Björn Löwenadler

CTA1-M2e-DD: A novel mucosal adjuvant targeted influenza vaccine

At present few vaccine candidates exists against potentially pandemic influenza virus infections. We provide compelling evidence that a targeted fusion protein based on the CTA1-DD adjuvant and containing tandem repeats of the matrix protein 2 (M2e) ectodomain epitope, CTA1-3M2e-DD, confers strong protective immunity against a potentially lethal challenge infection with influenza virus in mice. The formulation was highly effective for mucosal immunizations and promoted high M2e-specific serum IgG and mucosal IgA antibody titers and an hitherto unknown anti-M2e CD4 T cell immunity. This novel CTA1-3M2e-DD fusion protein combines adjuvant and a conserved influenza A antigen in a promising candidate for a universal anti-influenza vaccine.

CTA1-DD-Immune Stimulating Complexes: a Novel, Rationally Designed Combined Mucosal Vaccine Adjuvant Effective with Nanogram Doses of Antigen

Mucosally active vaccine adjuvants that will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin and lipophilic immune stimulating complexes (ISCOMS) containing Quil A can both act as adjuvants for orally administered Ags, possibly by targeting different APCs. Recently, we have been successful in separating the adjuvant and toxic effects of cholera toxin by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell-targeting moiety, D, derived from Staphylococcus aureus protein A. Here we have extended this work by combining CTA1-DD with ISCOMS, which normally target dendritic cells and/or macrophages. ISCOMS containing a fusion protein comprising the OVA323–339 peptide epitope linked to CTA1-DD were highly immunogenic when given in nanogram doses by the s.c., oral, or nasal routes, inducing a wide range of T cell-dependent immune responses. In contrast, ISCOMS containing the enzymatically inactive CTA1-R7K-DD mutant protein were much less effective, indicating that at least part of the activity of the combined vector requires the ADP-ribosylating property of CTA1. No toxicity was observed by any route. To our knowledge, this is the first report on the successful combination of two mechanistically different principles of adjuvant action. We conclude that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.

T and B cell responses to chimeric proteins containing heterologous T helper epitopes inserted at different positions.

The ability of a T helper (Th) epitope to induce help for B cells recognizing different determinants within a multideterminant antigen was investigated. Chimeric fusion proteins, containing inserts of single or multiple copies of the Th epitope ovalbumin 323-339 (ova) at two different positions, were compared with respect to their ability to induce specific antibody production and ova-specific T cell activation. The antibody responses against B cell determinants at the amino and carboxy terminus, respectively was differently influenced by the molecular positioning of the inserted Th determinant. All ova-containing fusion proteins induced antibody production against the B cell determinant at the amino terminal end irrespective of the positioning of ova. In addition, multiple copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against the determinant at the carboxy terminus was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. Furthermore a fusion protein, containing four copies of ova effectively elicited T cell help for high levels of antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. Immunodominant T cell recognition of ova in all chimeric peptides, independently of its position, was demonstrated by lymph node cell (LNC) proliferation of primed BALB/c mice. The level of ova-specific T cell proliferation was similar, irrespective of which chimeric peptide that had been used for priming, and thus did not reveal any differences in T cell priming efficiencies related to the number of ova copies in the fusion proteins. However, when the peptides were presented to a ova-specific T cell line by A20 B lymphoma cells, a close correlation between IL-2 production by the clonal T cells and the number of ova epitopes in the chimeric peptides was observed. Thus, increased cytokine production by ova-specific T cells may be important for the increased level of in vivo antibody production observed in response to multiple copies of ova in the chimeric antigens.

Tandem repeats of T helper epitopes enhance immunogenicity of fusion proteins by promoting processing and presentation.

Empirical findings have shown that recombinant chimeric proteins may be made more immunogenic if T helper epitopes are incorporated as tandem repeats. In the present study we investigated the mechanisms responsible for the enhanced immunogenicity of fusion proteins composed of the heat-stable enterotoxin of enterotoxigenic E. coli (STa) linked to multiple copies of the ovalbumin323-339 T helper epitope (ova) and a connecting dimer of an Ig-binding region of Staphylococcus aureus protein A (ZZ), which were previously shown to stimulate strong anti-STa titres in mice. We used B cell and macrophage cell lines as APC and IL-2 production by ova-specific T cells as our read-out system. Fusion proteins containing four repeated T helper epitopes were found to be the most immunogenic and resulted in 50-fold higher IL-2 production than constructs with a single T helper epitope. Under limiting APC conditions the construct with four epitopes was the best inducer of IL-2, indicating that this construct was most effectively processed by the APC. Analysis of IL-2R alpha expression by flow cytometry confirmed that four copies gave the highest frequency of activated T cells in culture, indicating a direct correlation between ability to activate T cells and IL-2 production in culture. Also in vivo, the fusion protein with four epitopes exhibited the strongest T cell priming effect. Moreover, both in vitro and in vivo, the ZZ construct was found to serve as an efficient means for targeting of the fusion proteins to B cells, thereby allowing access to the Ig receptor uptake pathway for Ag. The present study provides direct evidence that fusion proteins can be constructed to optimize processing in the individual APC and enhance activation of clonal T cells.

Fusion Proteins with Heterologous T Helper Epitopes. Recombinant E. coli Heat-Stable Enterotoxin Proteins

Fusion proteins containing specific B cell and T cell epitopes were used to examine how the intramolecular arrangement of T and B cell epitopes within a chimeric protein influences antigen-specific B cell antibody responses as well as specific T cell activation. Chimeric proteins, containing single or multiple copies of the Th epitope ovalbumin 323-339 (ova) linked at different positions to STa, the heat-stable enterotoxin of E. coli, were compared with respect to their ability to induce STa-specific antibody production and to induce ova-specific T cell activation. Chimeric proteins induced ova-dependent antibody production against STa at the amino terminal end, irrespective of the positioning of ova. Multiple tandem copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against a second B cell epitope at the carboxy terminus of the fusion proteins was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. A fusion protein, containing four copies of ova effectively elicited T cell help for antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. T cell recognition of ova in all chimeric peptides, independently of its position, following the same pattern of genetic restriction (i.e. immunodominant in H-2d and nonimmunogenic in H-2k) as in the native ovalbumin molecule.(ABSTRACT TRUNCATED AT 250 WORDS)