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ABSTRACT: 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.
Journal of proteomics 08/2012; · 5.07 Impact Factor
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ABSTRACT: The development of pre-erythrocytic Plasmodium vivax vaccines is hindered by the lack of in vitro culture systems or experimental rodent models. To help bypass these roadblocks, we exploited the fact that naturally exposed Fy- individuals who lack the Duffy blood antigen (Fy) receptor are less likely to develop blood-stage infections; therefore, they preferentially develop immune responses to pre-erythrocytic-stage parasites, whereas Fy+ individuals experience both liver- and blood-stage infections and develop immune responses to both pre-erythrocytic and erythrocytic parasites. We screened 60 endemic sera from P. vivax-exposed Fy+ or Fy- donors against a protein microarray containing 91 P. vivax proteins with P. falciparum orthologs that were up-regulated in sporozoites. Antibodies against 10 P. vivax antigens were identified in sera from P. vivax-exposed individuals but not unexposed controls. This technology has promising implications in the discovery of potential vaccine candidates against P. vivax malaria.
The American journal of tropical medicine and hygiene 07/2012; 87(3):460-9. · 2.59 Impact Factor
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ABSTRACT: Successful vaccination against smallpox with conventional vaccinia virus is usually determined by the development of a vesicular skin lesion at the site of vaccinia inoculation, called a "take." Although previous vaccination is known to be associated with attenuation of the take, the immunology that underlies a no-take in vaccinia-naïve individuals is not well understood. We hypothesized that antibody profiling of individuals before and after receiving vaccinia virus would reveal differences between takes and no-takes that may help better explain the phenomenon. Using vaccinia virus proteome microarrays and recombinant protein enzyme-linked immunosorbent assays (ELISAs), we first examined the antibody response in vaccinia-naïve individuals that failed to take after receiving different doses of the replication-competent DryVax and Aventis Pasteur (APSV) smallpox vaccines. Most that received diluted vaccine failed to respond, although four no-takes receiving diluted vaccine and four receiving undiluted vaccine mounted an antibody response. Interestingly, their antibody profiles were not significantly different from those of controls that did show a take. However, we did find elevated antibody titers in no-takes prior to receiving DryVax that were significantly different from those of takes. Although the sample size studied was small, we conclude the failure to take in responders correlates with preexisting immunity of unknown etiology that may attenuate the skin reaction in a way similar to previous smallpox vaccination.
Clinical and vaccine immunology: CVI 03/2012; 19(3):418-28. · 2.37 Impact Factor
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ABSTRACT: Modified Vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus that is being considered as a safer alternative to replicating vaccinia vaccine strains such as Dryvax(®) and ACAM2000. Its excellent safety profile and large genome also make it an attractive vector for the delivery of heterologous genes from other pathogens. MVA was attenuated by prolonged passage through chick embryonic fibroblasts in vitro. In human and most mammalian cells, production of infectious progeny is aborted in the late stage of infection. Despite this, MVA provides high-level gene expression and is immunogenic in humans and other animals. A key issue for vaccine developers is the ability to be able to monitor an immune response to MVA in both vaccinia naïve and previously vaccinated individuals. To this end we have used antibody profiling by proteome microarray to compare profiles before and after MVA and Dryvax vaccination to identify candidate serodiagnostic antigens. Six antigens with diagnostic utility, comprising three membrane and three non-membrane proteins from the intracellular mature virion, were purified and evaluated in ELISAs. The membrane protein WR113/D8L provided the best sensitivity and specificity of the six antigens tested for monitoring both MVA and Dryvax vaccination, whereas the A-type inclusion protein homolog, WR148, provided the best discrimination. The ratio of responses to membrane protein WR132/A13L and core protein WR070/I1L also provided good discrimination between primary and secondary responses to Dryvax, whereas membrane protein WR101/H3L and virion assembly protein WR118/D13L together provided the best sensitivity for detecting antibody in previously vaccinated individuals. These data will aid the development novel MVA-based vaccines.
Vaccine 11/2011; 30(3):614-25. · 3.77 Impact Factor
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Obinna N Nnedu,
Michael P O'Leary,
Daniel Mutua,
Beth Mutai,
Mina Kalantari-Dehaghi,
Al Jasinskas,
Rie Nakajima-Sasaki,
Grace John-Stewart,
Phelgona Otieno, Xiaowu Liang,
John Waitumbi,
Francis Kimani,
David Camerini,
Philip L Felgner,
Judd L Walson,
Adam Vigil
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ABSTRACT: Humoral immune responses play a pivotal role in naturally acquired immunity to malaria. Understanding which humoral responses are impaired among individuals at higher risk for malaria may improve our understanding of malaria immune control and contribute to vaccine development.
We compared humoral responses with 483 Plasmodium falciparum antigens between adults in, Kisumu (high, year-long malaria transmission leading to partial immunity), and adults in Kisii (low, seasonal malaria transmission). Then within each site, we compared malaria-specific humoral responses between those at higher risk for malaria (CD4(+) ≤500) and those at lower risk for malaria (CD4(+) >500). A protein microarray chip containing 483 P. falciparum antigens and 71 HIV antigens was used. Benjamini-Hochberg adjustments were made to control for multiple comparisons.
Fifty-seven antigens including CSP, MSP1, LSA1 and AMA1 were identified as significantly more reactive in Kisumu than in Kisii. Ten of these antigens had been identified as protective in an earlier study. CD4(+) T-cell count did not significantly impact humoral responses.
Protein microarrays are a useful method to screen multiple humoral responses simultaneously. This study provides useful clues for potential vaccine candidates. Modest decreases in CD4 counts may not significantly impact malaria-specific humoral immunity.
PROTEOMICS - CLINICAL APPLICATIONS 09/2011; 5(11-12):613-23. · 1.81 Impact Factor
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Li Liang,
Xiaolin Tan,
Silvia Juarez,
Homarh Villaverde,
Jozelyn Pablo,
Rie Nakajima-Sasaki,
Eduardo Gotuzzo,
Mayuko Saito,
Gary Hermanson,
Douglas Molina,
Scott Felgner,
W John W Morrow, Xiaowu Liang,
Robert H Gilman,
D Huw Davies,
Renée M Tsolis,
Joseph M Vinetz,
Philip L Felgner
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ABSTRACT: A complete understanding of the factors that determine selection of antigens recognized by the humoral immune response following infectious agent challenge is lacking. Here we illustrate a systems biology approach to identify the antibody signature associated with Brucella melitensis (Bm) infection in humans and predict proteomic features of serodiagnostic antigens. By taking advantage of a full proteome microarray expressing previously cloned 1406 and newly cloned 1640 Bm genes, we were able to identify 122 immunodominant antigens and 33 serodiagnostic antigens. The reactive antigens were then classified according to annotated functional features (COGs), computationally predicted features (e.g., subcellular localization, physical properties), and protein expression estimated by mass spectrometry (MS). Enrichment analyses indicated that membrane association and secretion were significant enriching features of the reactive antigens, as were proteins predicted to have a signal peptide, a single transmembrane domain, and outer membrane or periplasmic location. These features accounted for 67% of the serodiagnostic antigens. An overlay of the seroreactive antigen set with proteomic data sets generated by MS identified an additional 24%, suggesting that protein expression in bacteria is an additional determinant in the induction of Brucella-specific antibodies. This analysis indicates that one-third of the proteome contains enriching features that account for 91% of the antigens recognized, and after B. melitensis infection the immune system develops significant antibody titers against 10% of the proteins with these enriching features. This systems biology approach provides an empirical basis for understanding the breadth and specificity of the immune response to B. melitensis and a new framework for comparing the humoral responses against other microorganisms.
Journal of Proteome Research 08/2011; 10(10):4813-24. · 5.11 Impact Factor
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ABSTRACT: We performed partial evaluation of pemphigus vulgaris (PV) autoantibody profile using the protein array technology. The sera from seven patients with acute PV and five healthy donors were probed for the presence of autoantibodies characteristic of the organ-non-specific autoimmune disorders rheumatoid arthritis, lupus erythematosus, scleroderma, diabetes and some other autoimmune disorders, but not to desmosomal proteins. The array targeted 785 human genes amplified using Mammalian Gene Clone Collection with gene-specific primers containing 20-bp nucleotide extension complementary to ends of linear pXT7 vector. The array identified PV antibodies significantly (P<0.05) differentially reactive with 16 antigens, most of which were cell-surface proteins, such as CD2, CD31, CD33, CD36, CD37, CD40, CD54, CD66c and CD84 molecules, nicotinamide/nicotinic acid mononucleotide adenylyltransferase, immunoglobulin heavy chain constant region gamma 2 and others. Reactivity with Fc-IgG helps explain an ability of the chimeric desmoglein constructs to absorb out all disease-causing PV antibodies. Anti-M(1) muscarinic receptor antibody was also identified, consistent with the facts that while blockade of this receptor causes keratinocyte detachment, its activation is therapeutic in PV. Further proteomics analysis of PV antibodies should help elucidate the immunopathogenic mechanisms underlying keratinocyte detachment and blistering.
Experimental Dermatology 02/2011; 20(2):154-6. · 3.54 Impact Factor
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Maria I Cruz-Fisher,
Chunmei Cheng,
Guifeng Sun,
Sukumar Pal,
Andy Teng,
Douglas M Molina,
Matthew A Kayala,
Adam Vigil,
Pierre Baldi,
Philip L Felgner, Xiaowu Liang,
Luis M de la Maza
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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.
Infection and immunity 10/2010; 79(1):246-57. · 4.21 Impact Factor
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Shajo Kunnath-Velayudhan,
Hugh Salamon,
Hui-Yun Wang,
Amy L Davidow,
Douglas M Molina,
Vu T Huynh,
Daniela M Cirillo,
Gerd Michel,
Elizabeth A Talbot,
Mark D Perkins,
Philip L Felgner, Xiaowu Liang,
Maria L Gennaro
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ABSTRACT: Considerable effort has been directed toward controlling tuberculosis, which kills almost two million people yearly. High on the research agenda is the discovery of biomarkers of active tuberculosis (TB) for diagnosis and for monitoring treatment outcome. Rational biomarker discovery requires understanding host-pathogen interactions leading to biomarker expression. Here we report a systems immunology approach integrating clinical data and bacterial metabolic and regulatory information with high-throughput detection in human serum of antibodies to the entire Mycobacterium tuberculosis proteome. Sera from worldwide TB suspects recognized approximately 10% of the bacterial proteome. This result defines the M. tuberculosis immunoproteome, which is rich in membrane-associated and extracellular proteins. Additional analyses revealed that during active tuberculosis (i) antibody responses focused on an approximately 0.5% of the proteome enriched for extracellular proteins, (ii) relative target preference varied among patients, and (iii) responses correlated with bacillary burden. These results indicate that the B cell response tracks the evolution of infection and the pathogen burden and replicative state and suggest functions associated with B cell-rich foci seen in tuberculous lung granulomas. Our integrated proteome-scale approach is applicable to other chronic infections characterized by diverse antibody target recognition.
Proceedings of the National Academy of Sciences 08/2010; 107(33):14703-8. · 9.68 Impact Factor
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ABSTRACT: 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.
Vaccine 04/2010; 28(17):3014-24. · 3.77 Impact Factor
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03/2010; , ISBN: 9780470571224
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Peter D Crompton,
Matthew A Kayala,
Boubacar Traore,
Kassoum Kayentao,
Aissata Ongoiba,
Greta E Weiss,
Douglas M Molina,
Chad R Burk,
Michael Waisberg,
Algis Jasinskas, [......],
Didier Doumtabe,
Younoussou Kone,
David L Narum, Xiaowu Liang,
Ogobara K Doumbo,
Louis H Miller,
Denise L Doolan,
Pierre Baldi,
Philip L Felgner,
Susan K Pierce
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ABSTRACT: Abs are central to malaria immunity, which is only acquired after years of exposure to Plasmodium falciparum (Pf). Despite the enormous worldwide burden of malaria, the targets of protective Abs and the basis of their inefficient acquisition are unknown. Addressing these knowledge gaps could accelerate malaria vaccine development. To this end, we developed a protein microarray containing approximately 23% of the Pf 5,400-protein proteome and used this array to probe plasma from 220 individuals between the ages of 2-10 years and 18-25 years in Mali before and after the 6-month malaria season. Episodes of malaria were detected by passive surveillance over the 8-month study period. Ab reactivity to Pf proteins rose dramatically in children during the malaria season; however, most of this response appeared to be short-lived based on cross-sectional analysis before the malaria season, which revealed only modest incremental increases in Ab reactivity with age. Ab reactivities to 49 Pf proteins measured before the malaria season were significantly higher in 8-10-year-old children who were infected with Pf during the malaria season but did not experience malaria (n = 12) vs. those who experienced malaria (n = 29). This analysis also provided insight into patterns of Ab reactivity against Pf proteins based on the life cycle stage at which proteins are expressed, subcellular location, and other proteomic features. This approach, if validated in larger studies and in other epidemiological settings, could prove to be a useful strategy for better understanding fundamental properties of the human immune response to Pf and for identifying previously undescribed vaccine targets.
Proceedings of the National Academy of Sciences 03/2010; 107(15):6958-63. · 9.68 Impact Factor
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Li Liang,
Diana Leng,
Chad Burk,
Rie Nakajima-Sasaki,
Matthew A Kayala,
Vidya L Atluri,
Jozelyn Pablo,
Berkay Unal,
Thomas A Ficht,
Eduardo Gotuzzo,
Mayuko Saito,
W John W Morrow, Xiaowu Liang,
Pierre Baldi,
Robert H Gilman,
Joseph M Vinetz,
Renée M Tsolis,
Philip L Felgner
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ABSTRACT: 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.
PLoS Neglected Tropical Diseases 01/2010; 4(5):e673. · 4.69 Impact Factor
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Denise L Doolan,
Yunxiang Mu,
Berkay Unal,
Suman Sundaresh,
Siddiqua Hirst,
Conrad Valdez,
Arlo Randall,
Douglas Molina, Xiaowu Liang,
Daniel A Freilich,
J Aggrey Oloo,
Peter L Blair,
Joao C Aguiar,
Pierre Baldi,
D Huw Davies,
Philip L Felgner
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ABSTRACT: A complete description of the serological response following exposure of humans to complex pathogens is lacking and approaches suitable for accomplishing this are limited. Here we report, using malaria as a model, a method which elucidates the profile of antibodies that develop after natural or experimental infection or after vaccination with attenuated organisms, and which identifies immunoreactive antigens of interest for vaccine development or other applications. Expression vectors encoding 250 Plasmodium falciparum (Pf) proteins were generated by PCR/recombination cloning; the proteins were individually expressed with >90% efficiency in Escherichia coli cell-free in vitro transcription and translation reactions, and printed directly without purification onto microarray slides. The protein microarrays were probed with human sera from one of four groups which differed in immune status: sterile immunity or no immunity against experimental challenge following vaccination with radiation-attenuated Pf sporozoites, partial immunity acquired by natural exposure, and no previous exposure to Pf. Overall, 72 highly reactive Pf antigens were identified. Proteomic features associated with immunoreactivity were identified. Importantly, antibody profiles were distinct for each donor group. Information obtained from such analyses will facilitate identifying antigens for vaccine development, dissecting the molecular basis of immunity, monitoring the outcome of whole-organism vaccine trials, and identifying immune correlates of protection.
Proteomics 10/2008; 8(22):4680-94. · 4.43 Impact Factor
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David P Regis,
Carlota Dobaño,
Paola Quiñones-Olson, Xiaowu Liang,
Norma L Graber,
Maureen E Stefaniak,
Joseph J Campo,
Daniel J Carucci,
David A Roth,
Huaping He,
Philip L Felgner,
Denise L Doolan
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ABSTRACT: We have evaluated a technology called transcriptionally active PCR (TAP) for high throughput identification and prioritization of novel target antigens from genomic sequence data using the Plasmodium parasite, the causative agent of malaria, as a model. First, we adapted the TAP technology for the highly AT-rich Plasmodium genome, using well-characterized P. falciparum and P. yoelii antigens and a small panel of uncharacterized open reading frames from the P. falciparum genome sequence database. We demonstrated that TAP fragments encoding six well-characterized P. falciparum antigens and five well-characterized P. yoelii antigens could be amplified in an equivalent manner from both plasmid DNA and genomic DNA templates, and that uncharacterized open reading frames could also be amplified from genomic DNA template. Second, we showed that the in vitro expression of the TAP fragments was equivalent or superior to that of supercoiled plasmid DNA encoding the same antigen. Third, we evaluated the in vivo immunogenicity of TAP fragments encoding a subset of the model P. falciparum and P. yoelii antigens. We found that antigen-specific antibody and cellular immune responses induced by the TAP fragments in mice were equivalent or superior to those induced by the corresponding plasmid DNA vaccines. Finally, we developed and demonstrated proof-of-principle for an in vitro humoral immunoscreening assay for down-selection of novel target antigens. These data support the potential of a TAP approach for rapid high throughput functional screening and identification of potential candidate vaccine antigens from genomic sequence data.
Molecular and Biochemical Parasitology 04/2008; 158(1):32-45. · 2.55 Impact Factor
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D Huw Davies,
Douglas M Molina,
Jens Wrammert,
Joe Miller,
Siddiqua Hirst,
Yunxiang Mu,
Jozelyn Pablo,
Berkay Unal,
Rie Nakajima-Sasaki, Xiaowu Liang,
Shane Crotty,
Kevin L Karem,
Inger K Damon,
Rafi Ahmed,
Luis Villarreal,
Philip L Felgner
[show abstract]
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ABSTRACT: The eradication of smallpox by vaccination with vaccinia virus was probably one of the greatest achievements of vaccinology. However, the immunological basis of this protection is not fully understood. To this end, we have used protein microarrays of the vaccinia (Western Reserve, WR) proteome to profile antibody reactivities after primary infection or boosting with the licensed smallpox vaccine, Dryvax, and with archival convalescent smallpox sera. Some 25 antigens were consistently recognized by Dryvax sera, of which half were envelope proteins (notably, H3, A13, B5, and D8). The remainder consisted mainly of core proteins (e.g. A10, L4, and I1), proteins involved in intracellular morphogenesis (A11, D13), and the A-type inclusion protein, WR148. Convalescent smallpox sera also detected vaccinia antigens on the array, consistent with the notion that there is serological cross-reactivity between these two orthopox species that underlies protection. Moreover, the profiles of immunodominant antigens recognized by variola-infected individuals and Dryvax vaccinees were indistinguishable. This is the first description of antibody-specificity profiles induced after smallpox infection. The array data indicate that a significant component of the antibody response is not involved in virus neutralization, although these antigens should be considered alongside the envelope proteins as potential candidates for diagnostic and vaccine applications.
PROTEOMICS 06/2007; 7(10):1678-86. · 4.51 Impact Factor
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ABSTRACT: In this chapter, we describe an 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 transcribed. This strategy enables
investigators to “functionalize” their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars
or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to
circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery.
The proof-of-principle of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed
generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA
was used to identify critical parameters involved in naked DNA and nonviral gene delivery tech-nology. The greatest potential
of PDGC lies in the ability to attach specific ligands (e.g., pep-tides, proteins) to the plasmid DNA in order to overcome
cellular barriers of nonviral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described
and their effect on increasing the efficacy of gene therapy is presented.
12/2005: pages 195-211;
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D Huw Davies, Xiaowu Liang,
Jenny E Hernandez,
Arlo Randall,
Siddiqua Hirst,
Yunxiang Mu,
Kimberly M Romero,
Toai T Nguyen,
Mina Kalantari-Dehaghi,
Shane Crotty,
Pierre Baldi,
Luis P Villarreal,
Philip L Felgner
[show abstract]
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ABSTRACT: Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naive humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naive mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.
Proceedings of the National Academy of Sciences 02/2005; 102(3):547-52. · 9.68 Impact Factor
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ABSTRACT: An approach is described for making transcriptionally active PCR (TAP) fragments that were used directly in in vitro and in vivo expression 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 vivo homologous 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.
Journal of Biological Chemistry 03/2002; 277(5):3593-8. · 4.77 Impact Factor
