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Find video protocols related to scientific articles indexed in Pubmed.
T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and nonvirion association.
J. Immunol.
PUBLISHED: 07-14-2014
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Vaccinia virus (VACV) is a useful model system for understanding the immune response to a complex pathogen. Proteome-wide Ab profiling studies reveal the humoral response to be strongly biased toward virion-associated Ags, and several membrane proteins induce Ab-mediated protection against VACV challenge in mice. Some studies have indicated that the CD4 response is also skewed toward proteins with virion association, whereas the CD8 response is more biased toward proteins with early expression. In this study, we have leveraged a VACV strain Western Reserve (VACV-WR) plasmid expression library, produced previously for proteome microarrays for Ab profiling, to make a solubilized full VACV-WR proteome for T cell Ag profiling. Splenocytes from VACV-WR-infected mice were assayed without prior expansion against the soluble proteome in assays for Th1 and Th2 signature cytokines. The response to infection was polarized toward a Th1 response, with the distribution of reactive T cell Ags comprising both early and late VACV proteins. Interestingly, the proportions of different functional subsets were similar to that present in the whole proteome. In contrast, the targets of Abs from the same mice were enriched for membrane and other virion components, as described previously. We conclude that a "nonbiasing" approach to T cell Ag discovery reveals a T cell Ag profile in VACV that is broader and less skewed to virion association than the Ab profile. The T cell Ag mapping method developed in the present study should be applicable to other organisms where expressible "ORFeome" libraries are also available, and it is readily scalable for larger pathogens.
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Whole genome identification of C. trachomatis immunodominant antigens after genital tract infections and effect of antibiotic treatment of pigtailed macaques.
J Proteomics
PUBLISHED: 05-03-2014
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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.
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Identification of seroreactive proteins of Leptospira interrogans serovar copenhageni using a high-density protein microarray approach.
PLoS Negl Trop Dis
PUBLISHED: 10-01-2013
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Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease.
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Assessment of the role in protection and pathogenesis of the Chlamydia muridarum V-type ATP synthase subunit A (AtpA) (TC0582).
Microbes Infect.
PUBLISHED: 06-11-2013
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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.
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Protein Microarray Analysis of Antibody Responses to Plasmodium falciparum in Western Kenyan Highland Sites with Differing Transmission Levels.
PLoS ONE
PUBLISHED: 01-01-2013
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Malaria represents a major public health problem in Africa. In the East African highlands, the high-altitude areas were previously considered too cold to support vector population and parasite transmission, rendering the region particularly prone to epidemic malaria due to the lack of protective immunity of the population. Since the 1980s, frequent malaria epidemics have been reported and these successive outbreaks may have generated some immunity against Plasmodium falciparum amongst the highland residents. Serological studies reveal indirect evidence of human exposure to the parasite, and can reliably assess prevalence of exposure and transmission intensity in an endemic area. However, the vast majority of serological studies of malaria have been, hereto, limited to a small number of the parasites antigens. We surveyed and compared the antibody response profiles of age-stratified sera from residents of two endemic areas in the western Kenyan highlands with differing malaria transmission intensities, during two distinct seasons, against 854 polypeptides of P. falciparum using high-throughput proteomic microarray technology. We identified 107 proteins as serum antibody targets, which were then characterized for their gene ontology biological process and cellular component of the parasite, and showed significant enrichment for categories related to immune evasion, pathogenesis and expression on the hosts cell and parasites surface. Additionally, we calculated age-fitted annual seroconversion rates for the immunogenic proteins, and contrasted the age-dependent antibody acquisition for those antigens between the two sampling sites. We observed highly immunogenic antigens that produce stable antibody responses from early age in both sites, as well as less immunogenic proteins that require repeated exposure for stable responses to develop and produce different seroconversion rates between sites. We propose that a combination of highly and less immunogenic proteins could be used in serological surveys to detect differences in malaria transmission levels, distinguishing sites of unstable and stable transmission.
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Humoral immune responses to Plasmodium falciparum among HIV-1-infected Kenyan adults.
Proteomics Clin Appl
PUBLISHED: 09-30-2011
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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.
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Systems biology approach predicts antibody signature associated with Brucella melitensis infection in humans.
J. Proteome Res.
PUBLISHED: 09-08-2011
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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.
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Measurement of antibody responses to Modified Vaccinia virus Ankara (MVA) and Dryvax(®) using proteome microarrays and development of recombinant protein ELISAs.
Vaccine
PUBLISHED: 06-11-2011
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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.
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New targets of pemphigus vulgaris antibodies identified by protein array technology.
Exp. Dermatol.
PUBLISHED: 01-25-2011
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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.
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Identification of immunodominant antigens by probing a whole Chlamydia trachomatis open reading frame proteome microarray using sera from immunized mice.
Infect. Immun.
PUBLISHED: 10-18-2010
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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.
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Dynamic antibody responses to the Mycobacterium tuberculosis proteome.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-28-2010
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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.
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A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-29-2010
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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.
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Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensis antigens.
PLoS Negl Trop Dis
PUBLISHED: 03-19-2010
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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.
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Identification of immunodominant antigens of Chlamydia trachomatis using proteome microarrays.
Vaccine
PUBLISHED: 08-23-2009
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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.
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Proteomic identification of immunodominant chlamydial antigens in a mouse model.
J Proteomics
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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.
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Plasmodium vivax pre-erythrocytic-stage antigen discovery: exploiting naturally acquired humoral responses.
Am. J. Trop. Med. Hyg.
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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.
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Proteome-scale antibody responses and outcome of Mycobacterium tuberculosis infection in nonhuman primates and in tuberculosis patients.
J. Infect. Dis.
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Biomarkers of progression from latent Mycobacterium tuberculosis infection to active tuberculosis are needed. We assessed correlations between infection outcome and antibody responses in macaques and humans by high-throughput, proteome-scale serological studies.
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Failure of the smallpox vaccine to develop a skin lesion in vaccinia virus-naïve individuals is related to differences in antibody profiles before vaccination, not after.
Clin. Vaccine Immunol.
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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.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.