RTS,S vaccination is associated with serologic evidence of decreased exposure to Plasmodium falciparum liver- and blood-stage parasites.

The leading malaria vaccine candidate, RTS,S, targets the sporozoite and liver stages of the Plasmodium falciparum life 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. falciparum antigens by protein array in Mozambican children 6 months after receiving a full course of RTS,S (n = 291) versus comparator 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.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. 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. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.

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

BACKGROUND: 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. STUDY DESIGN AND METHODS: Herein, we take advantage of recent advances in B. microti genomic analyses, field surveys of the reservoir host, and human studies in endemic areas to apply a targeted immunomic approach to the discovery of B. microti antigens that serve as signatures of active or past babesiosis infections. Of 19 glycosylphosphatidylinositol (GPI)-anchored protein candidates (BmGPI1-19) identified in the B. microti proteome, 17 were successfully expressed, printed on a microarray chip, and used to screen sera from uninfected and B. microti-infected mice and humans to determine immune responses that are associated with active and past infection. RESULTS: Antibody responses to various B. microti BmGPI antigens were detected and BmGPI12 was identified as the best biomarker of infection that provided high sensitivity and specificity when used in a microarray antibody assay. CONCLUSION: BmGPI12 alone or in combination with other BmGPI proteins is a promising candidate biomarker for detection of B. microti antibodies that might be useful in blood screening to prevent transfusion-transmitted babesiosis.

Predicting antidisease immunity using proteome arrays and sera from children naturally exposed to malaria.

Malaria remains one of the most prevalent and lethal human infectious diseases worldwide. A comprehensive characterization of antibody responses to blood stage malaria is essential to support the development of future vaccines, sero-diagnostic tests, and sero-surveillance methods. We constructed a proteome array containing 4441 recombinant proteins expressed by the blood stages of the two most common human malaria parasites, P. falciparum (Pf) and P. vivax (Pv), and used this array to screen sera of Papua New Guinea children infected with Pf, Pv, or both (Pf/Pv) that were either symptomatic (febrile), or asymptomatic but had parasitemia detectable via microscopy or PCR. We hypothesized that asymptomatic children would develop antigen-specific antibody profiles associated with antidisease immunity, as compared with symptomatic children. The sera from these children recognized hundreds of the arrayed recombinant Pf and Pv proteins. In general, responses in asymptomatic children were highest in those with high parasitemia, suggesting that antibody levels are associated with parasite burden. In contrast, symptomatic children carried fewer antibodies than asymptomatic children with infections detectable by microscopy, particularly in Pv and Pf/Pv groups, suggesting that antibody production may be impaired during symptomatic infections. We used machine-learning algorithms to investigate the relationship between antibody responses and symptoms, and we identified antibody responses to sets of Plasmodium proteins that could predict clinical status of the donors. Several of these antibody responses were identified by multiple comparisons, including those against members of the serine enriched repeat antigen family and merozoite protein 4. Interestingly, both P. falciparum serine enriched repeat antigen-5 and merozoite protein 4 have been previously investigated for use in vaccines. This machine learning approach, never previously applied to proteome arrays, can be used to generate a list of potential seroprotective and/or diagnostic antigens candidates that can be further evaluated in longitudinal studies.

Genome-level determination of Plasmodium falciparum blood-stage targets of malarial clinical immunity in the Peruvian Amazon.

BACKGROUND: Persons with blood-stage Plasmodium falciparum parasitemia in the absence of symptoms are considered to be clinically immune. We hypothesized that asymptomatic subjects with P. falciparum parasitemia would differentially recognize a subset of P. falciparum proteins on a genomic scale. METHODS AND FINDINGS: Compared with symptomatic subjects, sera from clinically immune, asymptomatically infected individuals differentially recognized 51 P. falciparum proteins, including the established vaccine candidate PfMSP1. Novel, hitherto unstudied hypothetical proteins and other proteins not previously recognized as potential vaccine candidates were also differentially recognized. Genes encoding the proteins differentially recognized by the Peruvian clinically immune individuals exhibited a significant enrichment of nonsynonymous nucleotide variation, an observation consistent with these genes undergoing immune selection. CONCLUSIONS: A limited set of P. falciparum protein antigens was associated with the development of naturally acquired clinical immunity in the low-transmission setting of the Peruvian Amazon. These results imply that, even in a low-transmission setting, an asexual blood-stage vaccine designed to reduce clinical malaria symptoms will likely need to contain large numbers of often-polymorphic proteins, a finding at odds with many current efforts in the design of vaccines against asexual blood-stage P. falciparum.

Submicroscopic and asymptomatic Plasmodium falciparum and Plasmodium vivax infections are common in western Thailand - molecular and serological evidence.

BACKGROUND: Malaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels. METHODS: Three-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals. RESULTS: Of 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others. CONCLUSION: These findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.

Discovery of potential diagnostic and vaccine antigens in herpes simplex virus 1 and 2 by proteome-wide antibody profiling.

Routine serodiagnosis of herpes simplex virus (HSV) infections is currently performed using recombinant glycoprotein G (gG) antigens from herpes simplex virus 1 (HSV-1) and HSV-2. This is a single-antigen test and has only one diagnostic application. Relatively little is known about HSV antigenicity at the proteome-wide level, and the full potential of mining the antibody repertoire to identify antigens with other useful diagnostic properties and candidate vaccine antigens is yet to be realized. To this end we produced HSV-1 and -2 proteome microarrays in Escherichia coli and probed them against a panel of sera from patients serotyped using commercial gG-1 and gG-2 (gGs for HSV-1 and -2, respectively) enzyme-linked immunosorbent assays. We identified many reactive antigens in both HSV-1 and -2, some of which were type specific (i.e., recognized by HSV-1- or HSV-2-positive donors only) and others of which were nonspecific or cross-reactive (i.e., recognized by both HSV-1- and HSV-2-positive donors). Both membrane and nonmembrane virion proteins were antigenic, although type-specific antigens were enriched for membrane proteins, despite being expressed in E. coli.

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 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.

Dynamic antibody responses to the Mycobacterium tuberculosis proteome.

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.

A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray.

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.

Proteome-scale antibody responses and outcome of Mycobacterium tuberculosis infection in nonhuman primates and in tuberculosis patients.

BACKGROUND: 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. METHODS: Mycobacterium tuberculosis proteome microarrays were probed with serial sera from macaques representing various infection outcomes and with single-point human sera from tuberculosis suspects. Fluorescence intensity data were analyzed by calculating Z scores and associated P values. Temporal changes in macaque antibody responses were analyzed by polynomial regression. Correlations between human responses and sputum bacillary burden were assessed by quantile and hurdle regression. RESULTS: Macaque outcome groups exhibited distinct antibody profiles: early, transient responses in latent infection and stable antibody increase in active and reactivation disease. In humans, antibody levels and reactive protein numbers increased with bacillary burden. Responses to a subset of 10 proteins were more tightly associated with disease state than reactivity to the broader reactive proteome. CONCLUSIONS: Integration of macaque and human data reveals dynamic properties of antibody responses in relation to outcome and leads to actionable findings for translational research. These include the potential of antibody responses to detect acute infection and preclinical tuberculosis and to identify serodiagnostic proteins for the spectrum of bacillary burden in tuberculosis.

Vaccination with a UV-irradiated genetically attenuated mutant of Staphylococcus aureus provides protection against subsequent systemic infection.

Staphylococcus aureus are gram-positive bacteria that cause clinically significant infections in humans. Severe S. aureus infections are particularly problematic in hospitalized patients and reoccur despite therapeutic measures. The absence of natural protective immune responses and the lack of high-throughput approaches to identify S. aureus antigens have imposed constraints in the development of effective vaccines. Here, we showed that vaccination with the genetically attenuated S. aureus mutant, inactivated using UV irradiation rather than heat, significantly increased survival and diminished bacterial burden and kidney abscesses when mice were challenged with virulent methicillin-sensitive or methicillin-resistant S. aureus. Protection conferred by immunization could be transferred to the naive host and was not observed in B-cell-deficient mice. Using a novel S. aureus whole-proteome microarray, we show that immunoglobulin G antibody responses to 83 proteins were observed in the immunized mice. These results suggest that protection against S. aureus infections requires antibody responses to the wide repertoire of antigens/virulence factors. Vaccination using UV-irradiated genetically attenuated S. aureus induces humoral immunity and provides a vaccine strategy for pathogens that fail to induce protective immunity. We also describe a novel, high-throughput technology to easily identify S. aureus antigens for vaccine development.

A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens.

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.

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.

New targets of pemphigus vulgaris antibodies identified by protein array technology.

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.

Genome-wide profiling of humoral immune response to Coxiella burnetii infection by protein microarray.

Comprehensive evaluation of the humoral immune response to Coxiella burnetii may identify highly needed diagnostic antigens and potential subunit vaccine candidates. Here we report the construction of a protein microarray containing 1901 C. burnetii ORFs (84% of the entire proteome). This array was probed with Q-fever patient sera and naïve controls in order to discover C. burnetii-specific seroreactive antigens. Among the 21 seroreactive antigens identified, 13 were significantly more reactive in Q-fever cases than naïve controls. The remaining eight antigens were cross-reactive in both C. burnetii infected and naïve patient sera. An additional 64 antigens displayed variable seroreactivity in Q-fever patients, and underscore the diversity of the humoral immune response to C. burnetii. Nine of the differentially reactive antigens were validated on an alternative immunostrip platform, demonstrating proof-of-concept development of a consistent, safe, and inexpensive diagnostic assay alternative. Furthermore, we report here the identification of several new diagnostic antigens and potential subunit vaccine candidates for the highly infectious category B alphaproteobacteria, C. burnetii.

Antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of Dryvax.

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus that is under consideration as an alternative to the conventional smallpox vaccine Dryvax. MVA was attenuated by extensive passage of vaccinia virus Ankara in chicken embryo fibroblasts. Several immunomodulatory genes and genes that influence host range are deleted or mutated, and replication is aborted in the late stage of infection in most nonavian cells. The effect of these mutations on immunogenicity is not well understood. Since the structural genes appear to be intact in MVA, it is hypothesized that critical targets for antibody neutralization have been retained. To test this, we probed microarrays of the Western Reserve (WR) proteome with sera from humans and macaques after MVA and Dryvax vaccination. As most protein sequences of MVA are 97 to 99% identical to those of other vaccinia virus strains, extensive binding cross-reactivity is expected, except for those deleted or truncated. Despite different hosts and immunization regimens, the MVA and Dryvax antibody profiles were broadly similar, with antibodies against membrane and core proteins being the best conserved. The responses to nonstructural proteins were less well conserved, although these are not expected to influence virus neutralization. The broadest antibody response was obtained for hyperimmune rabbits with WR, which is pathogenic in rabbits. These data indicate that, despite the mutations and deletions in MVA, its overall immunogenicity is broadly comparable to that of Dryvax, particularly at the level of antibodies to membrane proteins. The work supports other information suggesting that MVA may be a useful alternative to Dryvax.

An extremely diverse CD4 response to vaccinia virus in humans is revealed by proteome-wide T-cell profiling.

CD4 T cells are required for the maintenance and recall of antiviral CD8 T cells and for antibody responses. Little is known concerning the overall architecture of the CD4 response to complex microbial pathogens. In a whole-proteome approach, 180 predicted open reading frames (ORFs) in the vaccinia virus genome were expressed and tested using responder cells from 20 blood samples from 11 vaccinees. Validation assays established the sensitivity and specificity of the system. Overall, CD4 responses were detected for 122 ORFs (68%). A mean of 39 ORFs were recognized per person (range, 13 to 63). The most frequently recognized ORFS were present in virions, including A3L and A10L (core proteins), WR148 (a fragmented homolog of an orthopoxvirus protein that forms inclusions in cells), H3L (a membrane protein), D13L (a membrane scaffold protein), and L4R (a nucleic acid binding protein). Serum immunoglobulin G profiling by proteome microarray detected responses to 45 (25%) of the ORFs and confirmed recent studies showing a diverse response directed to membrane and nonmembrane antigens. Our results provide the first empirical whole-proteome data set regarding the global CD4 response to full-length proteins in a complex virus and are consistent with the theory that abundant structural proteins are immunodominant.

A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1.

The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates. We show that mutations in a conserved docking site in these MAPKs (the CD/7m region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors. Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially competed for Fus3 binding. In vivo, some of the MAPK mutants displayed reduced Ste7-dependent phosphorylation, and all of them exhibited multiple defects in mating and pheromone response. The Kss1 mutants were also defective in Kss1-imposed repression of Ste12. We conclude that MAPKs contain a structurally and functionally conserved docking site that mediates an overall positively acting network of interactions with cognate docking sites on their regulators and substrates. Key features of this interaction network appear to have been conserved from yeast to humans.

Profiling of the antibody response to attenuated LC16m8 smallpox vaccine using protein array analysis.

Concerns about bioterrorism and outbreaks of zoonotic orthopoxvirus require safe and efficacious smallpox vaccines. We previously reported the clinical efficacy and safety profiles of LC16m8, a live, attenuated, cell culture-derived, smallpox vaccine, examined in over 3000 healthy Japanese adults with various vaccination histories. In this study, serum of approximately 200 subjects pre and post LC16m8 vaccination were subjected to a vaccinia virus-specific protein array to evaluate the proteome-wide immunogenicity. The relationships between antigen-specific antibodies and plaque reduction neutralization titers were analyzed. LC16m8 induced antibodies to multiple vaccinia antigens in primary-vaccinated individuals and yielded effective booster responses in previously vaccinated individuals, demonstrating similar antibody profiles to those reported for other vaccinia virus strains. Several immunodominant antigens were indicated to be important for neutralization of the intracellular mature virion. The similarity of antibody profiles between LC16m8 and other smallpox vaccine strains supports the immunogenicity and protective efficacy of LC16m8.