Mario Roggero

The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host

Conformational changes influence functional properties of circumsporozoite protein expressed on the surface of Plasmodium sporozoites.

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‐γ, 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.

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freunds adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection withLeishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), aLeishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser92 and Phe93, in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.

Mapping and comparison of the B-cell epitopes recognized on the Plasmodium vivax circumsporozoite protein by immune Colombians and immunized Aotus monkeys

Plasma samples of individuals from two malaria-endemic villages on the Colombian Pacific coast and synthetic peptides representing different fragments of the central and flanking regions of the Plasmodium vivax circumsporozoite protein (CSP) were used to perform a fine mapping of the B-cell epitopes on the whole CSP. In addition, the immunogenicity of long polypeptides corresponding to the amino (N) and carboxyl (C) regions was evaluated in Aotus monkeys. The epitopes recognized after natural infection of humans and after immunization of Aotus with these synthetic peptides were compared. Human samples more frequently contained specific antibodies to the central region. The type-I repeat region of the CSP was predominantly recognized by the human sera (by 68% of those from the village of Zacarías and 75% of those from Bajo Calima), a significantly smaller population reacting with the type-II repeat (20% and 11%, respectively). Most of the sera reacting with the type-I repeat recognized the minimal epitope AGDR. Although the N- and C-terminal polypeptides were both highly immunogenic in Aotus and induced long-lasting antibodies, titres of antibodies to the C-terminal polypeptide were higher than those of antibodies to the N-terminal. Competitive inhibition assays performed using human and monkey plasma allowed the identification of dominant B-cell epitopes on sequence 71-90 (p8) from the amino region and sequence 332-361 (p24/p25) from the carboxyl region. The high prevalence of naturally induced antibodies to the three epitopes, the possible functional role of the corresponding sequences, and the high immunogenicity of these epitopes in Aotus could be of great importance in the development of a malaria vaccine based on P. vivax CSP.

Extracellular processing and presentation of a 69-mer synthetic polypeptide to MHC class I-restricted T cells

The classical pathway for MHC class-I-restricted Ag presentation processes cytosolic Ag synthesized in or delivered into the cytosol for binding to MHC class I molecules in the ER. Alternatively, Ag may be processed and bind class I molecules in endocytic compartments or at the cell surface after regurgitation of processed peptides. We show that a 69-mer synthetic polypeptide that carries the optimal 9-mer Kd-restricted epitope from the Plasmodium berghei circumsporozoite protein, PbCS 245-253, is presented to CD8+ T cells after a short incubation (1-2 h) with target cells. The presentation kinetics correlate with the length of the peptides when shorter peptide analogues are used. This presentation is independent of the transporters associated with antigen processing and presentation (TAP), does not require newly synthesized proteins and does not proceed via regurgitation of intracellularly processed peptides. In contrast, it is substantially decreased in the absence of beta2 microglobulin or serum. Taken together, these data suggest that serum components, such as proteases and beta2 microglobulin, allow the processing and loading of exogenous polypeptides onto empty cell surface class I molecules for presentation to CTL.

The synthetic, oxidized C-terminal fragment of the Plasmodium berghei circumsporozoite protein elicits a high protective response

A polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid-phase peptide synthesis. The immunological and protective properties of peptide PbCS 242-310 were studied in BALB/c mice (H-2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti-peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd-restricted peptide PbCS 245-253 and (iii) partial CD8+-dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific CD8+ T cells were found by IFN-gamma ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN-gamma producing CD8 T cells and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection.

Synthetic polypeptides corresponding to the non-repeat regions from the circumsporozoite protein of Plasmodium falciparum: recognition by human T-cells and immunogenicity in owl monkeys

Synthetic polypeptides encompassing the non-repeated regions of the circumsporozoite protein (CSP) of Plasmodium falciparum are very immunogenic in mice and are recognized by sera from donors living in regions where malaria is endemic, both in Africa and South America. Long polypeptides, encompassing the N- or C-terminal regions, have now been used to demonstrate peptide-specific T cells in donors living in an endemic area of Colombia. Although the N-terminal peptide (22-125) was recognized almost exclusively by donors from the endemic area, the patterns of recognition of the C-terminal peptide (289-390) in donors from endemic and non-endemic areas were similar and like the pattern with smaller peptides. The availability of the long polypeptides made it possible to compare T-cell responses to the non-repeated regions of the CSP with the presence of peptide-specific antibodies. No correlation was found and no antibodies were detected in donors from non-endemic regions. The long polypeptides also elicited strong antibody and T-cell responses in owl monkeys (Aotus lemurinus). The antibodies generated against the synthetic peptides in such monkeys also recognized sporozoites, the natural infective form of the parasite. The results emphasise the potential of the peptides tested as malaria-vaccine candidates. Not only are they recognized by humans at both the B- and T-cell level but they also elicit strong responses in monkeys and encompass several distinct T-cell epitopes, thus overcoming the limitations of specific, major-histocompatibility-complex restriction.

Purification of recombinant proteins by chemical removal of the affinity tag.

The efficient removal of a N-or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and aPlasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag inEscherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.

Native-like, long synthetic peptides as components of sub-unit vaccines: practical and theoretical considerations for their use in humans.

Vaccines have been used as a successful tool in medicine by way of controlling many major diseases. In spite of this, vaccines today represent only a handful of all infectious diseases. Therefore, there is a pressing demand for improvements of existing vaccines with particular reference to higher efficacy and undisputed safety profiles. To this effect, as an alternative to available vaccine technologies, there has been a drive to develop vaccine candidate polypeptides by chemical synthesis. In our laboratory, we have recently developed a technology to manufacture long synthetic peptides of up to 130 residues, which are correctly folded and biologically active. This paper discusses the advantages of the molecularly defined, long synthetic peptide approach in the context of vaccine design, development and use in human vaccination.