Mucosal Immunization with a \u3cem\u3eStaphylococcus aureus\u3c/em\u3e IsdA-Cholera Toxin A\u3csub\u3e2\u3c/sub\u3e/B Chimera Induces Antigen-Specific Th2-Type Responses in Mice

Staphylococcus aureus is a leading cause of opportunistic infection worldwide and a significant public health threat. The iron-regulated surface determinant A (IsdA) adhesin is essential for S. aureus colonization on human nasal epithelial cells and plays an important role in iron acquisition and resistance to human skin defenses. Here we investigated the murine immune response to intranasal administration of a cholera toxin (CT) A2/B chimera containing IsdA. Plasmids were constructed to express the IsdA-CTA2/B chimera and control proteins in E. coli. Proper construction of the chimera was verified by SDS-PAGE, western blot, GM1 ELISA, and confocal microscopy. Groups of female BALB/c mice were immunized with IsdA-CTA2/B, IsdA mixed with CTA2/B, IsdA alone, or mock, followed by one booster immunization 10 days post-priming. Analysis of serum IgG and nasal, intestinal, and vaginal IgA suggested that mucosal immunization with IsdA-CTA2/B induces significant IsdA-specific humoral immunity. Functional in vitro assays revealed that α-IsdA immune serum significantly blocks the adherence of S. aureus to human epithelial cells. Splenocytes from mice immunized with IsdA-CTA2/B showed specific cellular proliferation and production of IL-4 after in vitro stimulation. Immunization with IsdA-CTA2/B drove isotype switching to IgG1, indicative of a Th2-type response. Our results suggest that the immunogenicity of the S. aureus IsdA-CTA2/B chimera merits further investigation as a potential mucosal vaccine candidate

Siglec-F is a novel intestinal M cell marker

Intestinal microfold (M) cells are epithelial cells primarily present on Peyer's patches (PPs) in the small intestine. The ability of M cells to shuttle antigens into the PP for appropriate immune responses makes M cells a target for next-generation oral vaccine delivery. In this regard, discovery of M cell specific receptors are of great interest, which could act as molecular tags for targeted delivery of cargo to M cells. Here, using a monoclonal antibody we generated to the Sialic acid-binding immunoglobulin-like lectin F (Siglec-F), we show that Siglec-F is expressed on mouse M cells in the small intestine. Immunohistochemical analysis of the PP tissue sections shows that Siglec-F is expressed on the surface of the M cell membrane exposed to the intestinal lumen. Anti-Siglec-F antibody injected into the mouse small intestine bound to M-cells, demonstrating the potential to target M cells via Siglec-F

Agonist Antibody Converts Stem Cells into Migrating Brown Adipocyte-Like Cells in Heart

We present data showing that Iodotyrosine Deiodinase (IYD) is a dual-function enzyme acting as a catalyst in metabolism and a receptor for cooperative stem cell differentiation. IYD is present both in thyroid cells where it is critical for scavenging iodine from halogenated by-products of thyroid hormone production and on hematopoietic stem cells. To close the cooperative loop, the mono- and di-Iodotyrosine (MIT and DIT) substrates of IYD in the thyroid are also agonists for IYD now acting as a receptor on bone marrow stem cells. While studying intracellular combinatorial antibody libraries, we discovered an agonist antibody, H3 Ab, of which the target is the enzyme IYD. When agonized by H3 Ab, IYD expressed on stem cells induces differentiation of the cells into brown adipocyte-like cells, which selectively migrate to mouse heart tissue. H3 Ab also binds to IYD expressed on human myocardium. Thus, one has a single enzyme acting in different ways on different cells for the cooperative purpose of enhancing thermogenesis or of regenerating damaged heart tissue

Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype owing to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine

An agonist antibody prefers relapsed AML for induction of cells that kill each other

Abstract Previously, we reported an agonist antibody to a cytokine receptor, Thrombopoietin receptor (TPOR) that effectively induces cytotoxic killer cells from precursor tumor cells isolated from newly diagnosed AML patients. Here, we show that the TPOR agonist antibody can induce even relapsed AML cells into killer cells more potently than newly diagnosed AML cells. After stimulation by the agonist antibody, these relapsed leukemic cells enter into a differentiation process of killer cells. The antibody-induced killer cells express, Granzyme B and Perforin that assault and kill other members of the AML cell population. Particularly, the agonist antibody showed potent efficacy on the AML xenograft model in mice using the NOD/LtSz-scid IL2Rγc null (NSG) mice. These results show that the TPOR agonist antibody that induces AML cells to kill each other is effective on both relapsed AML cells and in vivo. Therefore, this study suggests a new strategy for the treatment of cancer relapse after chemotherapy

Purification and Characterization of \u3cem\u3eYersinia enterocolitica\u3c/em\u3e and \u3cem\u3eYersinia pestis\u3c/em\u3e LcrV–Cholera Toxin A\u3csub\u3e2\u3c/sub\u3e/B Chimeras

Yersinia pestis is a virulent human pathogen and potential biological weapon. Despite a long history of research on this organism, there is no licensed vaccine to protect against pneumonic forms of Y. pestis disease. In the present study, plasmids were constructed to express cholera toxin A2/B chimeric molecules containing the LcrV protective antigen from Yersinia enterocolitica and Y. pestis. These chimeras were expressed and purified to high yields from the supernatant of transformed Escherichia coli. Western and GM1 ELISA assays were used to characterize the composition, receptor-binding and relative stability of the LcrV–CTA2/B chimera in comparison to cholera toxin. In addition, we investigated the ability of the Y. pestis LcrV–CTA2/B chimera to bind to and internalize into cultured epithelial cells and macrophages by confocal microscopy. These studies indicate that the uptake and trafficking of the LcrV antigen from the chimera is comparable to the trafficking of native toxin. Together these findings report that stable, receptor-binding, non-toxic LcrV–cholera toxin A2/B chimeras can be expressed at high levels in E. coli and purified from the supernatant. In addition, the internalization of antigen in vitro reported here supports the development of these molecules as novel mucosal vaccine candidates

Mucosal Immunization with a Staphylococcus aureus IsdA-Cholera Toxin A2/B Chimera Induces Antigen-Specific Th2-Type Responses in Mice▿

Staphylococcus aureus is a leading cause of opportunistic infection worldwide and a significant public health threat. The iron-regulated surface determinant A (IsdA) adhesin is essential for S. aureus colonization on human nasal epithelial cells and plays an important role in iron acquisition and resistance to human skin defenses. Here we investigated the murine immune response to intranasal administration of a cholera toxin A2/B (CTA2/B) chimera containing IsdA. Plasmids were constructed to express the IsdA-CTA2/B chimera and control proteins in Escherichia coli. Proper construction of the chimera was verified by SDS-PAGE, Western blotting, GM1 enzyme-linked immunosorbent assay (ELISA), and confocal microscopy. Groups of female BALB/c mice were mock immunized or immunized with IsdA-CTA2/B, IsdA mixed with CTA2/B, or IsdA alone, followed by one booster immunization at 10 days postpriming. Analysis of serum IgG and nasal, intestinal, and vaginal IgA suggested that mucosal immunization with IsdA-CTA2/B induces significant IsdA-specific humoral immunity. Functional in vitro assays revealed that immune serum significantly blocks the adherence of S. aureus to human epithelial cells. Splenocytes from mice immunized with IsdA-CTA2/B showed specific cellular proliferation and production of interleukin-4 (IL-4) after in vitro stimulation. Immunization with IsdA-CTA2/B drove isotype switching to IgG1, indicative of a Th2-type response. Our results suggest that the immunogenicity of the S. aureus IsdA-CTA2/B chimera merits further investigation as a potential mucosal vaccine candidate