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hierarchy of responses as well as identifi- cation of antibody isotypes, although the sensitivity of the array was lower than that of the ELISA by a significant factor (Supple- mentary Figure S2C). An expanded analysis of the array

revealed response differences among vaccination strategies. For many of the viruses represented, multiple forms of the hemagglutinin antigens are included within the array: HA0, globular head and stalk; HA1, globular head (Supplementary Table S1). Te responses from the sera of naïve and immunized mice against the HA0 subset of proteins within the microarray are shown in Figure 2. Immunization with the rH5 and saline elicited detectable responses that were largely constrained to the homologous subtype of the immunogen, H5 and were predominately of the IgG1 isotype. Vaccination with adjuvants altered the quality of responses and significantly increased responses to a number of proteins relative to rH5 and saline vaccination. Similar to the rH5 and saline immunization, the T2-biasing adjuvant, SE, produced mostly IgG1 responses, while the T1-biasing adjuvant, GLA-SE, produced a more balanced response between IgG1 and IgG2c antibodies (Figure 2). We also performed experiments on

ferret and human samples. We compared the response profiles of serum from an individual ferret before and after infection with the H5N1 A/Viet Nam/1203/2004 virus. Several responses were induced, particularly within the H5N1 subset of proteins (Supplementary Figure S3A). Next, we tested the capacity of the array to detect responses induced by vaccination with the 2012–2013 trivalent vaccine (A/ California/7/2009 (H1N1)-like virus, A/ Victoria /361/2011 (H3N2)-like virus, B/Wisconsin /1/2010-like virus) within a human subject (Supplementary Fig. S3B). As shown, the array demonstrated pre-existing responses as well as boosted and new responses to H1N1 and H3N2 strains. While the 2012–2013 trivalent vaccine contained no H5 antigens, it appeared that several H5N1 strains were recognized. The only limitation with regard to analysis of species is the avail- ability of secondary antibodies. Herein, we propose that our hemagglu-

tinin protein microarray provides a significant improvement over comparable measurements of serum antibody levels by techniques such as ELISA or bead based multiplexing. Cost and time savings are tremendous considering that an estimated 200 ELISA end point titration experiments would be required to acquire information equivalent to that from a single microarray slide.

Vol. 54 | No. 6 | 2013

Acknowledgments We thank Yeung Tutterow and Vanitha Raman for their thoughtful discussions as well as Patricia Hon and Blarney Hidsee for their contributions. We also thank Winston Wicomb and the vivarium staff at IDRI. We also thank Peter Palese and Florian Krammer for providing antibodies. Tis work is supported by a contract from BARDA #HHSO100201000039C and by a grant from the Bill and Melinda Gates Foundation #42387.

Competing interests Te authors declare no competing interests.

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Received 21 January 2013; accepted 13 May 2013.

Address correspondence to Darrick Carter, Infectious Disease Research Institute, Seattle, WA. E-mail:

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