Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor.

The subcellular localization and function of variant subtelomeric multigene families in Plasmodium vivax remain vastly unknown. Among them, the vir superfamily is putatively involved in antigenic variation and in mediating adherence to endothelial receptors. In the absence of a continuous in vitro culture system for P. vivax, we have generated P. falciparum transgenic lines expressing VIR proteins to infer location and function. We chose three proteins pertaining to subfamilies A (VIR17), C (VIR14) and D (VIR10), with domains and secondary structures that predictably traffic these proteins to different subcellular compartments. Here, we showed that VIR17 remained inside the parasite and around merozoites, whereas VIR14 and VIR10 were exported to the membrane of infected red blood cells (iRBCs) in an apparent independent pathway of Maurer's clefts. Remarkably, VIR14 was exposed at the surface of iRBCs and mediated adherence to different endothelial receptors expressed in CHO cells under static conditions. Under physiological flow conditions, however, cytoadherence was only observed to ICAM-1, which was the only receptor whose adherence was specifically and significantly inhibited by antibodies against conserved motifs of VIR proteins. Immunofluorescence studies using these antibodies also showed different subcellular localizations of VIR proteins in P. vivax-infected reticulocytes from natural infections. These data suggest that VIR proteins are trafficked to different cellular compartments and functionally demonstrates that VIR proteins can specifically mediate cytoadherence to the ICAM-1 endothelial receptor.

Vaccine potentials of an intrinsically unstructured fragment derived from the blood stage-associated Plasmodium falciparum protein PFF0165c.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

Processing of the circumsporozoite protein in infected hepatocytes is not dependent on aspartic proteases.

CD8(+) T cells play a major role in the protective immune response against the liver stage of malaria. It was previously shown that the circumsporozoite protein (CSP) is processed and presented to specific T cells by both traversed and infected hepatocytes, but their respective antigen processing requirements were not completely defined. In the present study, we show that in vitro processing of the Plasmodium berghei CSP by infected mouse primary hepatocytes is exclusively dependent on proteasomes, while aspartic proteases are also needed in the case of traversed hepatocytes.

Do antibody responses to malaria vaccine candidates influenced by the level of malaria transmission protect from malaria?

OBJECTIVES: To examine whether the humoural response to malaria vaccine candidate antigens, Plasmodium falciparum [circumsporozoite repetitive sequence (NANP)(5) GLURP fragments (R0 and R2) and MSP3] varies with the level of malaria transmission and to determine whether the antibodies (IgG) present at the beginning of the malaria transmission season protect against clinical malaria. METHODS: Cross-sectional surveys were conducted to measure antibody response before, at the peak and at the end of the transmission season in children aged 6 months to 10 years in two villages with different levels of malaria transmission. A cohort study was performed to estimate the incidence of clinical malaria. RESULTS: Antibodies to these antigens showed different seasonal patterns. IgG concentrations to any of the four antigens were higher in the village with high entomological inoculation rate. Multivariate analysis of combined data from the two villages indicated that children who were classified as responders to the selected antigens were at lower risk of clinical malaria than children classified as non-responders [(NANP)(5) (incidence rate ratio (IRR) = 0.65, 95% CI: 0.46-0.92; P = 0.016), R0 (IRR = 0.69, 95% CI: 0.48-0.97; P = 0.032), R2 (IRR = 0.73, 95% CI: 0.50-1.06; P = 0.09), MSP3 (IRR = 0.52, 95% CI: 0.32-0.85; P = 0.009)]. Fitting a model with all four antibody responses showed that MSP3 looked the best malaria vaccine candidate (IRR = 0.63; 95% CI: 0.38-1.05; P = 0.08). CONCLUSION: Antibody levels to the four antigens are affected by the intensity of malaria transmission and associated with protection against clinical malaria. It is worthwhile investing in the development of these antigens as potential malaria vaccine candidates.

IL-12p40-independent induction of protective immunity upon multiple Plasmodium berghei irradiated sporozoite immunizations.

Both IFN-gamma and IL-12 play critical roles in defence against malaria. In a previous study, using Plasmodium yoelii model, C57BL/6 IFN-gamma receptor deficient mice (IFN-gammaR-/-) failed to develop protective immunity after a single immunization with irradiated sporozoites, but were protected after multiple immunizations. In contrast, in another study, BALB/c IFN-gamma gene knockout mice (IFN-gamma-/-) and BALB/c IL-12-deficient mice (IL-12p40-/- and IL-12p35-/-) were unable to mount protective immune response even after multiple immunizations with the same irradiated parasites. To better define the role of IFN-gamma and IL-12p40 in sterile protection, we selected the C57BL/6 model. Wild-type and IL-12p40-/- mice were immunized with a single or multiple doses of P. berghei irradiated sporozoites. While the wild-type mice were able to rapidly produce IFN-gamma and mount a protective immune response after a single immunization with irradiated sporozoites, IL-12p40-/- mice were neither able to produce IFN-gamma nor were protected. However, both strains of mice were able to produce IFN-gamma and were protected after three doses of irradiated sporozoites. Protection was partially and largely mediated by CD4+ T cells and CD8+ T cells, respectively. Thus, IL-12p40 plays an important role in mediating early protection by irradiated sporozoite immunization but is dispensable for protective immunity induced by several immunizations with irradiated sporozoites. Moreover, treatment of hyperimmune IL-12p40-/- mice with rhIL-18 bp-Fc, an inhibitor of IL-18 activity, did not abrogate protection indicating that IL-18 is not required for the effector phase of the immune response; it remains possible, however, that IL-18 may compensate for the lack of IL-12p40 in the induction phase of the immune response.

Adjuvant activity of polymer microparticles and Montanide ISA 720 on immune responses to Plasmodium falciparum MSP2 long synthetic peptides in mice.

The purpose of this work was to test the immunogenicity in C57BL mice of two synthetic peptides derived from the constant region of 3D7 and FC27 Plasmodium falciparum MSP2 dimorphic proteins, either microencapsulated into poly-lactide-co-glycolide acid microparticles (PLGA MP) or delivered with the human compatible adjuvant Montanide ISA 720 for comparison. Potent and prolonged antibody responses were obtained for both peptides by using PLGA MP formulations after subcutaneous or intradermal injections. As compared to the subcutaneous route of immunization, the intradermal route induced greater immune responses. Montanide adjuvant was effective in eliciting antibodies against the 3D7 peptide but not against the FC27 peptide. Peptide-specific cytophilic antibodies (IgG2a) were detected after boosting with homologous peptide for all vaccine formulations. MP formulations elicited a lower IgE secretion as compared to that observed for both Montanide formulated vaccines. Our results demonstrate the ability of the polymer microparticles to overcome the lack of immunogenicity of FC27 MSP2 peptide in C57BL mice and their potential to induce desirable immune responses against malaria.

Cross-presentation of a human malaria CTL epitope is conformation dependent.

Little is known about the role of conformation on the antigen processing by antigen presenting cells. Using a well-defined antigen containing two disulfide bridges, the synthetic C-terminal fragment 282-383 derived from Plasmodium falciparum circumsporozoite protein (PfCS 282-383), we show that the reduced form is presented in vitro more efficiently than its oxidized counterpart, inducing stronger CTL recognition. In addition, only the reduced form can be presented by the TAP independent T2 cell line. Thus, the reduced form is processed by TAP dependent and independent pathways.

Natural antibody response to Plasmodium falciparum Exp-1, MSP-3 and GLURP long synthetic peptides and association with protection.

A longitudinal study was undertaken in Burkina Faso among 293 children aged 6 months to 9 years in order to determine the correlation between an antibody response to several individual malarial antigens and malarial infection. It was found that the presence of a positive antibody response at the beginning of the rainy season to three long synthetic peptides corresponding to Plasmodium falciparum Exp-1 101-162, MSP-3 154-249 and GLURP 801-920 but not to CSP 274-375 correlated with a statistically significant decrease in malarial infection during the ongoing transmission season. The simultaneous presence of an antibody response to more than one antigen is indicative of a lower frequency of malarial infection. This gives scientific credibility to the notion that a successful malaria vaccine should contain multiple antigens.

Effect on antibody and T-cell responses of mixing five GMP-produced DNA plasmids and administration with plasmid expressing GM-CSF.

One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.

Intranasal treatment with ovalbumin but not the major T cell epitope ovalbumin 323-339 generates interleukin-10 secreting T cells and results in the induction of allergen systemic tolerance.

BACKGROUND: Nasal administration of major peptide T cell epitopes gives contradictory data on the induction of peripheral tolerance. OBJECTIVE: To compare the prophylactic effect of intranasal treatment (INT) on the development of an allergic response, using either ovalbumin (OVA) or its major T cell epitope OVA 323-339 (OVAp). METHODS: BALB/c mice were treated intranasally with OVA or OVAp and subsequently immunized s.c. with OVA. Anti-OVA-specific antibody, T cell proliferation and cytokine responses were analysed. In an adoptive transfer model using OVAp specific TCR transgenic (Tg) T cells from D011.10 mice, in vivo tracking and characterization of transferred T cells in the cervical, inguinal and bronchial lymph nodes (BLN) and in the spleen were determined by FACS analysis. RESULTS: Prophylactic INT with OVA induced T cell tolerance towards subsequent OVA s.c. immunizations, inhibiting OVA specific T cell proliferation, IgE and IgG1 production, in contrast to INT with OVAp, which was unable to induce tolerance. In vivo analysis of transferred OVA-specific TCR Tg T cells showed that INT with OVA induced a preferential activation of T cells in BLN, as opposed to a broad, systemic activation with OVAp. In vivo, OVAp INT led to faster and more sustained cell division cycles than OVA INT. Ex vivo, tolerance to OVA was associated with the generation of IL-10 secreting CD4(+) T cells in BLN of OVA-treated mice only. CONCLUSION: INT with OVA but not with OVAp led to regional (as opposed to systemic) T cell activation and the induction of IL-10 secreting CD4(+) T cells in BLN, potentially critical steps in the induction of T cell-specific tolerance via the nasal route.

Humoral responses to defined malaria antigens in children living since birth under insecticide treated curtains in Burkina Faso.

Insecticide treated materials (ITM) are considered a useful malaria control measure for endemic countries, but whether they also delay the acquisition of immunity to malaria remains unclear. This study investigates plasma antibody levels in 160 children aged 3-6 years from five villages protected by insecticide treated curtains (ITC) over 6 years and in 184 children of the same age group from five villages in the same area never covered by ITC. The antigens to which antibodies were investigated were: the Plasmodium falciparum circumsporozoite protein (CSP) repetitive sequence (NANP)5; the C-terminal domain of the P. falciparum exported protein 1 (Cter-PfExp1); three fragments of the glutamate rich protein (GLURP), referred to as R0, R1 and R2; the merozoite surface protein 3 (MSP3). The level of antibodies was lower in children from the ITC area than in children from the non-ITC area for (NANP)5, R0, R2 and MSP3. Prevalence and intensity of P. falciparum infection were similar in the two groups of children. These findings suggest that reducing the level of malaria transmission over a long period may affect the level of antibodies in children to both sporozoite and blood stage malaria antigens.

Shift of fibril-forming ability of the designed alpha-helical coiled-coil peptides into the physiological pH region.

Recently, we designed a short alpha-helical fibril-forming peptide (alphaFFP) that can form alpha-helical nanofibrils at acid pH. The non-physiological conditions of the fibril formation hamper biomedical application of alphaFFP. It was hypothesized that electrostatic repulsion between glutamic acid residues present at positions (g) of the alphaFFP coiled-coil sequence prevent the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers axial growth of the fibril. To test this hypothesis, we synthesized alphaFFPs where all glutamic acid residues were substituted by glutamines or serines. The electron microscopy study confirmed that the modified alphaFFPs form nanofibrils in a wider range of pH (2.5-11). Circular dichroism spectroscopy, sedimentation, diffusion and differential scanning calorimetry showed that the fibrils are alpha-helical and have elongated and highly stable cooperative tertiary structures. This work leads to a better understanding of interactions that control the fibrillogenesis of the alphaFFPs and opens opportunities for their biomedical application.

In vivo kinetics of the immunoglobulin E response to allergen: bystander effect of coimmunization and relationship with anaphylaxis.

BACKGROUND: Murine models of hypersensitivity to allergens are useful tools for the evaluation of preclinical strategies to down-regulate the IgE response. OBJECTIVE: To monitor the long-term kinetics of T and B cell responses to allergen as a function of allergen dosage and to investigate the effect of parallel immunization with a second antigen; to correlate B cell response with anaphylaxis. METHODS: CBA/J mice were sensitized every other week by subcutaneous injections of phospholipase A2 (PLA2) and/or ovalbumin (OVA) adsorbed to alum. Specific antibody isotype responses, T cell proliferation, T cell cytokine production and anaphylaxis were assessed throughout the sensitization phase. RESULTS: Low-dose immunization with PLA2 (0.1 microg) favoured a long-term, specific T helper (Th)2 response with high IgE and IL-4 production in contrast to high-dose PLA2 (10 microg) immunization, which biased the immune response towards a Th1 response with high IgG2a and low IL-4 production. Parallel immunization with an unrelated antigen (ovalbumin) had a significant bystander effect on the immunization with PLA2, which was also dose-dependent. Finally, although anaphylaxis as measured by rectal temperature drop was allergen-specific, it could be induced in the high- and low-dose immunization groups, and was not solely dependent on IgE levels. CONCLUSION: Though low-dose allergen immunization appears to induce an efficient IgE response, the intensity and quality of this response may be modulated by bystander effects of parallel immunization and does not correlate strictly with anaphylaxis. This observation has relevance to the design of clinical immunotherapy protocols using murine model-based data.

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.

Selection of glutamate-rich protein long synthetic peptides for vaccine development: antigenicity and relationship with clinical protection and immunogenicity.

Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to each LSP was observed in all three areas of endemic infection. Levels of cytophilic immunoglobulin G (IgG) antibodies against both GLURP regions were significantly correlated with protection from clinical P. falciparum malaria. Protected children from the Ghana cohort possessed predominantly IgG1 antibodies against the nonrepeat epitope and IgG3 antibodies against the repeat epitope. T-cell proliferation responses, studied in the cohort from Senegal, revealed that T-helper-cell epitopes were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of different H-2 haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination against P. falciparum malaria.

A synthetic malaria vaccine elicits a potent CD8(+) and CD4(+) T lymphocyte immune response in humans. Implications for vaccination strategies.

We report the first synthetic peptide vaccine eliciting strong CD8(+) and CD4(+) T lymphocyte responses in humans. The vaccine, representing the C-terminal region of the circumsporozoite protein of Plasmodium falciparum (amino acids 282-383) was well tolerated and strong sporozoite-specific antibodies were elicited. In addition, robust lymphocyte proliferation responses were equally elicited with concomitant in vitro production of IFN-gamma, crucial in the elimination of the parasite. Most importantly, we also observed the development of CD8(+) T lymphocyte responses decisive in the immunity to malaria. The latter finding opens new, possibly safer, avenues for vaccination strategies when a CD8(+) T cell response is needed.

Long synthetic peptides encompassing the Plasmodium falciparum LSA3 are the target of human B and T cells and are potent inducers of B helper, T helper and cytolytic T cell responses in mice.

We synthesized 17 long synthetic peptides (LSP) spanning the whole 200-kDa Plasmodium falciparum liver stage antigen-3 (LSA3), an antigen that induces protection in chimpanzee, and analyzed their immunogenicity in BALB/c mice and their antigenicity in individuals living in a hyper-endemic malaria area. Our findings show that both specific antibodies and T cell proliferation against most LSA3-LSP develop in malaria-exposed adults. All individuals studied had detectable antibodies against a minimum of 6 and a maximum of 15 polypeptides. It is noteworthy that antibody prevalence and titers were as high against non-repeat as repeat regions. Although the extent of T cell reactivity was lower than that observed for B cells, most of the sequences contained at least one T helper epitope, indicating that the majority of LSA3-LSP contain both B and T cell epitopes within the same sequence. Injection of LSA3-LSP with SBSA2 adjuvant in mice, showed strong immunogenicity for most of them, eliciting both T cell responses and specific antibody production. While all the peptides were immunogenic for B cells, different patterns of T cell responses were induced. These peptides were thus classified in three sets according to the levels of the T cell proliferative and of the IFN-gamma-specific responses. Importantly, antibodies and T cells against some of the LSP were able to recognize LSA3 native protein on P. falciparum sporozoites. Additionally, some LSP (44-119, 1026-1095, 1601-1712) also contained epitopes recognized by H-2(d) class I-restricted T cells. These results led to the identification of numerous domains that are highly antigenic and immunogenic within the LSA3 protein, and underline the value of the LSP approach for vaccine development.

Induction of a cytotoxic T-cell response to HIV-1 proteins with short synthetic peptides and human compatible adjuvants.

The goal of this study was the induction of a strong CTL response against multiple CTL epitopes present in HIV proteins using short synthetic peptides. Four HLA-A2.1 restricted peptides (RT 476-484, p17 77-85, gp41 814-823, RT 956-964) that showed stable binding to the HLA-A2.1 molecule in an in vitro binding assay were able to elicit a strong specific immune response in HLA-A2.1 transgenic mice when injected with IFA or Montanide. The use of biodegradable microspheres (MS) as adjuvant was also successfully tested for all peptides. When the peptides were injected as a mixture the response was weaker as compared to individual injections of the peptides indicating the occurrence of immunodominance (ID). We are currently investigating whether ID can be overcome by a combined injection of peptide loaded MS with different release patterns. Taken together, it seems feasible to induce a specific CTL response in HLA-A2.1 transgenic mice against several HIV proteins using short synthetic peptides and human compatible adjuvants.

Api m 6: a new bee venom allergen.

BACKGROUND: Characterization of the primary structure of allergens is a prerequisite for the design of new diagnostic and therapeutic tools for allergic diseases. OBJECTIVE: The purpose of this study was the identification and characterization of a low-molecular-weight, IgE-binding, bee venom (BV) allergen. METHODS: BV proteins were separated by using size exclusion chromatography and HPLC. IgE antibody binding to purified proteins was analyzed by means of immunoblotting, and T-cell response was analyzed by means of proliferation assay. Amino acid sequence was determined with 2 approaches, namely Edman degradation and carboxy terminal analysis with mass spectrometry. RESULTS: Api m 6, which migrated as an 8-kd band in SDS-PAGE, was frequently (42%) recognized by IgE from BV-hypersensitive patients. In addition, PBMCs from BV-hypersensitive patients, as well as from a normal control subject, proliferated in response to this allergen. Api m 6 exists as 4 isoforms of 7190, 7400, 7598, and 7808 d, respectively. Amino acid sequences obtained from HPLC-purified preparations revealed that the isoforms were constituted of a common central core of 67 residues, only differing in the amino- and carboxy-terminal ends. Api m 6 showed no significant sequence homology with known proteins. CONCLUSIONS: We have identified and sequenced a new BV allergen that elicits a strong IgE and T-cell response in a large number of BV-hypersensitive patients. Api m 6 should be considered in the diagnostic and therapeutic approach of BV immunotherapy on the basis of peptides or recombinant proteins.

CD1d-restricted NK T cells are dispensable for specific antibody responses and protective immunity against liver stage malaria infection in mice.

Immunization with a single dose of irradiated sporozoites is sufficient to induce protection against malaria in wild-type mice. Although this protection is classically attributed to conventional CD4+ and CD8+ T cells, several recent reports have suggested an important role for CD1-restricted NK T cells in immunity to malaria. In this study, we directly compared the ability of C57BL/6 wild-type and CD1-deficient mice to mount a protective immune response against Plasmodium berghei sporozoites. Our data indicate that CD1-restricted NK T cells are not required for protection in this model system. Moreover, specific IgG antibody responses to the P. berghei circumsporozoite repeat sequence were also unaffected by CD1 deficiency. Collectively, our data demonstrate that CD1-restricted NK T cells are dispensable for protective immunity to liver stage P. berghei infection.