cell effector functions elicited by Plasmodium falciparum epitopes in malaria-exposed Africans tested by three T cell assays.

Natural immunity to malaria is characterized by low level CD4 T cell reactivity detected by either lymphoproliferation or IFN-gamma secretion. Here we show a doubling in the detection rate of responders to the carboxyl terminus of circumsporozoite protein (CS) of Plasmodium falciparum by employing three T cell assays simultaneously: rapid IFN-gamma secretion (ex vivo ELISPOT), IFN-gamma secretion after reactivation of memory T cells and expansion in vitro (cultured ELISPOT), and lymphoproliferation. Remarkably, for no individual peptide did a positive response for one T cell effector function correlate with any other. Thus these CS epitopes elicited unique T cell response patterns in malaria-exposed donors. Novel or important epitope responses may therefore be missed if only one T cell assay is employed. A borderline correlation was found between anti-CS Ab levels and proliferative responses, but no correlation was found with ex vivo or cultured IFN-gamma responses. This suggested that the proliferating population, but not the IFN-gamma-secreting cells, contained cells that provide help for Ab production. The data suggest that natural immunity to malaria is a complex function of T cell subgroups with different effector functions and has important implications for future studies of natural T cell immunity.

Identification of frequently recognized dimorphic T-cell epitopes in plasmodium falciparum merozoite surface protein-1 in West and East Africans: lack of correlation of immune recognition and allelic prevalence.

The merozoite surface protein-1 (MSP1) is the most studied malaria blood-stage vaccine candidate. Lymphokines such as interferon gamma (IFN-gamma) and interleukin 4 (IL-4) may mediate blood-stage specific protection. Here we identify Plasmodiumfalciparum MSP1 T-cell epitopes capable of rapid induction of IFN-gamma and/or IL-4 from peripheral blood mononuclear cells of East and West African donors. Both allelic forms of these novel MSP1 T-cell epitopes were stimulatory. An unusually high numbers of Gambian responders (> 80%) to these epitopes were observed, suggesting that MSPI reactivity may have been underestimated previously in this population. Surprisingly, IFN-gamma responses to allelic T-cell epitopes failed to correlate with differential antigenic exposure in The Gambia compared to Kenya. These results suggest an unexpected level of immunoregulation of IFN-gamma response with variable allelic T-cell reactivity independent of the level of antigenic exposure. Further analysis of the mechanisms determining this response pattern may be required if vaccines are to overcome this allelic reactivity bias in malaria-exposed populations.

Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells.

BACKGROUND: Identification of individuals latently infected with Mycobacterium tuberculosis is an important part of tuberculosis control. The current method, the tuberculin skin test (TST), has poor specificity because of the antigenic cross-reactivity of purified protein derivative (PPD) with M bovis BCG vaccine and environmental mycobacteria. ESAT-6 is a secreted antigen that is highly specific for M tuberculosis complex, but is absent from M bovis BCG. With an enzyme-linked immunospot (ELISPOT) assay for interferon gamma, we have identified ESAT-6-specific T cells as an accurate marker of M tuberculosis infection. METHODS: We did a prospective, masked study of 50 healthy contacts, with varying but well defined degrees of exposure to M tuberculosis, who attended an urban contact-tracing clinic. We assessed and compared the efficacy of our assay and TST for detection of symptomless infected individuals by correlation of test results with the degree of exposure to an infectious index case. FINDINGS: The ESAT-6 ELISPOT assay results had a strong positive relation with increasing intensity of exposure (odds ratio=9.0 per unit increase in level of exposure [95% CI 2.6--31.6], p=0.001), whereas TST results had a weaker relation with exposure (1.9 [1.0--3.5], p=0.05). By contrast, ELISPOT results were not correlated with BCG vaccination status (p=0.7), whereas TST results were significantly more likely to be positive in BCG-vaccinated contacts (12.1 [1.3--115.7], p=0.03). INTERPRETATION: This new antigen-specific T cell-based assay could allow more accurate identification of symptom-free individuals recently exposed to M tuberculosis, and thereby help to improve tuberculosis control.

Interleukin 10-mediated immunosuppression by a variant CD4 T cell epitope of Plasmodium falciparum.

The immunodominant CD4 T cell epitope region, Th2R, of the circumsporozoite protein of Plasmodium falciparum is highly polymorphic. Such variation might be utilized by the parasite to escape from or interfere with CD4 T cell effector functions. Here, we show that costimulation with naturally occurring altered peptide ligands (APL) can induce a rapid change from IFNgamma production to the immunosuppressive mediator interleukin 10 (IL-10). This mechanism may contribute to the low levels of T cell responses observed to this pathogen in malaria-endemic areas.

Broadly distributed T cell reactivity, with no immunodominant loci, to the pre-erythrocytic antigen thrombospondin-related adhesive protein of Plasmodium falciparum in West Africans.

Protective immunity to malaria has been achieved in human volunteers utilizing the pre-erythrocytic Plasmodium falciparum antigen, the circumsporozoite protein (CS). However, T cell reactivity to CS is focused on several highly polymorphic T cell epitope regions, potentially limiting the efficacy of any vaccine to specific malaria strains. Another important pre-erythrocytic malaria antigen, the thrombospondin-related adhesive protein (TRAP), can induce protection in animal models of malaria, but knowledge of human T cell responses is limited to the identification of CD8 T cell epitopes, with no CD4 epitopes identified to date. This comprehensive study assessed reactivity to overlapping peptides spanning almost the whole of P. falciparum TRAP (PfTRAP), as well as peptides selected on the basis of HLA class II-binding motifs. A total of 50 naturally exposed Gambian adults were assessed to define 26 T cell epitopes in PfTRAP capable of inducing rapid IFN-gamma or IL-4 production, as assessed by enzyme-linked immunospot assays. In contrast to the CS protein, this reactivity was broadly distributed along the length of TRAP. Moreover, of the 26 epitopes identified, 10 were found to be conserved in West Africa.