The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.

Due to the clear separation of innate immunity from adaptive responses, the externally developing zebrafish embryo represents a useful in vivo model for identification of innate host determinants of the response to bacterial infection. Here we performed a time-course transcriptome profiling study and gene ontology analysis of the embryonic innate immune response to infection with two model Salmonella strains that elicit either a lethal infection or an attenuated response. The transcriptional response to infection with both the lethal strain and the avirulent LPS O-Ag mutant strain showed clear conservation with host responses detected in other vertebrate models and human cells, including induction of genes encoding cell surface receptors, signaling intermediates, transcription factors, and inflammatory mediators. Furthermore, our study led to the identification of a large set of novel immune response genes and infection markers, the future functional characterization of which will support vertebrate genome annotation. From the time series and bacterial strain comparisons, matrix metalloproteinase genes, including mmp9, were among the most consistent infection-responsive genes. Purified Salmonella flagellin also strongly induced mmp9 expression. Using knockdown analysis, we showed that this gene was downstream of the zebrafish homologs of the flagellin receptor TLR5 and the adaptor MyD88. Additionally, flagellin-mediated induction of other inflammation markers, including il1b, il8, and cxcl-C1c, was reduced upon Tlr5 knockdown as well as expression of irak3, a putative negative TLR pathway regulator. Finally, we showed that induction of il1b, mmp9, and irak3 requires Myd88-dependent signaling, while ifn1 and il8 were induced Myd88 independently during Salmonella infection.

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The Spi1/Pu.1 transcription factor plays a crucial role in myeloid cell development in vertebrates. Despite extensive studies of Spi1, the controlled gene group remains largely unknown. To identify genes dependent on Spi1, we used a microarray strategy using a knockdown approach in zebrafish embryos combined with fluorescence-activated cell sorting of myeloid cells from transgenic embryos. This approach of using knockdowns with specific green fluorescent protein-marked cell types was highly successful in identifying macrophagespecific genes in Spi1-directed innate immunity. We found a gene group downregulated on spi1 knockdown, which is also enriched in fluorescence-activated cell-sorted embryonic myeloid cells of a spi1:GFP transgenic line. This gene group, representing putative myeloidspecific Spi1 target genes, contained all 5 previously identified Spi1-dependent zebrafish genes as well as a large set of novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6, and mfap4 were expressed specifically in early embryonic macrophages. In a functional approach, we demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, is involved in macrophage migration to the site of bacterial infection. Therefore, based on our combined transcriptome analyses, we discovered novel early macrophage-specific marker genes, including a signal transducer pivotal for macrophage migration in the innate immune response. (Blood. 2010;116(3):e1-e11)