Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery

Abstract

Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naive humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naive mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.

Fig. 1.

PCR amplification and recombination cloning of the vaccinia virus proteome. (A) A series of genes of increasing size from vaccinia were amplified according to the procedures described in the methods section. (B) The PCR products shown in A were transformed into competent E. coli. After transformation and overnight growth, the cells were lysed in phenol-chloroform and the total nucleic acid was isolated and run the agarose gel. Band 1 shows genomic DNA, and band 2 is supercoiled empty vector. Plasmids with insert migrate more slowly depending on the size of the insert. Bands 3 and 4 are 23S and 16S rRNAs. Routinely, purified DNA Miniprep DNA was prepared from the overnight culture without colony selection. The resulting plasmids are sequence verified and used directly in in vitro transcription/translation reactions.

Fig. 2.

The unpurified proteins expressed in vitro can be used for serology on immunodot blots or microarrays. (A) Immunodot blots of 112 different vaccinia proteins (encoded by 124 plasmids cloned by colony selection) expressed in vitro. The control reaction lacked plasmid template; empty vector controls generate a positive signal because of the expression of a small 10× His-positive fragment (data not shown). Volumes of 0.3 μl of each reaction were spotted directly onto nitrocellulose in duplicate and probed with either anti-His tag Ab (Upper Left), anti-HA tag Ab (Upper Right) VIG (Lower Left), or VIG plus 10% E. coli lysate (VIG+L; Lower Right). The identity of each pair of spots is given in Table 2, which is published as supporting information on the PNAS web site. Proteins marked with an asterisk in Upper were negative when probed with both anti-His and anti-HA Abs. E. coli lysate unmasks vaccinia proteins recognized by VIG; dots considered positive by visual estimation are indicated. (B) A pilot microarray constituting five vaccinia proteins expressed from DNA with colony selection and probed with VIG, preabsorbed with or without E. coli lysate. Interspot distance, 0.3 mm.

Fig. 3.

Protein microarray analysis of Abs generated during vaccinia infection. Shown are scans of a vaccinia virus proteome microarrays, probed with normal mouse serum (Upper Left) serum pooled from five vaccinia-immunized mice on day 19 after infection (Upper Right), vaccinia-naive human serum (Lower Left) or human VIG (Lower Right). Vaccinia-specific proteins recognized are indicated. Unannotated proteins in Lower are recognized by vaccinia-naïve human sera and, therefore, are considered nonspecific “background.” The identity of each pair of spots is given in Table 3, which is published as supporting information on the PNAS web site. All of the arrays shown were probed with serum preabsorbed with E. coli lysate. For mouse serum, which lacks anti-E. coli reactivity, there was no difference in the signal intensities against vaccinia antigens with or without lysate treatment (data not shown). No signals were seen by using secondary Ab alone.

Fig. 4.

Signal intensities of the microarrays shown in Fig. 3 probed with pooled mouse sera and VIG. Proteins annotated in parenthesis in Middle are the nonspecific proteins also seen by naïve human sera. Also shown are data from a Dryvax-immunized macaque on day 10 after monkeypox challenge (animal CH39 in ref. 14). Spots: 1–196, top left subarray; 197-392, top right, 393–588, bottom left, 589–784, bottom right. Proteins recognized by all three species are listed along the top. Relative intensities of a particular protein spot are proportional to the titer of the specific Abs in each serum.