A phase IIa, randomized, double-blind, safety, immunogenicity and efficacy trial of Plasmodium falciparum vaccine antigens merozoite surface protein 1 and RTS,S formulated with AS02 adjuvant in healthy, malaria-naïve adults.

BACKGROUND: To improve the efficacy of Plasmodium falciparum malaria vaccine RTS,S/AS02, we conducted a study in 2001 in healthy, malaria-naïve adults administered RTS,S/AS02 in combination with FMP1, a recombinant merozoite surface-protein-1, C-terminal 42kD fragment. METHODS: A double-blind Phase I/IIa study randomized N = 60 subjects 1:1:1:1 to one of four groups, N = 15/group, to evaluate safety, immunogenicity, and efficacy of intra-deltoid half-doses of RTS,S/AS02 and FMP1/AS02 administered in the contralateral (RTS,S + FMP1-separate) or same (RTS,S + FMP1-same) sites, or FMP1/AS02 alone (FMP1-alone), or RTS,S/AS02 alone (RTS,S-alone) on a 0-, 1-, 3-month schedule. Subjects receiving three doses of vaccine and non-immunized controls (N = 11) were infected with homologous P. falciparum 3D7 sporozoites by Controlled Human Malaria Infection (CHMI). RESULTS: Subjects in all vaccination groups experienced mostly mild or moderate local and general adverse events that resolved within eight days. Anti-circumsporozoite antibody levels were lower when FMP1 and RTS,S were co-administered at the same site (35.0 µg/mL: 95 % CI 20.3-63), versus separate arms (57.4 µg/mL: 95 % CI 32.3-102) or RTS,S alone (62.0 µg/mL: 95 % CI: 37.8-101.8). RTS,S-specific lymphoproliferative responses and ex vivo ELISpot CSP-specific interferon-gamma (IFN-γ) responses were indistinguishable among groups receiving RTS,S/AS02. There was no difference in antibody to FMP1 among groups receiving FMP1/AS02. After CHMI, groups immunized with a RTS,S-containing regimen had âˆ¼ 30 % sterile protection against parasitemia, and equivalent delays in time-to-parasitemia. The FMP1/AS02 alone group showed no sterile immunity or delay in parasitemia. CONCLUSION: Co-administration of RTS,S and FMP1/AS02 reduced anti-RTS,S antibody, but did not affect tolerability, cellular immunity, or efficacy in a stringent CHMI model. Absence of efficacy or delay of patency in the sporozoite challenge model in the FMP1/AS02 group did not rule out efficacy of FMP1/AS02 in an endemic population. However, a Phase IIb trial of FMP1/AS02 in children in malaria-endemic Kenya did not demonstrate efficacy against natural infection. CLINICALTRIALS: gov identifier: NCT01556945.

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. II. Only certain T helper or T suppressor cells are relevant in particular regulatory interactions.

11 cyanogen bromide (CB) peptides, comprising 70% of the large protein, Escherichia coli beta-galactosidase (GZ), were studied for their ability to induce T suppressor (Ts) cells capable of strongly suppressing the in vitro anti-fluorescein (FITC) response to GZ-FITC. Only CB-2 (amino acid residues 3-92) and CB-3 (residues 93-187) were found to bear such Ts-inducing epitopes. In examining the specificity of T helper cell (Th) targets susceptible to CB-2 and CB-3-specific Ts, it appeared that only nearly Th targets could be suppressed. Thus, CB-10-primed Th were not suppressed by either Ts; even CB-3-primed Ts did not suppress CB-2-specific Th, although CB-2-specific Ts were effective. Furthermore, analysis of the suppression pattern revealed a hierarchical use of potential epitopes on native GZ in triggering functional regulatory T cells. A dominant Th epitope near the amino terminus of GZ tops a hierarchy of potential Th, most of which are never engaged. The dominant determinant seems to exist on the peptide CB-2-3 (residues 3-187), and presumably is destroyed by its cleavage at Met 92; the Th cells that it induces are suppressible by each of the Ts-inducing peptides. In the GZ system, where the native antigen is quite large, the interactions between Th and Ts are highly circumscribed. This may be attributable to the topology of antigen fragments produced during processing; any relevant fragment must bear at least a Ts- and Th-reactive determinant to permit intercellular regulation. A final implication of these results is that, not only does the existence of a Th-inducing determinant depend on its being an appropriate distance from a B cell epitope, but the existence of Ts-inducing determinants likewise depends on the existence of a neighboring Th-B cell association.

An early commitment to expression of a particular TCRVbeta chain on CD8(+) T cells responding to attenuated Plasmodium berghei sporozoites is maintained following challenge with infectious sporozoites.

Protection induced by irradiated Plasmodium berghei sporozoites (Pbgamma-spz) in mice is linked to CD8(+) T cells specific for exo-erythrocytic-stage Ags, and intrahepatic memory CD8(+) T cells are associated with protracted protection. However, the Ag specificity of the protective CD8(+) T cells remains largely unknown. In this study, we characterized the TCR Vbeta usage by intrahepatic CD8(+) T cells during gamma-spz immunization and after the challenge with infectious Pb sporozoites. The repertoire of naïve (T(N)) and central memory (T(CM)) CD8(+) T cells was diverse and conserved between individual mice, and did not change with immunization. In contrast, preferential usage of one or more TCR Vbeta subset was observed in effector memory (T(EM)) CD8(+) T cells after immunization. The expanded TCR Vbeta varied between individual mice but Vbeta4, 6, 7, 8.3, 9 and 11 were the most frequently expressed. In addition, there was a correlation in the TCR Vbeta usage by gamma-spz-induced CD8(+) T(EM) in the liver and blood of individual mice. The expansion pattern of blood CD8(+) T(EM) did not change with challenge and remained the same for 8 weeks thereafter. These results demonstrate that immunization with gamma-spz skews the TCR Vbeta repertoire of CD8(+) T(EM), and commitment to a particular TCR Vbeta expression is maintained long-term.

The dissection of CD8 T cells during liver-stage infection.

Multiple injections of gamma-radiation-attenuated Plasmodium sporozoites (gamma-spz) can induce long-lived, sterile immunity against pre-erythrocytic stages of malaria. Malaria antigen (Ag)-specific CD8 T cells that produce IFN-gamma are key effector cells in this model of protection. Although there have been numerous reports dealing with gamma-spz-induced CD8 T cells in the spleen, CD8 T cells most likely confer protection by targeting infected hepatocytes. Consequently, in this chapter we discuss observations and hypotheses concerning CD8 T cell responses that occur in the liver after an encounter with the Plasmodium parasite. Protracted protection against pre-erythrocytic stages requires memory CD8 T cells and we discuss evidence that gamma-spz-induced immunity is indeed accompanied by the presence of intrahepatic CD44hi CD45RBlo CD62lo CD122lo effector memory (EM) CD8 T cells and CD44hi CD45RBhi CD621hi CD122hi central memory (CM) CD8 T cells. In addition, the EM CD8 T cells rapidly release IFN-gamma in response to spz challenge. The possible role of Kupffer cells in the processing of spz Ags and the production of cytokines is also considered. Finally, we discuss evidence that is consistent with a model whereby intrahepatic CM CD8 T cells are maintained by IL-15 mediated-homeostatic proliferation while the EM CD8 T cells are conscripted from the CM pool in response to a persisting depot of liver-stage Ag.

Correction: Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

[This corrects the article DOI: 10.1371/journal.pone.0159449.].

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine.

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.

Immune response to Plasmodium berghei sporozoite antigens. I. Evaluation of murine T cell repertoire following immunization with irradiated sporozoites.

The Plasmodium berghei sporozoite antigen-specific T cell repertoire was analyzed in C57BL/6 (H-2b), BALB/c (H-2d) and C3H/HeN (H-2k) mice following immunization with irradiated sporozoites. Proliferative responses were correlated with the protective status of each strain. Proliferative reactivities to sporozoite antigens were compared in cultures containing either CD4+ T cells, CD8+ T cells, or total splenic lymphocytes. CD8+ T cells had no proliferative activity to sporozoite antigens; CD4+ T cells and splenic lymphocytes responded to the priming antigen, but the responses varied according to the mouse strain tested. The proliferative activity diminished at the onset of protection, presumably due to the induction of regulatory or non-proliferative T cell subsets. Sporozoite-immune lymphocytes did not respond to P. berghei circumsporozoite synthetic peptides. The restricted utilization of T cell epitopes during anti-sporozoite responses can be interpreted as resulting in part from a limited processing of the CS protein antigen.

The distinctive specificity of antigen-specific suppressor T cells.

Although suppressor T cells have been cloned in only a few instances, the existence of a functional cadre of T cells that acts to downregulate the immune response is well documented. In this review Eli Sercarz and Urszula Krzych describe studies on suppressor T-cell (TS-cell) specificity that provide some support for the conclusion that the TS cell is a distinctive cell type with an expressed repertoire that is different from that expressed by helper T (TH) cells. They go on to explore the interaction between cells recognizing TS-cell-inducing determinants (SDs) and TH-cell-inducing determinants (HDs), and their relationship to immunogenicity and Ir gene effects.

The relationships between Ts-inducing and Th and Tp-inducing determinants on a large protein antigen.

The immunogenicity of several small monomeric protein antigens - lysozyme, myoglobin, cytochrome c and insulin - has been intensely studied during the past decade to try to learn the rules of the game. It is likely that before the stage is reached at which we can predict the nature of the determinants responded against by each lymphoid subpopulation, other types of molecules such as fibrous proteins, multimeric large proteins and viral capsid proteins will have to be examined, and such studies have commenced recently. In an effort to generalize our studies with lysozyme to one of these systems, we have been exploring the response of H-2k, CBA/J mice to E. coli B-galactosidase, GZ, a tetramer with a monomer molecular weight of 116,250. We feel that aspects of this system have important implications for the vaccine problem, and these will be summarized at the end of the article.

Plasmodium falciparum malaria blood stage parasites preferentially inhibit macrophages with high phagocytic activity.

Exposure to malaria blood stage antigens results in several defects of macrophages/monocytes one of which is an irreversible reduction of phagocytic activity. In the present study we analysed phagocytic activity of subpopulations of human monocyte-derived-macrophages (MDM) based on the capacity of individual cells to ingest FITC-labelled microbeads. The results demonstrate that malaria infection affected predominantly MDM subpopulations with high level of phagocytosis. This population decreased during parasitaemia, however, during recovery from the infection the highly phagocytic cells replaced the damaged cells. The exposure of MDM cultures to blood stage antigens showed that the highly active macrophages from persons with active malaria infection decreased further, while the population increased during recovery. Furthermore, we observed that while ingestion of a few parasitized RBC (3 schizonts) stimulated phagocytosis, larger amounts or longer exposure periods eventually paralysed the entire phagocytic system. Accordingly, by selectively blocking actively phagocytizing macrophages, the malaria parasite prevents both specific and non-specific immune responses, which are initiated by macrophages as phagocytes and professional antigen presenting cells.

The molecular context of determinants within the priming antigen establishes a hierarchy of T cell induction: T cell specificities induced by peptides of beta-galactosidase vs. the whole antigen.

The antibody response following priming with a macromolecule or a peptide will depend on the regulatory T cells that become activated by the antigenic determinants available. In this report, activation of T helper (Th) and T suppressor (Ts) cells by determinants on beta-galactosidase (GZ) was examined by comparing native GZ [1023 amino acid (a.a.) residues per monomer] with peptides from the immunodominant region encompassing residues 3 to 187. Each immunogen established its characteristic hierarchy of dominance of determinants within it: in particular, GZ and CB-2-3 (a.a. 3-187) each induced immunodominant Th cells which could not be induced by T8 (a.a. 60-140). Hierarchies of suppressor determinant are also created: T8-Ts suppresses all Th specificities and therefore can be deemed immunodominant: T8-2-Ts and T8-3-Ts have a more selective suppressor activity and can be considered subdominant. We conclude that the outcome of immunization shifts with a change in the nature of the immunogen and the context within which the determinant lies will crucially influence its expression. A particular "context" presumably determines the likely order of processing of that molecule which leads to a characteristic relationship among the Ts, Th and B cell determinants involved.

MHC class I-dependent presentation of exoerythrocytic antigens to CD8+ T lymphocytes is required for protective immunity against Plasmodium berghei.

T lymphocytes are believed to play a major role in protection against malaria. Previous experiments using in vivo depletion of CD8+ T cells, reconstitution with CD8+ T splenic cells, and adoptive transfer of CD8+ CTL clones demonstrated that protection against the exoerythrocytic stage of the murine strain, Plasmodium berghei malaria, was CD8+ T cell-dependent. Despite evidence for the critical role of CD8+ CTL, neither the cellular nor the molecular requirements for CD8+ T cell induction or for recognition of malaria Ags are known. In this study, we wished to define the role of CD8+ T cells and MHC class I molecules by using the P. berghei malaria attenuated sporozoites (SPZ) protection model in beta 2-microglobulin (beta 2m) knockout (-/-) mice. In contrast to observations that beta 2m-/- mice are resistant to many infectious diseases by compensatory mechanisms involving non-class I-restricted T cells, we found that beta 2m-/- mice failed to be protected against P. berghei SPZ, although immunization with attenuated SPZ induced production of IL-2, INF-gamma, anti-circumsporozoite protein IgG, and proliferative T cells. The lack of compensatory mechanisms involving non-CD8+ T cells was particularly evident in the failure to adoptively transfer protective immunity with wild-type SPZ-immune splenic T cells. From our data it can be concluded that CD8+ T cells induced during immunization with attenuated SPZ must recognize liver-expressed Ags presented by class I molecules to engage effectively in a response leading to destruction of the malaria parasites.

Detection of CD4+CD45RO+ T lymphocytes producing IL-4 in response to antigens on Plasmodium falciparum erythrocytes: an in vitro correlate of protective immunity induced with attenuated Plasmodium falciparum sporozoites.

Malaria is caused by Plasmodium spp. and is one of the major infectious diseases leading to morbidity and mortality in tropical areas of the world. The model of protective immunity induced by immunization with radiation-attenuated Plasmodia sporozoites (SPZ) has become the framework for the elucidation of protective immune mechanisms and the prototype for a promising vaccine strategy. We have previously reported that although considered stage specific based on antibody and CD8+ cytolytic T lymphocyte responses directed against preerythrocytic stage antigens, in particular, the circumsporozoite protein and sporozoite surface protein 2, protective immunity induced in humans by attenuated Plasmodium falciparum SPZ may also involve CD4+ T cell responding to antigens present on parasitized red blood cells (pRBC). In this study we examined the functional role of pRBC responding CD4+ T cells by comparing in vitro pRBC-stimulated responses of CD4+ T cells from persons during preimmunity to irradiated SPZ, during induction of protection, and infection induced with SPZ. The results reported herein corroborate previously published observations that antigens associated with pRBC induce proliferative CD4+ lymphocytes responses in subjects exposed to malaria parasite-derived antigens and not malaria-naive persons; however, now we demonstrate that pRBC-proliferative CD4+ T cells did not coincide with protective immunity. Similarly, pRBC-induced IFN-gamma levels did not distinguish malaria protected from susceptible persons, although IFN-gamma was observed only in lymphocyte cultures from malaria parasite-exposed volunteers and not in lymphocyte cultures from malaria-naive persons. In contrast, we noted an increase in the IL-4-producing CD4+ T cells that also exhibited the memory phenotype, CD45RO, and an upregulated expression of CD25 in cultures from malaria protected persons as compared to malaria naive persons and subjects who became parasitemic. Hence, these observations suggest that the induction of memory CD4+ T cell subset distinguished by the expression of CD45RO and CD25 and production of IL-4 coincides with protective immune responses generated by immunization with attenuated SPZ.

Immunization with irradiated Plasmodium berghei sporozoites induces IL-2 and IFN gamma but not IL-4.

Protective immunity against Plasmodium induced by immunization with irradiated sporozoites (SPZ) depends on both humoral and cellular responses. Although circumsporozoite protein (CSP)-specific cytolytic T lymphocyte responses have been established as an effector system, other cell types are required for protection. We have previously demonstrated that although protective immunity and T cell proliferative reactivity to SPZ are mouse strain- and SPZ dose-dependent, no correlation between the two responses could be found. Since protective immunity involves functionally diverse T cell subsets, we asked whether the discordance between proliferative responses to SPZ and protective immunity might have resulted from selective activation of either the Th1 or Th2 cell subset. Protective immunity, in vitro proliferative responses, and lymphokine production were tested in BALB/c, C57Bl/6, and C3H/HeN mice immunized according to different SPZ regimens. The levels of IL-2 paralleled the proliferative reactivities in each mouse strain examined. Although IFN gamma levels were present in the unprimed lymphocyte cultures, they increased following each SPZ immunization, in C57Bl/6, moderate in C3H/HeN, and lowest in BALB/c splenic cultures. Surprisingly, no IL-4 was detected in splenic cultures from any mouse strain during proliferative activity or protective immunity. In contrast, elevated IL-6 production was noted after each immunization, regardless of the protective status and it correlated with anti-CSP IgG serum levels. These data establish that lymphokine profiles corresponding primarily to the Th1 cells were induced by immunization with P. berghei SPZ and that IL-4 secreting T cells were not induced by the SPZ-stage berghei antigens.

Plasmodium berghei-specific T cells respond to non-processed sporozoites presented by B cells.

The mechanism of malaria protective immunity induced by immunization with radiation-attenuated Plasmodium sporozoites (SPZ) is only partially understood. For example, B and T cell responses specific for the circumsporozoite (CS) protein, a 46 kDa SPZ surface protein, have been characterized; however, events leading to SPZ-specific T cell activation, i.e., processing and presentation of SPZ by antigen-presenting cells have not been investigated. In the present study we describe the in vitro analysis of requirements for accessory cell function in the presentation of SPZ to SPZ-immune T cells. The results establish that SPZ-induced proliferative T cells are reactive to non-processed SPZ presented by activated B cells and, thus, imply that the non-processed form of the SPZ-associated CS protein restricts the induction of the potential CS protein T cell repertoire.

Memory phenotype CD8(+) T cells persist in livers of mice protected against malaria by immunization with attenuated Plasmodium berghei sporozoites.

Natural exposure to Plasmodium parasites induces short-lived protective immunity. In contrast, exposure to radiation-attenuated sporozoites (gamma spz) promotes long-lasting protection that is in part mediated by CD8(+) T cells that target exoerythrocytic stage antigens. The mechanisms underlying the maintenance of long-lasting protection are currently unclear. The liver is a repository of Plasmodium antigens and may support the development and / or homing of memory T cells. While activated CD8(+) T cells are presumed to die in the liver, the fate of anti-Plasmodium CD8(+) T cells remains unknown. We propose that inflammatory conditions in the liver caused by Plasmodium parasites may allow some effector CD8(+) T cells to survive and develop into memory cells. To support this hypothesis, in this initial study we demonstrate that liver mononuclear cells from P. berghei gamma spz-immune mice transferred protection to naive recipients and moreover, that CD4(+) and CD8(+) T cells responded to Plasmodium antigens by up-regulating activation / memory markers. While CD4(+) T cells under went a transient activation following immunization with gamma spz, CD8(+) T cells expanded robustly after spz challenge and exhibited stable expression of CD44(hi) and CD45RB(lo) during protracted protection. These results establish a key role for intrahepatic T cells in long-lasting protection against malaria.

Induction of helper and suppressor T cells by nonoverlapping determinants on the large protein antigen, beta-galactosidase.

The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppressor (Ts)-inducing determinants was examined with cyanogen bromide and tryptic peptides of Escherichia coli beta-galactosidase (GZ), a large tetrameric protein (monomer molecular weight = 116 kDa). Immunization with cyanogen bromide fragment 2 [CB-2, amino acids (a.a.) 3-92] induced both specific Th and Ts cells. Study of the induction of these functionally opposite T cell subpopulations with tryptic peptides of CB-2 indicated that Th and Ts were activated by separate, nonoverlapping determinants. Th-inducing activity resided in a nonapeptide, T6 (a.a. 44-52), whereas T4 (a.a. 27-37) induced Ts cells. The presence of distinct helper and suppressor determinants suggests that the specificity repertoire in these T cell subpopulations may differ, perhaps owing to the expression of antigen-recognizing receptors that are coded by unique gene families. Alternatively, antigen presentation structures may be physicochemically quite different, and bind to distinct parts of the peptide antigen.

Sex-related immunocompetence of BALB/c mice. I. Study of immunologic responsiveness of neonatal, weanling, and young adult mice.

The responses of lymphoid cells from the thymus, lymph nodes, and spleen of male and female BALB/c mice were evaluated to determine if sex-related variations in immune expression could be found. Immunologic assays used included blastogenic responses to mitogens, mixed lymphocyte responses, and direct and indirect measurement of plaque-forming cells against soluble and particulate antigens. The results indicated that responses of spleen cells from young adult female mice were higher than those of males in all comparative tests. Little or no differences between the sexes were observed in the mitogenesis of lymph nodes and thymuses. Newborn mice did not demonstrate the sex-associated immune differences. Among the weanling mice slight differences between male and female spleen cells responsiveness to mitogenic agents were observed.

Effects of sex hormones on some T and B cell functions, evidenced by differential immune expression between male and female mice and cyclic pattern of immune responsiveness during the estrous cycle in female mice.

The responses of spleen cells from male and female BALB/c mice were evaluated to determine if sex-related variations in immune expression could be found. The immunologic assays used included blastogenic responses to mitogens, and direct and indirect measurement of plaque-forming cells against particulate antigens. The results indicated that responses of spleen cells from young adult female mice were higher than those of males in all comparative tests. Newborn mice did not demonstrate the sex-associated immune differences; and among the weanling mice slight differences between male and female spleen cells responsiveness to mitogenic agents were observed. The blastogenic responsiveness of spleens from female BALB/c was greater at proestrus and metestrus, as compared to estrus and diestrus. The peaks of responsiveness corresponded to reported elevated levels of estrogen and pregnenolone during these phases of the cycle. Similar results were obtained with the IgM plaque-forming cell responses, which were also increased at proestrus and metestrus. This study supports a role of sex hormones in modulation of immune expression.