Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen.

Cross-linking of the B-cell receptors of an antigen-specific B-cell is the initial signal for B-cell activation, proliferation, and differentiation into antibody secreting plasma cells. Since multivalent particulate structures are efficient activators of antigen-specific B-cells, we developed biodegradable calcium phosphate nanoparticles displaying protein antigens on their surface and explored the efficacy of the B-cell activation after exposure to these nanoparticles. The calcium phosphate nanoparticles were functionalized with the model antigen Hen Egg Lysozyme (HEL) to take advantage of a HEL-specific B-cell receptor transgenic mouse model. The nanoparticles were characterized by scanning electron microscopy and dynamic light scattering. The functionalized calcium phosphate nanoparticles were preferentially bound and internalized by HEL-specific B-cells. Co-cultivation of HEL-specific B-cells with the functionalized nanoparticles also increased surface expression of B-cell activation markers. Functionalized nanoparticles were able to effectively cross-link B-cell receptors at the surface of antigen-matched B-cells and were 100-fold more efficient in the activation of B-cells than soluble HEL. Thus, calcium phosphate nanoparticles coated with protein antigens are promising vaccine candidates for induction humoral immunity.

HIV-derived lentiviral particles promote T-cell independent activation and differentiation of naïve cognate conventional B2-cells in vitro.

In animal models, lentiviral particles (LP) were shown to be promising HIV vaccine candidates. Since little is known about the direct impact of LP on antigen-specific B cells, we incorporated Hen Egg Lysozyme (HEL) into LP (HEL-LP) derived from HIV to study their effect on HEL-specific, B cell receptor-transgenic B-cells (HEL(+)B-cells) in vitro. We observed preferential binding of HEL-LP to HEL(+)B-cells and their efficient internalization. HEL-LP were able to effectively cross-link B-cell receptors as indicated by the loss of surface CD62L. In the absence of CD4(+) T-cells, other activation events induced by LP in cognate naïve B-cells included increased expression of activation and co-stimulatory molecules as well as an enhanced proliferative response. Additionally, the B-cell phenotype shifted toward a germinal center pattern with further differentiation into memory and IgG3- and IgA-producing cells. The observed CD4(+) T-cell independent activation and differentiation may be due to LP-induced expression of CD40L by a subset of cognate B-cells. Thus, even in the absence of CD4(+) T-cells LP provide strong direct activation signals to cognate naïve B-cells, which may contribute to the strong humoral immune responses observed after LP immunization.

Protective efficacy and immunogenicity of an adenoviral vector vaccine encoding the codon-optimized F protein of respiratory syncytial virus.

Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans.

Enhancement of immunostimulatory properties of exosomal vaccines by incorporation of fusion-competent G protein of vesicular stomatitis virus.

Exosomes have been proposed as candidates for therapeutic immunization. The present study demonstrates that incorporation of the G protein of vesicular stomatitis virus (VSV-G) into exosome-like vesicles (ELVs) enhances their uptake and induces the maturation of dendritic cells. Targeting of VSV-G and ovalbumin as a model antigen to the same ELVs increased the cross-presentation of ovalbumin via an endosomal acidification mechanism. Immunization of mice with VSV-G and ovalbumin containing ELVs led to an increased IgG2a antibody response, expansion of antigen-specific CD8 T cells, strong in vivo CTL responses, and protection from challenge with ovalbumin expressing tumor cells. Thus, incorporation of VSV-G and targeting of antigens to ELVs are attractive strategies to improve exosomal vaccines.

Transcriptional signatures of immune cells in aryl hydrocarbon receptor (AHR)-proficient and AHR-deficient mice.

The ligand-activated aryl hydrocarbon receptor (AHR) is known to modulate many genes in a highly cell-specific manner, either directly or indirectly via secondary effects. In contrast, little is known about the effects of AHR deficiency on gene expression balance. We compared the transcriptome of CD4 T cells from AHR-/- mice and wild-type mice; 390 genes, many of them immunotypic, were deregulated in AHR-deficient CD4 cells. TCDD-induced transcriptome changes correlated with the AHR expression level in immune cells. However, there was little overlap in AHR-dependent transcripts found in T lineage cells or dendritic cells. Our results demonstrate flexible gene accessibility for the AHR in immune cells. The idea of a universal battery of AHR-responsive genes is not tenable.

Role of the aryl hydrocarbon receptor in thymocyte emigration in vivo.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent member of the PAS-bHLH-family of nuclear receptors. Anthropogenic ligands include environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Over-activation of the AHR causes thymus atrophy and immunosuppression. Signaling via the AHR changes the thymocyte differentiation program at several checkpoints, in particular within the CD4-CD8- double-negative (DN) thymocyte subset. Here, we show that AHR over-activation led to the preferential emigration of DN thymocytes to the periphery and accumulation in the spleen. Some of these recent thymic emigrants (RTE) had a novel "activated immature" phenotype (CD3-TCRbeta-CD25+/intCD44-CD45RB+/intCD62L+CD69- cells). Gene expression profiling of DN RTE revealed 15 genes that were up-regulated more than threefold by TCDD, including the S100A9 gene. Exposure of S100A9 null mice to TCDD showed a role for this protein in AHR-mediated thymic egress.

The influence of conditioned medium from mouse intestinal epithelial cells on the proliferative activity of crypt cells: role of granulocyte-macrophage colony-stimulating factor.

BACKGROUND: The aim of this work was to study the influence of soluble factors produced by native mouse intestinal epithelial cells (IECs) on the proliferative activity of freshly isolated intestinal crypt cells. METHODS: The crypt cells were cultured with either conditioned medium and its ultrafiltrates or recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence or absence of neutralizing anti-GM-CSF antibodies. GM-CSF in culture medium was identified by the electrochemiluminescence method. RESULTS: It was demonstrated that the IEC conditioned medium contained GM-CSF. This cytokine led to both the upregulation and downregulation of crypt cell proliferative activity, depending on its concentration in the culture medium. The effect of native GM-CSF was reproduced with recombinant mouse GM-CSF: 25 and 5 ng/ml inhibited the proliferative activity, whereas 1 ng/ml led to its significant stimulation. CONCLUSIONS: Freshly isolated murine IECs produce GM-CSF, which plays a critical role in crypt cell proliferative activity in vitro. These results suggest the involvement of this factor in the regulation of the crypt proliferative zone, in an autocrine and/or paracrine manner.