An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. in Rabbit polyclonal to ANKRD50. HIV-1-infected humans and HIV-1-vaccinated humans rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity particularly regarding the depth of epitope variants acknowledged at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research. Keywords: HIV peptide microarray diversity antibody vaccine 1 Introduction One of the fundamental challenges in HIV-1 vaccine development is the huge diversity of HIV-1 strains worldwide (Korber et al. 2001 Gaschen et al. 2002 Taylor et al. 2008 Barouch and Korber 2009 Korber et al. 2009 Walker et al. 2011 Ndung’u and Weiss 2012 Picker et al. 2012 Stephenson and Barouch 2013 Globally there are more than a dozen Bortezomib (Velcade) HIV-1 subtypes and hundreds of circulating HIV-1 recombinant forms (CRFs) and between-subtype variation can be as large as 35% (Hemelaar et al. 2006 Taylor et al. 2008 Ndung��u and Weiss 2012 Several HIV-1 vaccine candidates under development aim to overcome the challenge of HIV-1 genetic diversity either through Bortezomib (Velcade) the choice of HIV-1 antigen sequence or the method of antigen delivery (Stephenson and Barouch 2013 However most tools used to assess the immunogenicity of these vaccines focus on measuring the magnitude of HIV-1-specific antibody responses rather than the epitope diversity and specificity of these responses. Peptide microarrays are a potential tool for the measurement of antibody diversity against linear epitopes in HIV-1 vaccine studies. This platform has been utilized to characterize antibody binding to linear sequences in multiple fields including HIV-1 vaccine research (Nahtman et al. 2007 Cerecedo et al. 2008 Gaseitsiwe et al. 2008 Lorenz et al. 2009 Tomaras et al. 2011 Haynes et al. 2012 HIV-1-specific microarrays to date however have not included extensive coverage of variable sequences (Karasavvas et al. 2012 Gottardo et al. 2013 Imholte et al. 2013 Here we describe the development of a global HIV-1 peptide microarray that includes 6 564 overlapping linear HIV-1 peptides covering most common HIV-1 variants in the HIV-1 sequence database at Los Alamos National Laboratory (LANL). This microarray includes 6 564 peptides including an average of 7 peptide variants for each 15 amino acid position in HIV-1 Env Gag Nef Pol Rev Tat and Vif with up to 95 peptide variants per location within the most variable regions of HIV-1 Env. This epitope diversity around the microarray allows for more precise measurements of the magnitude breadth and depth of HIV-1-specific binding IgG responses. 2 Methods 2.1 of peptide library In collaboration with JPT Peptide Technologies (Berlin Germany) we designed a library of HIV-1 linear peptides that provided optimal coverage of HIV-1 global sequence diversity. We began by downloading Bortezomib (Velcade) the sequence alignment for HIV-1 genes ENV GAG NEF POL REV TAT and VIF from the website of the LANL HIV-1 sequence database (Theoretical Biology and Biophysics 2009 using the following settings: Alignment type: Web Alignment (all complete sequences); 12 months: 2009; Region: Pre-defined region of the genome; Subtype: All M Group (A-K + Recombinants); DNA/Protein: Protein; Format: FASTA. Full length proteins of gp120 gp41 p17 p24 Tat and Nef were used as were the immunogenic fragments of p2p7p1p6 protease reverse transcriptase integrase Vif and Ref Bortezomib (Velcade) as published by LANL (Theoretical Biology and Biophysics 2010 (Table 1). Table 1 Composition of global HIV-1 peptide microarray. From the global sequence database we selected the individual sequences to be used as peptides that would provide optimal coverage of sequence diversity using the program package MosaicVaccines.1.2.11 from LANL (ftp://ftp-t10.lanl.gov/pub/btk/mosaic/) (Fischer et al. 2007 Thurmond et al. 2008 Parameters for the generation of MOSAIC sequences were -s 20 -d=true -T 20 -p 100. Sequence manipulation and processing were performed in R 2.11.1 (http://www.r-project.org/) using the package Biostrings (http:www.bioconductor.org/packages/2.2/bioc/html/Biostrings.html) or using bespoke scripts in python (http:www.python.org/). Since our goal was to cover the seven most frequent clades (A B C.