Large Scale Immune Profiling of Infected Humans and Goats Reveals Differential Recognition of Brucella melitensis Antigens

Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host

Serological Profiling of a Candida albicans Protein Microarray Reveals Permanent Host-Pathogen Interplay and Stage-Specific Responses during Candidemia

Candida albicans in the immunocompetent host is a benign member of the human microbiota. Though, when host physiology is disrupted, this commensal-host interaction can degenerate and lead to an opportunistic infection. Relatively little is known regarding the dynamics of C. albicans colonization and pathogenesis. We developed a C. albicans cell surface protein microarray to profile the immunoglobulin G response during commensal colonization and candidemia. The antibody response from the sera of patients with candidemia and our negative control groups indicate that the immunocompetent host exists in permanent host-pathogen interplay with commensal C. albicans. This report also identifies cell surface antigens that are specific to different phases (i.e. acute, early and mid convalescence) of candidemia. We identified a set of thirteen cell surface antigens capable of distinguishing acute candidemia from healthy individuals and uninfected hospital patients with commensal colonization. Interestingly, a large proportion of these cell surface antigens are involved in either oxidative stress or drug resistance. In addition, we identified 33 antigenic proteins that are enriched in convalescent sera of the candidemia patients. Intriguingly, we found within this subset an increase in antigens associated with heme-associated iron acquisition. These findings have important implications for the mechanisms of C. albicans colonization as well as the development of systemic infection

High-throughput prediction of protein antigenicity using protein microarray data

Motivation: Discovery of novel protective antigens is fundamental to the development of vaccines for existing and emerging pathogens. Most computational methods for predicting protein antigenicity rely directly on homology with previously characterized protective antigens; however, homology-based methods will fail to discover truly novel protective antigens. Thus, there is a significant need for homology-free methods capable of screening entire proteomes for the antigens most likely to generate a protective humoral immune response

A Genome-Wide Proteome Array Reveals a Limited Set of Immunogens in Natural Infections of Humans and White-Footed Mice with Borrelia burgdorferi▿ †

Humans 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. burgdorferi in 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 leucopus mice, a major reservoir. The high-ranking set included several B. burgdorferi proteins 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. burgdorferi do not elicit antibody responses during infection and that the limited sets of immunogens are similar for two different host species

Mitochondrial Akt Signaling Modulated Reprogramming of Somatic Cells.

The signaling mechanisms controlling somatic cell reprogramming are not fully understood. In this study, we report a novel role for mitochondrial Akt1 signaling that enhanced somatic cell reprogramming efficiency. The role of mitochondrial Akt1 in somatic cell reprogramming was investigated by transducing fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, c-Myc) in conjunction with Mito-Akt1, Mito-dnAkt1, or control virus. Mito-Akt1 enhanced reprogramming efficiency whereas Mito-dnAkt1 inhibited reprogramming. The resulting iPSCs formed embryoid bodies in vitro and teratomas in vivo. Moreover, Oct4 and Nanog promoter methylation was reduced in the iPSCs generated in the presence of Mito-Akt1. Akt1 was activated and translocated into mitochondria after growth factor stimulation in embryonic stem cells (ESCs). To study the effect of mitochondrial Akt in ESCs, a mitochondria-targeting constitutively active Akt1 (Mito-Akt1) was expressed in ESCs. Gene expression profiling showed upregulation of genes that promote stem cell proliferation and survival and down-regulation of genes that promote differentiation. Analysis of cellular respiration indicated similar metabolic profile in the resulting iPSCs and ESCs, suggesting comparable bioenergetics. These findings showed that activation of mitochondrial Akt1 signaling was required during somatic cell reprogramming

Sterile protective immunity to malaria is associated with a panel of novel P. falciparum antigens

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 immunizationwith 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

Identification of Immunodominant Antigens by Probing a Whole Chlamydia trachomatis Open Reading Frame Proteome Microarray Using Sera from Immunized Mice ▿ †

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