Search

Vaccination has been playing an important role in treating both infectious and cancerous diseases. Nevertheless, many diseases still lack proper vaccines due to the difficulty to generate sufficient amounts of antigen-specific antibodies or T cells. Adjuvants provide an important route to improve and direct immune responses. However, there are few adjuvants approved clinically and many of them lack the clear structure/adjuvanticity relationship. Here, we synthesized and evaluated a series of dendronized polypeptides (denpols) functionalized with varying tryptophan/histidine (W/H) molar ratios of 0/100, 25/75, 50/50, 75/25, and 100/0 as tunable synthetic adjuvants. The denpols showed structure-dependent inflammasome activation in THP1 monocytic cells and structure-related activation and antigen cross-presentation in vitroin bone marrow-derived dendritic cells. We used the denpols with bacterial pathogen Coxiella burnetiiantigens in vivo, which showed both high and tunable adjuvating activities, as demonstrated by the antigen-specific antibody and T cell responses. The denpols are easy to make and scalable, biodegradable, and have highly adjustable chemical structures. Taken together, denpols show great potential as a new and versatile adjuvant platform that allows us to adjust adjuvanticity based on structure–activity correlation with the aim to fine-tune the immune response, thus advancing vaccine development.

Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.

We have established a predictive modelling framework to systematically analyze IgG antibody responses against a large panel of P. falciparum-specific antigens and identify a predictive signature of naturally acquired immunity to malaria. Our results show that an individual's immune status can be accurately predicted by measuring IgG antibody responses to a parsimonious set of 15 target antigens. The identified immune signature is highly versatile and capable of providing precise and accurate estimates of clinical protection from malaria in demographically distinct populations.

Examines nonviral gene delivery systems for gene therapy. Problems encountered when using viruses as gene transfer vectors; Electrical problems in gene transfection techniques; Transfection of cells with genes through the exposure of cells to gene-containing liposomes; Modification of lipoplexes before gene delivery; Uses of naked DNA in vaccine development.

Coxiella burnetiiis the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.

ABSTRACTTularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensissubsp. tularensis(type A) and F. tularensissubsp. holarctica(type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensisinfections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.

We present the synthesis of novel adjuvants for vaccine development using multivalent scaffolds and bioconjugation chemistry to spatially manipulate Toll-like receptor (TLR) agonists. TLRs are primary receptors for activation of the innate immune system during vaccination. Vaccines that contain a combination of small and macromolecule TLR agonists elicit more directed immune responses and prolong responses against foreign pathogens. In addition, immune activation is enhanced upon stimulation of two distinct TLRs. Here, we synthesized combinations of TLR agonists as spatially defined tri- and di-agonists to understand how specific TLR agonist combinations contribute to the overall immune response. We covalently conjugated three TLR agonists (TLR4, 7, and 9) to a small molecule core to probe the spatial arrangement of the agonists. Treating immune cells with the linked agonists increased activation of the transcription factor NF-κB and enhanced and directed immune related cytokine production and gene expression beyond cells treated with an unconjugated mixture of the same three agonists. The use of TLR signaling inhibitors and knockout studies confirmed that the tri-agonist molecule activated multiple signaling pathways leading to the observed higher activity. To validate that the TLR4, 7, and 9 agonist combination would activate the immune response to a greater extent, we performed in vivostudies using a vaccinia vaccination model. Mice vaccinated with the linked TLR agonists showed an increase in antibody depth and breadth compared to mice vaccinated with the unconjugated mixture. These studies demonstrate how activation of multiple TLRs through chemically and spatially defined organization assists in guiding immune responses, providing the potential to use chemical tools to design and develop more effective vaccines.

Malaria-specific antibody responses are short lived in children, leaving them susceptible to repeated bouts of febrile malaria. The cellular and molecular mechanisms underlying this apparent immune deficiency are poorly understood. Recently, T follicular helper (Tfh) cells have been shown to play a critical role in generating long-lived antibody responses. We show that Malian children have resting PD-1+CXCR5+CD4+Tfh cells in circulation that resemble germinal center Tfh cells phenotypically and functionally. Within this population, PD-1+CXCR5+CXCR3−Tfh cells are superior to Th1-polarized PD-1+CXCR5+CXCR3+Tfh cells in helping B cells. Longitudinally, we observed that malaria drives Th1 cytokine responses, and accordingly, the less-functional Th1-polarized Tfh subset was preferentially activated and its activation did not correlate with antibody responses. These data provide insights into the Tfh cell biology underlying suboptimal antibody responses to malaria in children and suggest that vaccine strategies that promote CXCR3−Tfh cell responses may improve malaria vaccine efficacy.

ABSTRACTMalaria elimination efforts would benefit from vaccines that block transmission of Plasmodium falciparumgametocytes from humans to mosquitoes. A clear understanding of gametocyte-specific antibody responses in exposed populations could help determine whether transmission-blocking vaccines (TBV) would be boosted by natural gametocyte exposure, and also inform the development of serologic tools to monitor gametocyte exposure in populations targeted for malaria elimination. To this end, plasma was collected from Malian children and adults before and after the 6-month malaria season and probed against a microarray containing 1,204 P. falciparumproteins. Using publicly available proteomic data, we classified 91 proteins as gametocyte specific and 69 as proteins not expressed by gametocytes. The overall breadth and magnitude of gametocyte-specific IgG responses increased during the malaria season, although they were consistently lower than IgG responses to nongametocyte antigens. Notably, IgG specific for the TBV candidates Pfs48/45 and Pfs230 increased during the malaria season. In addition, IgGs specific for the gametocyte proteins Pfmdv1, Pfs16, PF3D7_1346400, and PF3D7_1024800 were detected in nearly all subjects, suggesting that seroconversion to these proteins may be a sensitive indicator of gametocyte exposure, although further studies are needed to determine the specificity and kinetics of these potential serologic markers. These findings suggest that TBV-induced immunity would be boosted through natural gametocyte exposure, and that antibody responses to particular antigens may reliably indicate gametocyte exposure.

Acquisition of acute toxoplasmosis during the first trimester of pregnancy can have catastrophic consequences for the foetus. Diagnosis is routinely based on the detection of maternal Toxoplasma gondii – antibodies using whole parasite extracts as detection antigen. While such assays are sensitive, they show no specificity for the stage of infection. We hypothesized diagnosis might be improved using recombinant antigens for detection, particularly if antibodies to certain antigen(s) were associated with early or late stages of infection. To address this, protein microarrays comprising 1513 T. gondii exon products were probed with well-characterized sera from seronegative (‘N’) controls, and acute (‘A’), chronic/IgM-persisting (‘C/M’) and chronic (‘C’) toxoplasmosis cases from Turkey. Three reactive exon products recognized preferentially in A infections, and three recognized preferentially in C/M infections, were expressed in Escherichia coli and tested for discrimination in IgG ELISAs. The best discriminators were exon 1 of TGME49_086450 (GRA5) which detected C/M infections with 70·6% sensitivity and 81·8% specificity, and exon 6 of TGME49_095700 (ubiquitin transferase domain-containing protein) which detected A infections with 84·8% sensitivity and 82·4% specificity. Overall, the data support a recombinant protein approach for the development of improved serodiagnostic tests for toxoplasmosis. [ABSTRACT FROM AUTHOR]

The leading malaria vaccine candidate, RTS,S, targets the sporozoite and liver stages of the Plasmodium falciparumlife cycle, yet it provides partial protection against disease associated with the subsequent blood stage of infection. Antibodies against the vaccine target, the circumsporozoite protein, have not shown sufficient correlation with risk of clinical malaria to serve as a surrogate for protection. The mechanism by which a vaccine that targets the asymptomatic sporozoite and liver stages protects against disease caused by blood-stage parasites remains unclear. We hypothesized that vaccination with RTS,S protects from blood-stage disease by reducing the number of parasites emerging from the liver, leading to prolonged exposure to subclinical levels of blood-stage parasites that go undetected and untreated, which in turn boosts pre-existing antibody-mediated blood-stage immunity. To test this hypothesis, we compared antibody responses to 824 P. falciparumantigens by protein array in Mozambican children 6 months after receiving a full course of RTS,S (n= 291) versuscomparator vaccine (n= 297) in a Phase IIb trial. Moreover, we used a nested case-control design to compare antibody responses of children who did or did not experience febrile malaria. Unexpectedly, we found that the breadth and magnitude of the antibody response to both liver and asexual blood-stage antigens was significantly lower in RTS,S vaccinees, with the exception of only four antigens, including the RTS,S circumsporozoite antigen. Contrary to our initial hypothesis, these findings suggest that RTS,S confers protection against clinical malaria by blocking sporozoite invasion of hepatocytes, thereby reducing exposure to the blood-stage parasites that cause disease. We also found that antibody profiles 6 months after vaccination did not distinguish protected and susceptible children during the subsequent 12-month follow-up period but were strongly associated with exposure. Together, these data provide insight into the mechanism by which RTS,S protects from malaria.

With increasing efficiency, accuracy, and speed we can access complete genome sequences from thousands of infectious microorganisms; however, the ability to predict antigenic targets of the immune system based on amino acid sequence alone is still needed. Here we use a Leptospira interrogansmicroarray expressing 91% (3359) of all leptospiral predicted ORFs (3667) and make an empirical accounting of all antibody reactive antigens recognized in sera from naturally infected humans; 191 antigens elicited an IgM or IgG response, representing 5% of the whole proteome. We classified the reactive antigens into 26 annotated COGs (clusters of orthologous groups), 26 JCVI Mainrole annotations, and 11 computationally predicted proteomic features. Altogether, 14 significantly enriched categories were identified, which are associated with immune recognition including mass spectrometry evidence of in vitro expression and in vivo mRNA up-regulation. Together, this group of 14 enriched categories accounts for just 25% of the leptospiral proteome but contains 50% of the immunoreactive antigens. These findings are consistent with our previous studies of other Gram-negative bacteria. This genome-wide approach provides an empirical basis to predict and classify antibody reactive antigens based on structural, physical–chemical, and functional proteomic features and a framework for understanding the breadth and specificity of the immune response to L. interrogans.

ABSTRACTWe have previously shown that an assay based on detection of anti-Salmonella entericaserotype Typhi antibodies in supernatant of lymphocytes harvested from patients presenting with typhoid fever (antibody in lymphocyte supernatant [ALS] assay) can identify 100% of patients with blood culture-confirmed typhoid fever in Bangladesh. In order to define immunodominant proteins within the S. Typhi membrane preparation used as antigen in these prior studies and to identify potential biomarkers unique to S. Typhi bacteremic patients, we probed microarrays containing 2,724 S. Typhi proteins with ALS collected at the time of clinical presentation from 10 Bangladeshis with acute typhoid fever. We identified 62 immunoreactive antigens when evaluating both the IgG and IgA responses. Immune responses to 10 of these antigens discriminated between individuals with acute typhoid infection and healthy control individuals from areas where typhoid infection is endemic, as well as Bangladeshi patients presenting with fever who were subsequently confirmed to have a nontyphoid illness. Using an ALS enzyme-linked immunosorbent assay (ELISA) format and purified antigen, we then confirmed that immune responses against the antigen with the highest immunoreactivity (hemolysin E [HlyE]) correctly identified individuals with acute typhoid or paratyphoid fever in Dhaka, Bangladesh. These observations suggest that purified antigens could be used with ALS and corresponding acute-phase activated B lymphocytes in diagnostic platforms to identify acutely infected patients, even in areas where enteric fever is endemic.

ABSTRACTBrucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensisinfection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4+T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensisprotein microarray. Gamma interferon (IFN-?) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n= 9) and from patients who relapsed (n= 5). Four peptide epitopes derived from 3 B. melitensisproteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-? ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensisMHC-II-restricted CD4+T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen.

ABSTRACTHuman brucellosis is a common zoonosis worldwide. Here we present a case of focal vertebral brucellosis in a 71-year-old Mexican-American woman who contracted infection from unpasteurized goat milk. Standard agglutination serology was negative; the diagnosis was established by the isolation of Brucella melitensisfrom abscess fluid. A B. melitensisprotein microarray comprised of nearly all proteins encoded by the bacterial genome was used to determine the kinetics of this patient's antibody responses to the complete collection of open reading frames existing in the genome (ORFeome). Three patterns of antibody responses against B. melitensisantigens were seen for serum samples obtained on days 0 (pretreatment), 14, 49, 100, and 180: (i) stable titers over time, (ii) a steady fall in titers, and (iii) an initial rise in titers followed by declining titers. Sera from this patient with chronic brucellosis recognized some of the same B. melitensisproteins as those recognized by sera from acute/subacute, blood culture-positive brucellosis patients but also recognized a distinct set of proteins. This study is the first to determine the kinetics of the human antibody responses to the complete repertoire of proteins encoded by a bacterial genome and demonstrates fundamentally different immunopathogenetic mechanisms between acute human brucellosis and chronic human brucellosis. While an extension of these findings to a larger patient population is necessary, these findings have important clinical and diagnostic implications and lead toward new insights into the fundamental immunopathogenesis of brucellosis.

Burkholderia pseudomalleiis the etiological agent of human melioidosis, a disease with a broad spectrum of clinical manifestations ranging from fatal septicemia to chronic localized infection or asymptomatic latent infection. Most clinical and immunological studies to date have focused on the acute disease process; however, little is known about pathology and immune response in chronic melioidosis. Here, we have developed a murine model of chronic disease by challenging C57BL/6 mice intranasally with a low dose of B. pseudomalleiand monitoring them up to 100 days postinfection. Bacterial burdens were heterogeneous in different animals at all time points, consistent with the spectrum of clinical severity observed in humans. Proinflammatory cytokines such as gamma interferon (IFN-γ), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were induced during chronic infection, and histopathological analysis showed features in common with human melioidosis. Interestingly, many of these features were similar to those induced by Mycobacterium tuberculosisin humans, such as development of a collagen cord that encapsulates the lesions, the presence of multinucleated giant cells, and granulomas with a caseous necrotic center, which may explain why chronic melioidosis is often misdiagnosed as tuberculosis. Our model now provides a relevant and practical tool to define the immunological features of chronic melioidosis and aid in the development of more effective treatment of this disease in humans.

ABSTRACTMelioidosis is a severe infectious disease caused by the saprophytic facultative intracellular pathogen Burkholderia pseudomallei. The disease is endemic in Southeast Asia and Northern Australia, and no effective vaccine exists. To describe human cell-mediated immune responses to B. pseudomalleiand to identify candidate antigens for vaccine development, the ability of antigen-pulsed monocyte-derived dendritic cells (moDCs) to trigger autologous T-cell responses to B. pseudomalleiand its products was tested. moDCs were prepared from healthy individuals exposed or not exposed to B. pseudomallei, based on serological evidence. These were pulsed with heat-killed B. pseudomalleior purified antigens, including ABC transporters (LolC, OppA, and PotF), Bsa type III secreted proteins (BipD and BopE), tandem repeat sequence-containing proteins (Rp1 and Rp2), flagellin, and heat shock proteins (Hsp60 and Hsp70), prior to being mixed with autologous T-cell populations. After pulsing of cells with either heat-killed B. pseudomallei, LolC, or Rp2, coculturing the antigen-pulsed moDCs with T cells elicited gamma interferon production from CD4+T cells from seropositive donors at levels greater than those for seronegative donors. These antigens also induced granzyme B (cytotoxic) responses from CD8+T cells. Activation of antigen-specific CD4+T cells required direct contact with moDCs and was therefore not dependent on soluble mediators. Rp peptide epitopes recognized by T cells in healthy individuals were identified. Our study provides valuable novel data on the induction of human cell-mediated immune responses to B. pseudomalleiand its protein antigens that may be exploited in the rational development of vaccines to combat melioidosis.

Chlamydia trachomatis infections can lead to severe chronic complications, including trachoma, ectopic pregnancy, and infertility. The only effective approach to disease control is vaccination. The goal of this work was to identify new potential vaccine candidates through a proteomics approach. We constructed a protein chip array (Antigen Discovery, Inc.) by expressing the open reading frames (ORFs) from C. trachomatis mouse pneumonitis (MoPn) genomic and plasmid DNA and tested it with serum samples from MoPn-immunized mice. Two groups of BALB/c female mice were immunized either intranasally or intravaginally with live elementary bodies (EB). Another two groups were immunized by a combination of the intramuscular and subcutaneous routes with UV-treated EB (UV-EB), using either CpG and Montanide as adjuvants to favor a Th1 response or alum to elicit a Th2 response. Serum samples collected at regular intervals postimmunization were tested in the proteome array. The microarray included the expression products of 909 proteins from a total of 921 ORFs of the Chlamydia MoPn genome and plasmid. A total of 185 immunodominant proteins elicited an early and sustained antibody response in the mice immunized with live EB, and of these, 71 were also recognized by the sera from mice immunized with UV-EB. The reactive antigens included some proteins that were previously described as immunogenic, such as the major outer membrane protein, OmpB, Hsp60, and IncA and proteins from the type III secretion system. In addition, we identified in mice several new immunogens, including 75 hypothetical proteins. In summary, we have identified a new group of immunodominant chlamydial proteins that can be tested for their ability to induce protection.

Melioidosis is a severe infectious disease caused by the saprophytic facultative intracellular pathogen Burkholderia pseudomallei. The disease is endemic in Southeast Asia and Northern Australia, and no effective vaccine exists. To describe human cell-mediated immune responses to B. pseudomallei and to identify candidate antigens for vaccine development, the ability of antigen-pulsed monocyte-derived dendritic cells (moDCs) to trigger autologous T-cell responses to B. pseudomallei and its products was tested. moDCs were prepared from healthy individuals exposed or not exposed to B. pseudomallei, based on serological evidence. These were pulsed with heat-killed B. pseudomallei or purified antigens, including ABC transporters (LolC, OppA, and PotF), Bsa type III secreted proteins (BipD and BopE), tandem repeat sequence-containing proteins (Rp1 and Rp2), flagellin, and heat shock proteins (Hsp60 and Hsp70), prior to being mixed with autologous T-cell populations. After pulsing of cells with either heat-killed B. pseudomallei, LolC, or Rp2, coculturing the antigen-pulsed moDCs with T cells elicited gamma interferon production from CD4+T cells from seropositive donors at levels greater than those for seronegative donors. These antigens also induced granzyme B (cytotoxic) responses from CD8+T cells. Activation of antigen-specific CD4+T cells required direct contact with moDCs and was therefore not dependent on soluble mediators. Rp peptide epitopes recognized by T cells in healthy individuals were identified. Our study provides valuable novel data on the induction of human cell-mediated immune responses to B. pseudomallei and its protein antigens that may be exploited in the rational development of vaccines to combat melioidosis.

ABSTRACTChlamydia trachomatisinfections can lead to severe chronic complications, including trachoma, ectopic pregnancy, and infertility. The only effective approach to disease control is vaccination. The goal of this work was to identify new potential vaccine candidates through a proteomics approach. We constructed a protein chip array (Antigen Discovery, Inc.) by expressing the open reading frames (ORFs) from C. trachomatismouse pneumonitis (MoPn) genomic and plasmid DNA and tested it with serum samples from MoPn-immunized mice. Two groups of BALB/c female mice were immunized either intranasally or intravaginally with live elementary bodies (EB). Another two groups were immunized by a combination of the intramuscular and subcutaneous routes with UV-treated EB (UV-EB), using either CpG and Montanide as adjuvants to favor a Th1 response or alum to elicit a Th2 response. Serum samples collected at regular intervals postimmunization were tested in the proteome array. The microarray included the expression products of 909 proteins from a total of 921 ORFs of the ChlamydiaMoPn genome and plasmid. A total of 185 immunodominant proteins elicited an early and sustained antibody response in the mice immunized with live EB, and of these, 71 were also recognized by the sera from mice immunized with UV-EB. The reactive antigens included some proteins that were previously described as immunogenic, such as the major outer membrane protein, OmpB, Hsp60, and IncA and proteins from the type III secretion system. In addition, we identified in mice several new immunogens, including 75 hypothetical proteins. In summary, we have identified a new group of immunodominant chlamydial proteins that can be tested for their ability to induce protection.

ABSTRACTQ fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish between C. burnetiistrains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetiiprotein(s). Here we describe the use of a C. burnetiiprotein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetiiinfection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetiiopen reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetiiproteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetiiproteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.

ABSTRACTHumans and other animals with Lyme borreliosis produce antibodies to a number of components of the agent Borrelia burgdorferi, but a full accounting of the immunogens during natural infections has not been achieved. Employing a protein array produced in vitro from 1,292 DNA fragments representing ∼80% of the genome, we compared the antibody reactivities of sera from patients with early or later Lyme borreliosis to the antibody reactivities of sera from controls. Overall, ∼15% of the open reading frame (ORF) products (Orfs) of B. burgdorferiin the array detectably elicited an antibody response in humans with natural infections. Among the immunogens, 103 stood out on the basis of statistical criteria. The majority of these Orfs were also immunogenic with sera obtained from naturally infected Peromyscus leucopusmice, a major reservoir. The high-ranking set included several B. burgdorferiproteins hitherto unrecognized as immunogens, as well as several proteins that have been established as antigens. The high-ranking immunogens were more likely than nonreactive Orfs to have the following characteristics: (i) plasmid-encoded rather than chromosome-encoded proteins, (ii) a predicted lipoprotein, and (iii) a member of a paralogous family of proteins, notably the Bdr and Erp proteins. The newly discovered antigens included Orfs encoded by several ORFs of the lp36 linear plasmid, such as BBK07 and BBK19, and proteins of the flagellar apparatus, such as FliL. These results indicate that the majority of deduced proteins of B. burgdorferido not elicit antibody responses during infection and that the limited sets of immunogens are similar for two different host species.

An approach is described for makingtranscriptionally active PCR (TAP) fragments that were used directly in in vitroand in vivoexpression experiments. TAP fragments encoding reporter genes were amplified in 1 day using typical PCR methodology and were expressed in cultured cells and in mice at levels comparable with a widely used cytomegalovirus promoter-based plasmid expression vector. Following intramuscular injection, a TAP fragment encoding hepatitis B surface antigen (HBsAg) induced anti-HBsAg antibody titers comparable with those induced by supercoiled plasmid encoding the same antigen. Epitope-tagged TAP fragments were generated and transfected into cells for rapid, high throughput immunocytochemical analysis of the tagged gene products. TAP fragments were also transferred directly into expression vectors by in vivohomologous recombination without conventional cloning, affording a high throughput cloning approach that does not require restriction enzyme digestion, ligations, or thymidine adenine complementation cloning. The methodology has been adapted to a robotic work station enabling the high throughput generation of transcriptionally active genes at the rate of more than 400 different genes per day. This technology offers a practical approach to directly utilize genome sequence data to generate functional proteomes.

There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited. We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells. This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE). This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (β-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells. Two systems were used to demonstrate that the proteins were delivered in a functionally active form. First, intracellular β-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme. Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis. Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells. This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.

We describe an effective approach using a peptide nucleic acid (PNA) "clamp" to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently tran- scribed. To demonstrate this approach a highly fluorescent preparation of plasmid DNA was generated by hybridizing a fluorescently labeled PNA to the plasmid. Fluorescent plasmid prepared in this way was nei- ther functionally nor conformationally altered. PNA binding was sequence specific, saturable, extremely stadistribution. ble, and did not influence the nucleic acid intracellular This method was utilized for the first time to study the biodistribution of conformationally and functionally intact plasmid DNA in living cells af- ter cationic lipid-mediated transfection. A fluorescent plasmid expressing green fluorescent protein (GFP) en- abled simultaneous colocalization of both plasmid and expressed protein in living cells and in real time. GFP was shown to be expressed in cells containing detectable nuclear fluorescent plasmid. The fluorescent PNA- labeled plasmid revealed a marked difference in the nuclear uptake between oligonucleotide and plasmid, sug- gesting that nuclear entry of plasmid may require cell division. This detection method provides a way to si- multaneously monitor the intracellular localization and expression of plasmid DNA in living cells, and to elucidate the mechanism of plasmid delivery and its nuclear import with synthetic gene delivery systems.

Summary Protocols and techniques are presented describing the use of cationic lipid-mediated transfection, applicable for the functional delivery of plasmid DNA, in vitro-transcribed messenger RNA or small antisense oligonucleotides into cultured cells. Methodology for transfection in microtiter plates and in larger petri dishes is presented. The procedure and reagents are known for their efficacy and simple implementation. The aqueous cationic lipid reagent is mixed with nucleic acid molecules and the complexes are introduced into culture cells under serum-reduced conditions. Ten percent of bovine serum is added after 4 h of incubation with complexes. Cells are harvested and assayed for DNA or RNA expression at 2 days or 12 h, respectively. The technique was originally developed for Lipofectin (DOTMA/DOPE at 1:1 molar ratio, from GIBCO/BRL). Several commercial reagents are available, such as TransfectAce from GIBCO/BRL, Transfectam from Promega, DOTAP from Boeringer-Mannheim, and LipofectAMINE (GIBCO/BRL), and can be used similarly.

ABSTRACTAn important goal of gene therapy for cardiovascular diseases and cancer is the development of effective vectors for catheter-based gene delivery. Although adenoviral vectors have proven effective for this purpose in animal models, the ability to achieve comparable gene transfer with nonviral vectors would provide potentially desirable safety and toxicity features for clinical studies. In this report, we describe the use of a new cationic DNAliposome complex using an improved expression vector and lipid, N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaniminium bromide/dioleoyl phosphatidylethanolamine (GAP-DLRIE/DOPE) to optimize catheter-mediated gene transfer in porcine arteries. The efficiency of this vector was compared to DNA alone, DNA with a previously described cationic liposome complex, ( )-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE/DOPE), and a replication-defective adenoviral vector in a porcine artery gene transfer model. When used in optimal ratios, GAP-DLRIE/DOPE liposomes provided a 15-fold higher level of gene expression in arteries compared to DNA alone or DMRIE/DOPE. Gene expression was observed in intimal and medial cells. However, when compared to adenoviral vectors (1010pfu/ml), gene expression following GAP-DLRIE/DOPE transfection was ~20-fold lower. Following intravenous injection of GAP-DLRIE/DOPE in mice, biochemical, hematological, and histopathological abnormalities were not observed. Significant improvements in the efficacy of arterial gene expression can be achieved by optimization of transfection conditions with DNAliposome complexes in vivothat may prove useful for arterial gene delivery in cardiovascular diseases and cancer.

The hexokinase: fumarase ratios of mitochondria isolated from ten tissues of the rat were determined, and compared with the tissue content of phosphoglucomutase and phosphorylase, taken as representatives of enzymes concerned with glycogen metabolism. A generally inverse relationship was found between the mitochondrial hexokinase: fumarase ratio and phosphoglucomutase levels. The cytochrome: fumarase ratios were relatively invariant in these same mitochondria. The results are interpreted as indicating a specialization of mitochondria, with increased amounts of hexokinase being associated with the mitochondria in tissues exhibiting less dependence on glycogen metabolism, as judged from phosphoglucomutase levels.

A cavitary glioblastoma model was created by injection of RT-2 cells, which express endogenous wild type p53, into the peritoneal cavity of nude mice. This model developed multiple layers of tumor cells invading the peritoneal surface and was used to mimic the postoperative surgical cavity remaining after glioblastoma (GBM) excision in patients. Rhodamine labeled DMRIE/DOPE + DNA complexes were found to penetrate at least 20 tumor cell layers. Injection of p53 gene/liposome complexes into the intraperitoneal cavity after the tumor was established resulted in massive tumor necrosis. Prominent staining of human p53 protein using the DO-1 antibody was found in tumor cells near the necrotic lesions. Tumor explants expressed human p53 protein and showed a 54 % growth reduction in an in vitro growth assay. Further, DMRIE/DOPE mediated p53 gene transfection significantly increased the mean survival time of tumor bearing mice compared to vector control. These results demonstrate the efficiency of using exogenous wild type p53 to suppress glioblastoma cell with endogenous wild type p53 in vivo through liposome mediated transfection method.

ABSTRACTAmong the potential nonviral vectors for human gene therapy are DNAliposome complexes. In a recent clinical study, this delivery system has been utilized. In this report, a novel cationic lipid, dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), has been substituted into the DNAliposome complex with dioleoyl phosphatidylethanolamine (DOPE), which both improves transfection efficiencies and allows increased doses of DNA to be delivered in vivo. The safety and toxicity of this DNAliposome complex has been evaluated in two species, mice and pigs. The efficacy of DMRIE/DOPE in inducing an antitumor response in mice after transfer of a foreign MHC has been confirmed. No abnormalities were detected after administration of up to 1,000-fold higher concentrations of DNA and lipid than could be tolerated in vivopreviously. Examination of serum biochemical enzymes, pathologic examination of tissue, and analysis of cardiac function in mice and pigs revealed no toxicities related to this treatment. This improved cationic lipid formulation is well-tolerated in vivoand could therefore allow higher dose administration and potentially greater efficiency of gene transfer for gene therapy.

A growing number of cationic lipid reagents are being used to transfect tissue culture cells and to direct in vivo delivery of functional gene sequences. Lipofection transfection is a simple method whereby reagent is introduced into a polynucleotide solution, and the mixture is added onto tissue culture cells. We describe the features of this transfection methodology, which is convenient, reproducible, efficient, and effective in a wide variety of tissue culture cell types.Copyright 1993, 1999 Academic Press

ABSTRACTFive analytical assays are described that provide a platform for systematically evaluating the effect of formulation variables on the physical properties of cationic lipidDNA complexes (lipoplexes). The assays are for (i) lipid recovery, (ii) total DNA, (iii) free DNA, (iv) nuclease sensitivity, and (v) physical stability by filtration. Lipid recovery was determined by measuring lipid primary amino groups labeled with the fluorescamine reagent in the presence of the detergent Zwittergent. Zwittergent was effective at disrupting lipoplexes, making the primary amine accessible to the fluorescamine reagent. Total DNA was determined with the PicoGreen reagent, also in the presence of Zwittergent. The PicoGreen assay in the absence of Zwittergent gave the percentage of the total DNA that was not complexed with cationic lipid. The results of this assay for free DNA agreed well with the amount of DNA that could be separated from complexes by centrifugation as well as with the amount of DNA that was accessible to DNase I digestion. Monitoring the lipid and DNA recoveries after filtration through polycarbonate membranes provided a quantitative method for assessing changes in lipoplex physical characteristics. Together, these assays provide a convenient high-throughput approach to assess physical properties of lipoplexes, allowing systematic evaluation of different formulations.

ABSTRACTMaternal antibodies may play a role in protecting newborns against malaria disease. Plasmodium falciparumparasite surface antigens are diverse, and protection from infection requires allele-specific immunity. Although malaria-specific antibodies have been shown to cross the placenta, the extent to which antibodies that respond to the full repertoire of diverse antigens are transferred from the mother to the infant has not been explored. Understanding the breadth of maternal antibody responses and to what extent these antibodies are transferred to the child can inform vaccine design and evaluation. We probed plasma from cord blood and serum from mothers at delivery using a customized protein microarray that included variants of malaria vaccine target antigens to assess the intensity and breadth of seroreactivity to three malaria vaccine candidate antigens in mother-newborn pairs in Malawi. Among the 33 paired specimens that were assessed, mothers and newborns had similar intensity and repertoire of seroreactivity. Maternal antibody levels against vaccine candidate antigens were the strongest predictors of infant antibody levels. Placental malaria did not significantly impair transplacental antibody transfer. However, mothers with placental malaria had significantly higher antibody levels against these blood-stage antigens than mothers without placental malaria. The repertoire and levels of infant antibodies against a wide range of malaria vaccine candidate antigen variants closely mirror maternal levels in breadth and magnitude regardless of evidence of placental malaria. Vaccinating mothers with an effective malaria vaccine during pregnancy may induce high and potentially protective antibody repertoires in newborns.

Individuals that are exposed to malaria eventually develop immunity to the disease with one possible mechanism being the gradual acquisition of antibodies to the range of parasite variant surface antigens in their local area. Major antibody targets include the large and highly polymorphic Plasmodium falciparumErythrocyte Membrane Protein 1 (PfEMP1) family of proteins. Here, we use a protein microarray containing 123 recombinant PfEMP1-DBLα domains (VAR) from Papua New Guinea to seroprofile 38 nonimmune children (<4 years) and 29 hyperimmune adults (≥15 years) from the same local area. The overall magnitude, prevalence and breadth of antibody response to VAR was limited at <2 years and 2–2.9 years, peaked at 3–4 years and decreased for adults compared with the oldest children. An increasing proportion of individuals recognized large numbers of VAR proteins (>20) with age, consistent with the breadth of response stabilizing with age. In addition, the antibody response was limited in uninfected children compared with infected children but was similar in adults irrespective of infection status. Analysis of the variant-specific response confirmed that the antibody signature expands with age and infection. This also revealed that the antibody signatures of the youngest children overlapped substantially, suggesting that they are exposed to the same subset of PfEMP1 variants. VAR proteins were either seroprevalent from early in life, (<3 years), from later in childhood (≥3 years) or rarely recognized. Group 2 VAR proteins (Cys2/MFK-REY+) were serodominant in infants (<1-year-old) and all other sequence subgroups became more seroprevalent with age. The results confirm that the anti-PfEMP1-DBLα antibody responses increase in magnitude and prevalence with age and further demonstrate that they increase in stability and complexity. The protein microarray approach provides a unique platform to rapidly profile variant-specific antibodies to malaria and suggests novel insights into the acquisition of immunity to malaria.

Individuals that are exposed to malaria eventually develop immunity to the disease with one possible mechanism being the gradual acquisition of antibodies to the range of parasite variant surface antigens in their local area. Major antibody targets include the large and highly polymorphic Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of proteins. Here, we use a protein microarray containing 123 recombinant PfEMP1-DBLα domains (VAR) from Papua New Guinea to seroprofile 38 nonimmune children (<4 years) and 29 hyperimmune adults (≥15 years) from the same local area. The overall magnitude, prevalence and breadth of antibody response to VAR was limited at <2 years and 2–2.9 years, peaked at 3–4 years and decreased for adults compared with the oldest children. An increasing proportion of individuals recognized large numbers of VAR proteins (>20) with age, consistent with the breadth of response stabilizing with age. In addition, the antibody response was limited in uninfected children compared with infected children but was similar in adults irrespective of infection status. Analysis of the variant-specific response confirmed that the antibody signature expands with age and infection. This also revealed that the antibody signatures of the youngest children overlapped substantially, suggesting that they are exposed to the same subset of PfEMP1 variants. VAR proteins were either seroprevalent from early in life, (<3 years), from later in childhood (≥3 years) or rarely recognized. Group 2 VAR proteins (Cys2/MFK-REY+) were serodominant in infants (<1-year-old) and all other sequence subgroups became more seroprevalent with age. The results confirm that the anti-PfEMP1-DBLα antibody responses increase in magnitude and prevalence with age and further demonstrate that they increase in stability and complexity. The protein microarray approach provides a unique platform to rapidly profile variant-specific antibodies to malaria and suggests novel insights into the acquisition of immunity to malaria.

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.

The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized.

ABSTRACTDetection of microbial antigens in clinical samples can lead to rapid diagnosis of an infection and administration of appropriate therapeutics. A major barrier in diagnostics development is determining which of the potentially hundreds or thousands of antigens produced by a microbe are actually present in patient samples in detectable amounts against a background of innumerable host proteins. In this report, we describe a strategy, termed in vivomicrobial antigen discovery (InMAD), that we used to identify circulating bacterial antigens. This technique starts with “InMAD serum,” which is filtered serum that has been harvested from BALB/c mice infected with a bacterial pathogen. The InMAD serum, which is free of whole bacterial cells, is used to immunize syngeneic BALB/c mice. The resulting “InMAD immune serum” contains antibodies specific for the soluble microbial antigens present in sera from the infected mice. The InMAD immune serum is then used to probe blots of bacterial lysates or bacterial proteome arrays. Bacterial antigens that are reactive with the InMAD immune serum are precisely the antigens to target in an antigen immunoassay. By employing InMAD, we identified multiple circulating antigens that are secreted or shed during infection using Burkholderia pseudomalleiand Francisella tularensisas model organisms. Potential diagnostic targets identified by the InMAD approach included bacterial proteins, capsular polysaccharide, and lipopolysaccharide. The InMAD technique makes no assumptions other than immunogenicity and has the potential to be a broad discovery platform to identify diagnostic targets from microbial pathogens.IMPORTANCEEffective treatment of microbial infection is critically dependent on early diagnosis and identification of the etiological agent. One means for rapid diagnosis is immunoassay for antigens that are shed into body fluids during infection. Immunoassays can be inexpensive, rapid, and adaptable to a point-of-care format. A major impediment to immunoassay for diagnosis of infectious disease is identification of appropriate antigen targets. This report describes a strategy that can be used for identification of microbial antigens that are shed into serum during infection by the biothreats Burkholderia pseudomalleiand Francisella tularensis. Termed InMAD (in vivomicrobial antigen discovery), the strategy has the potential for application to a broad spectrum of microbial pathogens.

Toxoplasmosis, caused by infection of the protozoan parasite Toxoplasma gondii, is associated with mild disease in healthy individuals, whereas individuals with depressed immunity may develop encephalitis, neurologic disorders, and other organ diseases. Women who develop acute toxoplasmosis during pregnancy are at risk of transmitting the infection to the fetus, which may lead to fetal damage. A diagnosis is usually confirmed by measuring IgG, or IgM where it is important to determine the onset of infection. A negative IgM result essentially excludes acute infection, whereas a positive IgM test is largely uninterpretable because IgM can persist for up to 18 months after infection. To identify antigens for improved diagnosis of acute infection, we probed protein microarrays displaying the polypeptide products of 1357 Toxoplasmaexons with well-characterized sera from Turkey. The sera were classified according to conventional assays into (1) seronegative individuals with no history of T. gondiiinfection; (2) acute infections defined by clinical symptoms, high IgM titers, and low avidity IgG; (3) chronic/convalescent cases with high avidity IgG but persisting IgM; (iv) true chronic infections, defined by high avidity IgG and no IgM. We have identified 38 IgG target antigens and 108 IgM target antigens that can discriminate infected patients from healthy controls, one or more of which could form the basis of a ‘tier-1′ test to determine current or previous exposure. Of these, three IgG antigens and five IgM antigens have the potential to discriminate chronic/IgM persisting or true chronics from recent acutely infected patients (a ‘tier-2′ test). Our analysis of the antigens revealed several enriched features relative to the whole proteome, which include transmembrane domains, signal peptides, or predicted localization at the outer membrane. This is the first protein microarray survey of the antibody response to T. gondii, and will help in the development of improved serodiagnostics and vaccines.

Motivation: We present a study of antigen expression signals from a newly developed high-throughput protein microarray technique. These signals are a measure of antibody–antigen binding activity and provide a basis for understanding humoral immune responses to various infectious agents and supporting vaccine and diagnostic development. Results: We investigate the characteristics of these expression profiles and show that noise models, normalization, variance estimation and differential expression analysis techniques developed in the context of DNA microarray analysis can be adapted and applied to these protein arrays. Using a high-dimensional dataset containing measurements of expression profiles of antibody reactivity against each protein (295 antigens and 9 controls) in 42 malaria (Plasmodium falciparum) protein arrays derived from 22 donors with various clinical presentations of malaria, we present a methodology for the analysis and identification of significantly expressed antigens targeted by immune responses for individual sera, groups of sera and across stages of infection. We also conduct a short study highlighting the top immunoreactive antigens where we identify three novel high priority antigens for future evaluation. Availability: All software programs (in R) used for the analysis described in this paper are freely available for academic purposes at www.igb.uci.edu/servers/servers.html Contact: pfbaldi@uci.edu