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Background Screening malaria-specific antibody responses on protein microarrays can help identify immune factors that mediate protection against malaria infection, disease, and transmission, as well as markers of past exposure to both malaria parasites and mosquito vectors. Most malaria protein microarray work has used serum as the sample matrix, requiring prompt laboratory processing and a continuous cold chain, thus limiting applications in remote locations. Dried blood spots (DBS) pose minimal biohazard, do not require immediate laboratory processing, and are stable at room temperature for transport, making them potentially superior alternatives to serum. The goals of this study were to assess the viability of DBS as a source for antibody profiling and to use DBS to identify serological signatures of low-density Plasmodium falciparum infections in malaria-endemic regions of Myanmar. Methods Matched DBS and serum samples from a cross-sectional study in Ingapu Township, Myanmar were probed on protein microarrays populated with P. falciparum antigen fragments. Signal and trends in both sample matrices were compared. A case-control study was then performed using banked DBS samples from malaria-endemic regions of Myanmar, and a regularized logistic regression model was used to identify antibody signatures of ultrasensitive PCR-positive P. falciparum infections. Results Approximately 30% of serum IgG activity was recovered from DBS. Despite this loss of antibody activity, antigen and population trends were well-matched between the two sample matrices. Responses to 18 protein fragments were associated with the odds of asymptomatic P. falciparum infection, albeit with modest diagnostic characteristics (sensitivity 58%, specificity 85%, negative predictive value 88%, and positive predictive value 52%). Conclusions Malaria-specific antibody responses can be reliably detected, quantified, and analysed from DBS, opening the door to serological studies in populations where serum collection, transport, and storage would otherwise be impossible. While test characteristics of antibody signatures were insufficient for individual diagnosis, serological testing may be useful for identifying exposure to asymptomatic, low-density malaria infections, particularly if sero-surveillance strategies target individuals with low previous exposure as sentinels for population exposure.

Knowledge of the Plasmodium falciparum antigens that comprise the human liver stage immunoproteome is important for pre-erythrocytic vaccine development, but, compared with the erythrocytic stage immunoproteome, more challenging to classify. Previous studies of P. falciparum antibody responses report IgG and rarely IgA responses. We assessed IgG and IgA antibody responses in adult sera collected during two controlled human malaria infection (CHMI) studies in malaria-naïve volunteers and in 1- to 6-year-old malaria-exposed Malian children on a 251 P. falciparum antigen protein microarray. IgG profiles in the two CHMI groups were equivalent and differed from Malian children. IgA profiles were robust in the CHMI groups and a subset of Malian children. We describe immunoproteome differences in naïve vs. exposed individuals and report pre-erythrocytic proteins recognized by the immune system. IgA responses detected in this study expand the list of pre-erythrocytic antigens for further characterization as potential vaccine candidates.

Background Shigella is a major diarrheal pathogen for which there is presently no vaccine. Whole genome sequencing provides the ability to predict and derive novel antigens for use as vaccines. Here, we aimed to identify novel immunogenic Shigella antigens that could serve as Shigella vaccine candidates, either alone, or when conjugated to Shigella O-antigen. Methods Using a reverse vaccinology approach, where genomic analysis informed the Shigella immunome via an antigen microarray, we aimed to identify novel immunogenic Shigella antigens. A core genome analysis of Shigella species, pathogenic and non-pathogenic Escherichia coli, led to the selection of 234 predicted immunogenic Shigella antigens. These antigens were expressed and probed with acute and convalescent serum from microbiologically confirmed Shigella infections. Results Several Shigella antigens displayed IgG and IgA seroconversion, with no difference in sero-reactivity across by sex or age. IgG sero-reactivity to key Shigella antigens was observed at birth, indicating transplacental antibody transfer. Six antigens (FepA, EmrK, FhuA, MdtA, NlpB, and CjrA) were identified in in vivo testing as capable of producing binding IgG and complement-mediated bactericidal antibody. Conclusions These findings provide six novel immunogenic Shigella proteins that could serve as candidate vaccine antigens, species-specific carrier proteins, or targeted adjuvants.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens. This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera. Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

Seasonal influenza is a serious public health problem because the viral infection spreads easily from person to person and because of antigenic drift in neutralizing epitopes. Influenza vaccination is the most effective way to prevent the disease, although challenging because of the constant evolution of influenza virus subtypes. Our high-throughput protein microarrays allow for interrogation of subunit-specific IgG and IgA responses to 283 different HA proteins comprised of HA1 and HA2 domains as well as full-length HA proteins. This provides a tool that allows for novel insights into the response to exposure to influenza virus antigens. Data generated with our technology will enhance our understanding of the factors that improve the strength, breadth, and durability of vaccine-mediated immune responses and develop more effective vaccines., Current seasonal influenza virus vaccines engender antibody-mediated protection that is hemagglutinin (HA) subtype specific and relatively short-lived. Coverage for other subtypes or even variants within a subtype could be improved from a better understanding of the factors that promote HA-specific antibody cross-reactivity. Current assays to evaluate cross-reactivity, such as the ELISA, require a separate test for each antigen and are neither high-throughput nor sample-sparing. To address this need, we produced an array of 283 purified HA proteins from influenza A virus subtypes H1 to H16 and H18 and influenza B virus. To evaluate performance, arrays were probed with sera from individuals before and after a booster dose of inactivated heterologous H5N1 vaccine and naturally infected cases at presentation and follow-up during the 2010 to 2011 influenza season, when H3N2 was prevalent. The response to the H5 vaccine boost was IgG only and confined to H5 variants. The response to natural H3N2 infection consisted of IgG and IgA and was reactive with all H3 variants displayed, as well as against other group 2 HA subtypes. In both groups, responses to HA1 proteins were subtype specific. In contrast, baseline signals were higher, and responses broader, against full-length HA proteins (HA1+HA2) compared to HA1 alone. We propose that these elevated baseline signals and breadth come from the recognition of conserved epitopes in the stalk domain by cross-reactive antibodies accumulated from previous exposure(s) to seasonal influenza virus. This array is a valuable high-throughput alternative to the ELISA for monitoring specificity and cross-reactivity of HA antibodies and has many applications in vaccine development. IMPORTANCE Seasonal influenza is a serious public health problem because the viral infection spreads easily from person to person and because of antigenic drift in neutralizing epitopes. Influenza vaccination is the most effective way to prevent the disease, although challenging because of the constant evolution of influenza virus subtypes. Our high-throughput protein microarrays allow for interrogation of subunit-specific IgG and IgA responses to 283 different HA proteins comprised of HA1 and HA2 domains as well as full-length HA proteins. This provides a tool that allows for novel insights into the response to exposure to influenza virus antigens. Data generated with our technology will enhance our understanding of the factors that improve the strength, breadth, and durability of vaccine-mediated immune responses and develop more effective vaccines.

The development of vaccines against malaria and serodiagnostic tests for detecting recent exposure requires tools for antigen discovery and suitable animal models. The protein microarray is a high throughput, sample sparing technique, with applications in infectious disease research, clinical diagnostics, epidemiology, and vaccine development. We recently demonstrated Qdot®-based indirect immunofluorescence together with a portable optical imager ArrayCAM® using single isotype detection could replicate data using the conventional laser confocal scanner system, opening up the opportunity for deployment of protein microarray-based immunoassays in more laboratories worldwide. We developed a multiplexing protocol for simultaneous detection of IgG, IgA and IgM and compared samples from a controlled human malaria infection model with those from controlled malaria infections of Aotus nancymaae, a widely-used non-human primate model of human malaria. IgG profiles showed the highest concordance in number of reactive antigens. Thus, of the 139 antigens recognized by human IgG antibody, 111 were also recognized by Aotus monkeys. Interestingly, IgA profiles were largely non-overlapping. Finally, on the path toward wider deployment of the portable platform, we show excellent correlations between array data obtained in 5 independent laboratories around the U.S. using the multiplexing protocol (R(2): 0.60 – 0.92).This study supports the use of this platform for wider deployment, particularly in endemic areas where such a tool will have the greatest impact on global human health.

We describe a novel nanobiotechnology platform based on subviral particles derived from Infectious bursal disease virus (IBD-SVPs). The major virus coat protein VP2 assembles into spherical, 23 nm SVPs when expressed as a heterologous protein in the yeast Pichia pastoris. We recovered up to 38 mg of IBD-SVPs at . 95% purity from 1 L of recombinant yeast culture. The purified particles were able to tolerate organic solvents up to 20% concentration (ethanol or dimethylsulfoxide), they resisted temperatures up to 65 uC and remained stable over a wide pH range (2.5-9.0). We achieved bioconjugation to the amine groups of lysine residues and to the carboxyl groups of aspartic and glutamic acid residues, allowing the functionalization of IBD-SVPs with biotin. The accessibility of surface amine groups was measured using Alexa Fluor 488 N-hydroxysuccinimide (NHS) ester, an amine-selective fluorescent dye, revealing that approximately 60 dye molecules were attached to the surface of each particle. IBD-SVPs can therefore be exploited as a robust and versatile nanoscaffold to display diverse functional ligands.

Background Despite largely successful control efforts, malaria remains a significant public health problem in Thailand. Based on microscopy, the northwestern province of Tak, once Thailand’s highest burden area, is now considered a low-transmission region. However, microscopy is insensitive to detect low-level parasitaemia, causing gross underestimation of parasite prevalence in areas where most infections are subpatent. The objective of this study was to assess the current epidemiology of malaria prevalence using molecular and serological detection methods, and to profile the antibody responses against Plasmodium as it relates to age, seasonal changes and clinical manifestations during infection. Three comprehensive cross-sectional surveys were performed in a sentinel village and from febrile hospital patients, and whole blood samples were collected from infants to elderly adults. Genomic DNA isolated from cellular fraction was screened by quantitative-PCR for the presence of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasma samples were probed on protein microarray to obtain antibody response profiles from the same individuals. Results Within the studied community, 90.2 % of Plasmodium infections were submicroscopic and asymptomatic, including a large number of mixed-species infections. Amongst febrile patients, mixed-species infections comprised 68 % of positive cases, all of which went misdiagnosed and undertreated. All samples tested showed serological reactivity to Plasmodium antigens. There were significant differences in the rates of antibody acquisition against P. falciparum and P. vivax, and age-related differences in species-specific immunodominance of response. Antibodies against Plasmodium increased along the ten-month study period. Febrile patients had stronger antibody responses than asymptomatic carriers. Conclusions Despite a great decline in malaria prevalence, transmission is still ongoing at levels undetectable by traditional methods. As current surveillance methods focus on case management, malaria transmission in Thailand will not be interrupted if asymptomatic submicroscopic infections are not detected and treated. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1393-4) contains supplementary material, which is available to authorized users.

Organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. However, the detection equipment is often expensive, fragile, and hard to deploy widely. Quantum dots (Qdot) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. Here, we have evaluated a small benchtop "personal" optical imager (ArrayCAM) developed for quantification of protein arrays probed by Qdot-based indirect immunofluorescence. The aim was to determine if the Qdot imager system provides equivalent data to the conventional organic dye-labeled antibody/laser scanner system. To do this, duplicate proteome microarrays of Vaccinia virus, Brucella melitensis and Plasmodium falciparum were probed with identical samples of immune sera, and IgG, IgA, and IgM profiles visualized using biotinylated secondary antibodies followed by a tertiary reagent of streptavidin coupled to either P3 (an organic cyanine dye typically used for microarrays) or Q800 (Qdot). The data show excellent correlation for all samples tested (R > 0.8) with no significant change of antibody reactivity profiles. We conclude that Qdot detection provides data equivalent to that obtained using conventional organic dye detection. The portable imager offers an economical, more robust, and deployable alternative to conventional laser array scanners.

PLoS one 8(12), e83210 (2013). doi:10.1371/journal.pone.0083210, Published by PLoS [u.a.], Lawrence, Kan.