Giampietro Corradin

Malarial hemozoin is a Nalp3 inflammasome activating danger signal.

Background Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents. Methodology/Principal Findings We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1β. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K+ efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged. Significance/Conclusions The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria.

A single administration of tetanus toxoid in biodegradable microspheres elicits T cell and antibody responses similar or superior to those obtained with aluminum hydroxide

The use of biodegradable polymer microspheres as a single dose vaccine delivery system was investigated by using tetanus toxoid (TT). In order to compare the immunogenicity of TT-microspheres (TT-MS) with aluminum hydroxide (alum)-based TT, BALB/c mice were immunized with TT in different formulations including individual or mixtures of MS and TT-alum. All TT-MS formulations elicited high proliferative and antibody responses comparable to those obtained with TT-alum formulation. Antibody levels remained elevated over a long period of time. Certain individual MS preparations elicited lower antibody titers than the MS mixtures. More importantly, the proliferative and antibody responses induced by a single injection of three TT-MS mixtures with different particle sizes and degradation rates were similar to those obtained with three injections of TT-alum. In addition, TT-MS induce similar isotypesubclass antibodies to those TT-alum induced. These results raise the possibility to obtain optimal and long-lasting immune responses by single administration of the three TT-MS mixture formulations alone.

Tetanus toxoid and synthetic malaria antigen containing poly(lactide)/poly(lactide-co-glycolide) microspheres: importance of polymer degradation and antigen release for immune response

Abstract The importance of in vitro degradation of poly(lactide)/poly(lactide-co-glycolide) (PLA/PLGA) microspheres and of the concomitant in vitro release of a natural and a synthetic antigen for eliciting immune response was studied in mice. A variety of PLAs and PLGAs differing in molecular weight ( M w of 14–130 kDa) and in polymer composition (lactic/glycolic acid ratio of 100:00, 75:25, and 50:50) were examined for their in vitro degradation, which ranged from approximately 4 to 20 weeks. Three specific polymers were then selected for microencapsulation of the two antigens tetanus toxoid (TT) and a weakly immunogenic synthetic branched malaria peptide (P30B2). The in vitro release data showed that antigen delivery correlates fairly well with polymer degradation giving rise to a distinct burst release during the first 24 h and an additional release pulse towards the end of polymer degradation. After single subcutaneous administration in mice, long lasting high antibody titers were obtained with the antigen containing microspheres, as compared to TT adsorbed on alum or to P30B2 in Incomplete Freunds Adjuvant. Moreover, the immune responses induced by microspheres were clearly influenced by the antigen release kinetics, the polymer type and the size of the microspheres. The results demonstrate the immunopotentiating properties of the biodegradable microspheres and their potential to elicit long-lasting immune responses after single administration when tailoring in vitro release characteristics and particle size.

Improving Stability and Release Kinetics of Microencapsulated Tetanus Toxoid by Co-Encapsulation of Additives

AbstractPurpose. Tetanus toxoid (Ttxd) encapsulated in polyester microspheres (MS) for single injection immunization have so far given pulsatile in vitro release and strong immune response in animals, but no boosting effect. This has been ascribed to insufficient toxoid stability within the MS exposed to in vivo conditions over a prolonged time period. This study examined the effect of co-encapsulated putative stabilizing additives. Methods. Two different Ttxd were encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA 50:50) and poly(D,L-lactic acid) (PLA) MS by spray-drying. The influence of co-encapsulated additives on toxoid stability, loading in and release from the MS, was studied by fluorimetry and ELISA. Results. Co-encapsulated albumin, trehalose and γ-hydroxypropyl cyclodextrin all improved the toxoid encapsulation efficiency in PLGA 50:50 MS. Albumin increased the encapsulation efficiency of antigenic Ttxd by one to two orders of magnitude. Further, with albumin or a mixture of albumin and trehalose ELISA responsive Ttxd was released over 1−2 months following a pulsatile pattern. Conclusions. Optimized Ttxd containing MS may be valuable for a single-dose vaccine delivery system.

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.

Phase I malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein 3 antigen.

ABSTRACT The C-terminal conserved region of Plasmodium falciparum merozoite surface protein 3 (MSP3) is the trigger antigen of a protective immune response mediated by cytophilic antibodies. In an open, randomized, two-adjuvant (Montanide ISA 720, aluminum hydroxide) phase I clinical trial we evaluated the safety and immunogenicity of increasing doses of a long synthetic peptide construct spanning the conserved region of MSP3 targeted by biologically active antibodies (MSP3-LSP). Thirty-five healthy volunteers were randomized to receive three subcutaneous injections on days 0, 30, and 120. Of the 100 injections given, 10 caused severe local reactions, 62 caused transient mild to moderate local reactions, and 28 caused no reaction. On the basis of preestablished exclusion criteria, use of the Montanide formulation led to withdrawal of five volunteers after the second injection. This led to a reduction in the subsequent vaccine doses in four of the groups. No vaccine-related serious adverse events occurred throughout the trial. After the third injection, volunteers displayed a marked specific anti-MSP3-LSP antibody response (23/30 individuals, compared with 29/34 individuals for plasma from an area where malaria is endemic), an anti-native MSP3 antibody response (19/30 individuals), a T-cell-antigen-specific proliferative response (26/30 individuals), and gamma interferon production (25/30 individuals). In conclusion, the MSP3-LSP vaccine was immunogenic with both adjuvants, although it was unacceptably reactogenic when it was combined with Montanide. The potential usefulness of the candidate vaccine is supported by the induction of a strong cytophilic response (i.e., the type of anti-MSP3 antibodies involved in antibody-dependent, monocyte-mediated protective mechanisms in areas where malaria is endemic).

MHC class I- and class II-restricted processing and presentation of microencapsulated antigens

Macrophages were found of having a strong capacity of phagocytosing small size microcapsules (MS) and presenting microencapsulated antigens to either CD4+ and CD8- T cells. The class I-restricted presentation of microencapsulated tetanus toxoid by macrophages requires an intracellular processing which might follow the phagosome-to-cytosol route to enter the classical MHC class I presentation pathway. In contrast, presentation of microencapsulated cytotoxic peptide PbCS252-260 to specific CD8+ T cells has been observed with different APC and is not blocked by cytochalasin D, suggesting that peptide released from MS may directly bind to MHC class I molecules on the cell surface. In the case of MHC class II-restricted T cells, prefixation or treatment of macrophages with chloroquine, brefeldin A and cycloheximide inhibits the presentation of microencapsulated and soluble tetanus toxoid. These findings illustrate the capacity of microencapsulated antigens to enter different presentation pathways and should facilitate the development of subunit vaccines.

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

Abstract Background: The α-helical coiled coil structures formed by 25–50 residues long peptides are recognized as one of Natures 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 α-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 α-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 α-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.

T helper epitopes enhance the cytotoxic response of mice immunized with MHC class I-restricted malaria peptides

We have previously derived MHC class I (H-2Kd) restricted cytotoxic T lymphocytes (CTL) from BALB/c mice immunized with irradiated sporozoites from Plasmodium (P.) berghei and P. yoelii. The CTL recognize synthetic peptides corresponding to a region of the circumsporozoite (CS) protein that is homologous in the two species. In the present study, we have attempted to induce CS-specific CTL by immunization with those peptides in incomplete Freunds adjuvant. Only a low level CTL response was detected in BALB/c mice immunized with synthetic peptides corresponding to the Pb or Py CTL epitopes. In contrast, CS-specific CTL responses could be readily detected in mice injected with mixtures of peptides that combined the P. berghei or P. yoelii CTL epitopes with previously defined T helper epitopes. Several different T helper epitopes were shown to enhance the response when injected as separate peptides in a mixture, or when covalently linked to a CTL epitope. These results may have general implications for the elicitation of CTL responses to defined CTL epitopes and for the design of peptide-based synthetic vaccines.

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.