Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS(280-288) epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS(280-288) peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations.
Type 1 herpes virus (HSV-1) glycoprotein D (gD) enhances antigen-specific immune responses, particularly CD8(+) T cell responses, in mice immunized with DNA vaccines encoding hybrid proteins genetically fused with the target antigen at a site near the C-terminal end. These effects are attributed to the interaction of gD with the herpes virus entry mediator (HVEM) and the concomitant blockade of a coinhibitory mechanism mediated by the B- and T-lymphocyte attenuator (BTLA). However, questions concerning the requirement for endogenous synthesis of the antigen or the adjuvant/antigen fusion itself have not been addressed so far. In the present study, we investigated these points using purified recombinant gDs, genetically fused or not with type 16 papilloma virus (HPV-16) E7 oncoprotein. Soluble recombinant gDs, but not denatured forms, retained the ability to bind surface-exposed cellular receptors of HVEM-expressing U937 cells. In addition, in vivo administration of the recombinant proteins, particularly gD genetically fused with E7 (gDE7), promoted the activation of dendritic cells (DC) and antigen-specific cytotoxic CD8(+) T cells. More relevantly, mice immunized with the gDE7 protein developed complete preventive and partial therapeutic antitumor protection, as measured in mice following the implantation of TC-1 cells expressing HPV-16 oncoproteins. Collectively, these results demonstrate that the T cell adjuvant effects of the HSV-1 gD protein did not require endogenous synthesis and could be demonstrated in mice immunized with purified recombinant proteins.
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungusParacoccidioidomycosis (PCM) is a granulomatous disease caused by the thermodimorphic fungus Paracoccidioides brasiliensis, which is prevalent in Brazil and other Latin American countries (5, 6, 33). Some individuals develop one of the two main clinical forms of PCM. The acute form is characterized by impaired cellular immunity, negative delayed-type hypersensitivity reactions, increased systemic proliferation of the fungus, and high mortality rate. The chronic form, ranging from mild to severe chronic disease, shows exacerbated host cellular immune responses and formation of granulomas containing fungal cells and may evolve to develop extensive sequelae, including fibrotic lesions and impairment of lung function (33, 39).Vaccines against PCM are still not available for human use, but promising formulations have been experimentally tested during the last few years. Irradiated P. brasiliensis or cellular antigens fractionated by anion-exchange chromatography conferred partial protection against fungal proliferation in the murine model (11). The extracellular gp43 glycoprotein, the major diagnostic antigen of P. brasiliensis, is the most intensively studied component aimed at a vaccine for PCM control. Previous reports have shown that mice immunized with the purified protein, DNA, or anti-idiotypic monoclonal antibody were partially protected against challenges by P. brasiliensis (28,36,37,40). A 15-amino-acid peptide (QTLIAIHTLAIRY AN), designated P10, contains the gp43 immunodominant CD4 ϩ T-cell-specific epitope presented by major histocompatibility complex class II molecules from three different mouse haplotypes (37) and most human HLA-DR alleles (17, 18). Indeed, parenteral immunization with P10 in complete Freund adjuvant (CFA), or in the form of a truncated multiple-antigen peptide (MAP) complex, induced protective Th1 cellular immune responses in mice against intratracheal (i.t.) challenge with a virulent P. brasiliensis isolate (37,38,41).The rational use of vaccines has been significantly improved after elucidation of innate immune mechanisms in mammalian cells. The recognition of distinct pathogen-associated molecular patterns by members of the Toll-like receptor (TLR) family initiates a signaling cascade mediated by adaptor proteins, including MyD88 and interleukin-1 (IL-1) receptor-associated kinase, that culminates in the production of proinflammatory cytokines, such as tumor necrosis factor alpha and IL-12, and increased expression of cell surface molecules involved in epitope presentation by antigen-presenting cells (APC) (1,19). Proper APC activation by TLR agonists represents a key step for an effective adaptive immune response induced by pathogens or vaccines and explains, at least in part, the marked adjuvant effects of several bacterial molecules, including lipopolysaccharides, lipoproteins, peptidoglycan fragments, and flagellins (2).Flagellin, the structural subunit of bacterial flagellum, is a
In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His(6)FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund's adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP1(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity.
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