Andy Teng

Identification of immunodominant antigens of Chlamydia trachomatis using proteome microarrays.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen in the world. In order to control this infection there is an urgent need to formulate a vaccine. Identification of protective antigens is required to implement a subunit vaccine. To identify potential antigen vaccine candidates, three strains of mice, BALB/c, C3H/HeN and C57BL/6, were inoculated with live and inactivated C. trachomatis mouse pneumonitis (MoPn) by different routes of immunization. Using a protein microarray, serum samples collected after immunization were tested for the presence of antibodies against specific chlamydial antigens. A total of 225 open reading frames (ORF) of the C. trachomatis genome were cloned, expressed, and printed in the microarray. Using this protein microarray, a total of seven C. trachomatis dominant antigens were identified (TC0052, TC0189, TC0582, TC0660, TC0726, TC0816 and, TC0828) as recognized by IgG antibodies from all three strains of animals after immunization. In addition, the microarray was probed to determine if the antibody response exhibited a Th1 or Th2 bias. Animals immunized with live organisms mounted a predominant Th1 response against most of the chlamydial antigens while mice immunized with inactivated Chlamydia mounted a Th2-biased response. In conclusion, using a high throughput protein microarray we have identified a set of novel proteins that can be tested for their ability to protect against a chlamydial infection.

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

ABSTRACT 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.

Proteomic identification of immunodominant chlamydial antigens in a mouse model

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen in the world. To identify new vaccine candidates a protein microarray was constructed by expressing the open reading frames (ORFs) from Chlamydia mouse pneumonitis (MoPn). C57BL/6, C3H/HeN and BALB/c mice were immunized either intranasally or intravaginally with live MoPn elementary bodies (EB). Two additional groups were immunized by the intramuscular plus subcutaneous routes with UV-treated EB, using CpG and Montanide as adjuvants to favor a Th1 response, or Alum, to elicit a Th2 response. Serum samples collected from the three strains of mice were tested in the microarray. The array included the expression of 909 proteins from the 921 ORFs of the MoPn genome and plasmid. A total of 530 ORFs were recognized by at least one serum sample. Of these, 36 reacted with sera from the three strains of mice immunized with live EB. These antigens included proteins that were previously described as immunogenic such as MOMP and HSP60. In addition, we uncovered new immunogens, including 11 hypothetical proteins. In summary, we have identified new immunodominant chlamydial proteins that can be tested for their ability to induce protection in animal models and subsequently in humans.

Whole genome identification of C. trachomatis immunodominant antigens after genital tract infections and effect of antibiotic treatment of pigtailed macaques

UNLABELLED The cervix and/or fallopian tubes of pigtailed macaques were experimentally infected with Chlamydia trachomatis. Their sera were collected at varying time points and screened for identification of immunodominant antigens using a whole-genome protein microarray. The effect of doxycycline treatment on the antibody response generated in these macaques was also investigated. Twenty-five female macaques were infected with C. trachomatis serovars D or E in the cervix and/or fallopian tubes. Bloods were collected at baseline and at various intervals after challenge. Serum samples were tested for antibodies using a C. trachomatis serovar D protein microarray. Twenty chlamydial antigens reacted with sera from at least 68% (17/25) of the macaques. In addition to some well-known chlamydial antigens, nine different proteins, not previously recognized as immunodominant, including four hypothetical proteins (CT005, CT066, CT360 and CT578), were identified. Monkeys infected in the fallopian tubes developed a more robust antibody response than animals inoculated in the cervix. Treatment with doxycycline significantly decreased Chlamydia-specific antibody levels. In summary, using protein microarray serum samples from experimentally infected pigtailed macaques were screened for immunodominant chlamydial antigens. These antigens can now be tested in animal models for their ability to protect and as markers of disease progression. BIOLOGICAL SIGNIFICANCE This is the first time that Chlamydia trachomatis immunodominant antigens have been identified in pigtailed macaques following a uterine cervix or a fallopian tubes infection. These immunodominant antigens can now be used to vaccinate non-human primates and determine their ability to protect against a C. trachomatis genital infection. Proteins that are protective can subsequently be tested in humans. Amongst the immunodominant antigens some were predominantly recognized by sera from macaques inoculated in the fallopian tubes rather than in the cervix and therefore, may be markers for upper genital tract pathology. In addition, treatment with doxycycline following infection significantly decreased Chlamydia-specific antibody levels. This information can be used to evaluate the efficacy of antibiotic treatment and potentially susceptibility to reinfection.

Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes.Naturally acquired antibodies may inhibit Plasmodium survival in mosquitoes, alter malaria transmission dynamics, and form the basis for transmission-blocking vaccines. Here, using sera from malaria-exposed individuals, Stone et al. reveal novel antibody correlates of transmission-reducing activity.

A targeted immunomic approach identifies diagnostic antigens in the human pathogen Babesia microti

Babesia microti is a protozoan parasite responsible for the majority of reported cases of human babesiosis and a major risk to the blood supply. Laboratory screening of blood donors may help prevent transfusion‐transmitted babesiosis but there is no Food and Drug Administration–approved screening method yet available. Development of a sensitive, specific, and highly automated B. microti antibody assay for diagnosis of acute babesiosis and blood screening could have an important impact on decreasing the health burden of B. microti infection.

Assessment of the role in protection and pathogenesis of the Chlamydia muridarum V-type ATP synthase subunit A (AtpA) (TC0582)

A novel Chlamydia muridarum antigen (TC0582) was used to vaccinate BALB/c mice. Mice were also immunized with other components of the ATP synthase complex (TC0580, TC0581, and TC0584), or with the major outer membrane protein (MOMP). TC0582 was also formulated in combination with TC0580, TC0581 or MOMP. TC0582 alone, or in combination with the other antigens, elicited strong Chlamydia-specific humoral and cellular immune responses. Vaccinated animals were challenged intranasally and the course of the infection was followed for 10 days. Based on percentage change in body weight, lung weight, and number of Chlamydia inclusion forming units recovered from the lungs, mice immunized with TC0582, TC0581 or MOMP, as single antigens, showed significant protection. Mice immunized with combinations of two antigens were also protected but the level of protection was not additive. TC0582 has sequence homology with the eukaryotic ATP synthase subunit A (AtpA). Therefore, to determine if immunization with TC0582, or with Chlamydia, elicited antibodies that cross-reacted with the mouse AtpA, the two proteins were printed on a microarray. Sera from mice immunized with TC0582 and/or live Chlamydia, strongly reacted with TC0582 but did not recognize the mouse AtpA. In conclusion, TC0582 may be considered as a Chlamydia vaccine candidate.

Identification of Novel Serodiagnostic Signatures of Typhoid Fever Using a Salmonella Proteome Array

Current diagnostic tests for typhoid fever, the disease caused by Salmonella Typhi, are poor. We aimed to identify serodiagnostic signatures of typhoid fever by assessing microarray signals to 4,445 S. Typhi antigens in sera from 41 participants challenged with oral S. Typhi. We found broad, heterogeneous antibody responses with increasing IgM/IgA signals at diagnosis. In down-selected 250-antigen arrays we validated responses in a second challenge cohort (n = 30), and selected diagnostic signatures using machine learning and multivariable modeling. In four models containing responses to antigens including flagellin, OmpA, HlyE, sipC, and LPS, multi-antigen signatures discriminated typhoid (n = 100) from other febrile bacteremia (n = 52) in Nepal. These models contained combinatorial IgM, IgA, and IgG responses to 5 antigens (ROC AUC, 0.67 and 0.71) or 3 antigens (0.87), although IgA responses to LPS also performed well (0.88). Using a novel systematic approach we have identified and validated optimal serological diagnostic signatures of typhoid fever.

Identification of a novel, EBV-based antibody risk stratification signature for early detection of nasopharyngeal carcinoma in Taiwan

Background. Epstein–Barr virus (EBV) is necessary for the development of nasopharyngeal carcinoma (NPC). By adulthood, approximately 90% of individuals test EBV-positive, but only a fraction develop cancer. Factors that identify which individuals are most likely to develop disease, including differential antibody response to the virus, could facilitate detection at early stages when treatment is most effective. Methods. We measured anti-EBV IgG and IgA antibody responses in 607 Taiwanese individuals. Antibodies were measured using a custom protein microarray targeting 199 sequences from 86 EBV proteins. Variation in response patterns between NPC cases and controls was used to develop an antibody-based risk score for predicting NPC. The overall accuracy [area under the curve (AUC)] of this risk score, and its performance relative to currently used biomarkers, was evaluated in two independent Taiwanese cohorts. Findings. Levels of 60 IgA and 73 IgG anti-EBV antibodies differed between stage I/IIa NPC cases and controls (P < 0.0002). Risk prediction analyses identified antibody targets that best discriminated NPC status—BXLF1, LF2,BZLF1, BRLF1, EAd, BGLF2, BPLF1, BFRF1, and BORF1. When combined with currently used VCA/EBNA1 IgA biomarkers, the resulting risk score predicted NPC with 93% accuracy (95% CI, 87%–98%) in the general Taiwanese population, a significant improvement beyond current biomarkers alone (82%; 95% CI, 75%–90%, P ≤ 0.01). This EBV-based risk score also improved NPC prediction in genetically high-risk families (89%; 95% CI, 82%–96%) compared with current biomarkers (78%; 95% CI, 66%–90%, P ≤ 0.03). Interpretation. We identified NPC-related differences in 133 anti-EBV antibodies and developed a risk score using this microarray dataset that targeted immune responses against EBV proteins from all stages of the viral life cycle, significantly improving the ability to predict NPC. Clin Cancer Res; 24(6); 1305–14. ©2017 AACR.

Genome-wide profiling of humoral immunity and pathogen genes under selection identifies immune evasion tactics of Chlamydia trachomatis during ocular infection.

The frequency and duration of Chlamydia trachomatis (Ct) ocular infections decrease with age, suggesting development of partial immunity. However, there is a lack of clear correlates of immunity to Ct infection in humans. We screened sera from a cohort of Gambian children followed for six-months against a Ct-proteome microarray. At genome sequence level, we detected signatures of selection from a population of ocular Ct isolates from Guinea-Bissau. Together these approaches allowed us to highlight the focus of humoral responses and hypothesise new modes of pathogen immune evasion. Children who were susceptible to frequent and/or prolonged Ct infection had a less focussed antibody response, including preferential recognition of forty-two antigens. There was evidence of positive and purifying selection across the genome, but little balancing selection. In contrast, most antigens that were associated with susceptibility were under neutral selection. These data suggest an evasion strategy in which Ct presents a large panel of irrelevant antigens to the immune system to block or misdirect protective responses. Development of a focused immune response, possibly induced through vaccination, may be an effective strategy to promote protection to Ct infection.