François Spertini

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).

Targeting of Secretory IgA to Peyer’s Patch Dendritic and T Cells after Transport by Intestinal M Cells

In addition to being instrumental to the protection of mucosal epithelia, secretory IgA (SIgA) adheres to and is transported by intestinal Peyer’s patch (PP) M cells. The possible functional reason for this transport is unknown. We have thus examined in mice the outcome of SIgA delivered from the intestinal lumen to the cells present in the underlying organized mucosa-associated lymphoreticular tissue. We show selective association of SIgA with dendritic cells and CD4+ T and B lymphocytes recovered from PP in vitro. In vivo, exogenously delivered SIgA is able to enter into multiple PP lining the intestine. In PP, SIgA associates with and is internalized by dendritic cells in the subepithelial dome region, whereas the interaction with CD4+ T cells is limited to surface binding. Interaction between cells and SIgA is mediated by the IgA moiety and occurs for polymeric and monomeric molecular forms. Thus, although immune exclusion represents the main function of SIgA, transport of the Ab by M cells might promote Ag sampling under neutralizing conditions essential to the homeostasis of mucosal surfaces.

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.

Secretory IgA Possesses Intrinsic Modulatory Properties Stimulating Mucosal and Systemic Immune Responses

Secretory IgA (SIgA) is essential in protecting mucosal surfaces by ensuring immune exclusion. In addition, SIgA binds selectively to M cells in Peyer’s patches (PP), resulting in transport across the epithelium and targeting of dendritic cells (DC) in the dome region. The immunological consequences of such an interaction are unknown. In this study, we find that oral delivery of SIgA comprising human secretory component and mouse IgA induces human secretory component-specific Ab and cellular responses in mucosal and peripheral tissues in mice. This takes place in the absence of coaddition of cholera toxin, identifying so far unraveled properties in SIgA. Specific immune responses are accompanied by sustained IL-10 and TGF-β expression in draining mesenteric lymph nodes and spleen. SIgA also triggers migration of DC to the T cell-rich regions of PP, and regulates expression of CD80 and CD86 on DC in PP, mesenteric lymph nodes, and spleen. These results provide evidence that mucosal SIgA re-entering the body exerts a function of Ag delivery that contributes to effector and/or regulatory pathways characteristic of the intestinal mucosal compartment.

Allergen‐derived long peptide immunotherapy down‐regulates specific IgE response and protects from anaphylaxis

To evaluate a long peptide‐based allergy vaccine in a murine model, CBA/J mice were sensitized with low dose alum‐adsorbed phospholipase A2 (PLA2), a major bee venom allergen. Presensitized mice were treated by daily i.p. injections of a mixture of three long overlapping peptides (44‐ to 60‐mer) spanning the entire PLA2 molecule (100 μ g/peptide) for 6 consecutive days. This therapeutic approach induced a sharp drop in PLA2‐specific IgE, an increase in specific IgG2a, and a marked T cell hyporesponsiveness. T cell cytokine secretion was characterized by a shift from a Th2 to a Th1 profile. Prophylactic treatment of naive mice with long peptides prior to sensitization with PLA2 induced a comparable modulation of B and T cell responses. Upon i.p. challenge with native PLA2, presensitized mice treated with the long peptide mixture were fully protected from anaphylaxis. This indicated that allergen‐derived long overlapping peptides were safe and able to modulate an established Th2 response or to prevent its development. Furthermore, long peptide‐based immunotherapy provided clinical protection against anaphylaxis, thus appearing as a promising approach of the therapy of allergic diseases.

Antigen-Independent Suppression of the Allergic Immune Response to Bee Venom Phospholipase A2 by DNA Vaccination in CBA/J Mice

Phospholipase A2 (PLA2) is one of the major honey bee venom allergens for humans. To assess the long-term prevention of allergic reactions by DNA vaccination, a PLA2-CBA/J mouse model was employed using empty or PLA2 sequence-carrying DNA plasmids. Early skin application of either DNA construct before (prophylactic approach) or after (therapeutic approach) sensitization with PLA2/alum led to reduced PLA2-specific IgE and IgG1 titers at 7 mo, with concomitant rise in IgG2a and IgG3. Splenocytes recovered at 5–6 mo after the last DNA administration exhibited a sustained IFN-γ and IL-10 secretion and reduced IL-4 production. Recall challenge with PLA2 boosted IFN-γ and IL-10 secretion, suggesting the reactivation of quiescent memory Th1 lymphocytes. Mice from the prophylactic groups were fully protected against anaphylaxis, whereas 65% of the animals recovered in the therapeutic groups. Th1-polarized immune responses were also active in mice vaccinated with an empty plasmid 32 wk before sensitization with another Ag (OVA). This is the first demonstration that the Ag-coding sequence in DNA vaccine is not necessary to promote immune modulation in naive and sensitized animals for a prolonged period, and has relevance for the understanding of the innate and induced mechanisms underlying gene immunotherapy in long-term treatment of allergy.

Inducing Tolerance by Intranasal Administration of Long Peptides in Naive and Primed CBA/J Mice

To assess the capacity of a peptide-based immunotherapy to induce systemic tolerance via the nasal route, we designed three long overlapping peptides of 44–60 aa covering the entire sequence of phospholipase A2 (PLA2), a major bee venom allergen. Both prophylactic and therapeutic intranasal administrations of long peptides to PLA2-hypersensitive CBA/J mice induced specific T cell tolerance to the native allergen. In prophylactic conditions, this tolerance was marked by a suppression of subsequent specific IgE response, whereas the therapeutic approach in presensitized mice induced a more than 60% decrease in PLA2-specific IgE. This decline was associated with a shift in the cytokine response toward a Th1 profile, as demonstrated by decreased PLA2-specific IgG1 and enhanced IgG2a levels, and by a decline in the specific IL-4/IFN-γ ratios. T cell transfer from long peptide-tolerized mice to naive animals abrogated the expected anti-PLA2 IgE and IgG1 Ab response, as well as specific T cell proliferation, but enhanced specific IgG2a response upon sensitization with PLA2. These events were strongly suggestive of a clonal anergy affecting more profoundly Th2 than the Th1 subsets. In conclusion, these results demonstrate that allergen-derived long peptides delivered via the nasal mucosa may offer an alternative to immunotherapy with native allergens without the inherent risk of systemic anaphylactic reactions. Moreover, long peptides, in contrast to immunotherapy strategies based on short peptides, have the advantage of covering all potential T cell epitopes, and may represent novel and safe tools for the therapy of allergic diseases.

Individualising HIV treatment: pharmacogenetics and immunogenetics

In this issue of The Lancet Simon Mallal and colleagues report that the genetic susceptibility to abacavir hypersensitivity is carried on the major histocompatibility complex (MHC) 57.1 ancestral haplotype. Immunogenetics was used to investigate early and sometimes deadly hypersensitivity to the reverse transcriptase inhibitor abacavir. The findings also promise to clarify the basic mechanisms of hypersensitivity reactions. However the genetic approach to drug hypersensitivity is not new and it has so far not led to a striking breakthrough in the prevention of immune-mediated drug reactions especially because MHC associations are difficult to reproduce. Thus further discussion indicates that MHC typing could with analysis of other markers of progression of HIV disease improve decisions about the need and optimal timing for starting antiretroviral therapy. Moreover the decreased risk of abacavir hypersensitivity associated with Black race raises the issue of ethnicity and therapeutics.

The candidate tuberculosis vaccine Mtb72F/AS02 in PPD positive adults: A randomized controlled phase I/II study

UNLABELLED Prevention of tuberculosis (TB) through vaccination would substantially reduce the global TB burden. Mtb72F/AS02 is a candidate TB vaccine shown to be immunogenic and well tolerated in PPD-negative adults. We evaluated the safety and immunogenicity of Mtb72F/AS02 in Mycobacterium-primed adults (BCG-vaccinated, or infected adults who had received post-exposure chemoprophylaxis or treatment for pulmonary TB disease). In this observer-blind controlled trial, 20 BCG-vaccinated adults and 18 adults previously infected with Mycobacterium tuberculosis (Mtb), were randomized 3:1 to receive three doses of Mtb72F/AS02 or AS02 at one-month intervals, and followed for 6 months post third vaccination. Mtb72F/AS02 was well tolerated in BCG-vaccinated adults, and tended to be more reactogenic in Mtb-infected adults. Adverse events were mainly self-limiting, resolving without sequelae. No serious adverse events were reported. The adverse events in Mtb72F/AS02 vaccinees were not clearly associated with vaccine-induced responses (as assessed by proinflammatory cytokines, total IgE and C-reactive protein levels). No Th2 T-cell responses, or vaccine-induced T-cell responses to Mtb antigens (CFP-10/PPD/ESAT-6) were detected by ICS. In both cohorts, Mtb72F/AS02 induced persistent polyfunctional Mtb72F-specific CD4(+) T-cell responses and anti-Mtb72F humoral responses. IFN-γ was detectable in serum one day post each vaccination. Further evaluation of the candidate vaccine, Mtb72F/AS02, is warranted. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT00146744.

Two‐dimensional electrophoretic analysis of cryoproteins: A report of 335 samples

Cryoproteins are defined as proteins precipitating at low temperature. Most frequently, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Three types of cryoglobulins have been described: type I contains a single monoclonal Ig, whereas type II is a mixture of a monoclonal Ig with polyclonal Igs, and type III is a mixture of polyclonal Igs of different isotypes, most frequently IgG and IgM. Type II and type III are also called mixed cryoglobulins. A new type of cryoglobulins, containing polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM has recently been described after two‐dimensional polyacrylamide gel electrophoresis (2‐DE). This type of cryoglobulin has been called type II–III cryoglobulin. In this study, we report on 2‐DE analysis of 335 cryoproteins from patients with heterogeneous clinical conditions. In 69 out of 335 samples (20.7%), 2‐DE revealed patterns that were inadequate to characterize the cryoproteins. Out of 335 (79.3%) cryoproteins, 266 were identified according to their two‐dimensional patterns: 265 samples contained Igs and were diagnosed as cryoglobulins, and one sample consisted of fibrinogen, and was identified as cryofibrinogen. Among the 265 cryoglobulins, types I, II, and III were observed in 9 (3.4%), 69 (26%), and 116 (43.8%) cases, respectively, whereas type II–III was detected in 71 (26.8%) cases. Eleven of the latter consisted of oligoclonal Igs (IgM in 10 cases, IgA in 1 case) mixed with traces of polyclonal IgG. These cryoproteins were tentatively named type II–IIIvariant cryoglobulins. Taken together, our result clearly show that 2‐DE is a suitable technique to analyze cryoproteins.