From Functional Genomics to Functional Immunomics: New Challenges, Old Problems, Big Rewards

The development of DNA microarray technology a decade ago led to the establishment of functional genomics as one of the most active and successful scientific disciplines today. With the ongoing development of immunomic microarray technology—a spatially addressable, large-scale technology for measurement of specific immunological response—the new challenge of functional immunomics is emerging, which bears similarities to but is also significantly different from functional genomics. Immunonic data has been successfully used to identify biological markers involved in autoimmune diseases, allergies, viral infections such as human immunodeficiency virus (HIV), influenza, diabetes, and responses to cancer vaccines. This review intends to provide a coherent vision of this nascent scientific field, and speculate on future research directions. We discuss at some length issues such as epitope prediction, immunomic microarray technology and its applications, and computation and statistical challenges related to functional immunomics. Based on the recent discovery of regulation mechanisms in T cell responses, we envision the use of immunomic microarrays as a tool for advances in systems biology of cellular immune responses, by means of immunomic regulatory network models

Conservation and Diversity of Influenza A H1N1 HLA-Restricted T Cell Epitope Candidates for Epitope-Based Vaccines

Background: The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated. Methodology/Principal Findings: HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007-2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes. Conclusions/Significance: Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus. © 2010 Tan et al

Dendritic Cell Mediated Delivery of Plasmid DNA Encoding LAMP/HIV-1 Gag Fusion Immunogen Enhances T Cell Epitope Responses in HLA DR4 Transgenic Mice

This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d) and HLA-DR4 (DRA1*0101, DRB1*0401) transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag) chimera antigen. Three immunization protocols were compared: 1) primary subcutaneous immunization with 1×105 immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2) primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3) immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-γ ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a) the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b) the value of HLA transgenic mice as a model system for the identification and evaluation of epitope-based vaccine strategies, and c) the application of variability analysis across reported sequences in public databases for selection of historically conserved HIV epitopes as vaccine targets

Human Leukocyte Antigen (HLA) Class I Restricted Epitope Discovery in Yellow Fewer and Dengue Viruses: Importance of HLA Binding Strength

Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV epitopes were selected using the EpiSelect algorithm to allow for optimal coverage of viral strains. The selected predicted epitopes were synthesized and approximately 75% were found to bind the predicted restricting HLA molecule with an affinity, KD, stronger than 500 nM. The immunogenicity of 25 HLA-A*02:01, 28 HLA-A*24:02 and 28 HLA-B*07:02 binding peptides was tested in three HLA-transgenic mice models and led to the identification of 17 HLA-A*02:01, 4 HLA-A*2402 and 4 HLA-B*07:02 immunogenic peptides. The immunogenic peptides bound HLA significantly stronger than the non-immunogenic peptides. All except one of the immunogenic peptides had KD below 100 nM and the peptides with KD below 5 nM were more likely to be immunogenic. In addition, all the immunogenic peptides that were identified as having a high functional avidity had KD below 20 nM. A*02:01 transgenic mice were also inoculated twice with the 17DD YFV vaccine strain. Three of the YFV A*02:01 restricted peptides activated T-cells from the infected mice in vitro. All three peptides that elicited responses had an HLA binding affinity of 2 nM or less. The results indicate the importance of the strength of HLA binding in shaping the immune response

Lipid droplet levels vary heterogeneously in response to simulated gastrointestinal stresses in different probiotic Saccharomyces cerevisiae strains

AbstractTo exert their therapeutic action, probiotic Saccharomyces cerevisiae strains must survive harsh digestive environments. Lipid droplets accumulate in cells which undergo stress-inducing situations, supposedly having a protective role. We assessed lipid droplet levels, either naturally accumulated or induced in response to digestive challenges, of probiotic strains S. boulardii, S. cerevisiae A-905, S. cerevisiae Sc47 and S. cerevisiae L11, and of non-probiotic strains S. cerevisiae BY4741 and S. cerevisiae BY4743. Strains 905 and Sc47 had lower and higher lipid droplet levels, respectively, when compared to the remaining strains, showing that higher accumulationof these neutral lipids is not a feature shared by all probiotic Saccharomyces strains. When submitted to simulated gastric or bile salts environments, lipid droplet levels increase in all tested probiotic strains, at least for one to the induced stresses, suggesting that lipid droplets participate in the protective mechanisms against gastrointestinal stresses in probiotic Saccharomyces yeasts

T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4+ and CD8+ T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, "promiscuous" T-cell antigens might contribute to the high efficacy of the yellow fever vaccines. © 2013 de Melo et al

From Re-Emergence to Hyperendemicity: The Natural History of the Dengue Epidemic in Brazil

The spread of dengue virus is a major public health problem. Though the burden of dengue has historically been concentrated in Southeast Asian countries, Brazil has become the country that reports the largest number of cases in the world. While prior to 2007 the disease affected mostly adults, during the 2007 epidemic the number of dengue hemorrhagic fever cases more than doubled, and over 53% of cases were in children under 15 years of age. In this paper, we propose that the conditions for the shift were being set gradually since the re-introduction of dengue in 1986 and that they represent the transition from re-emergence to hyperendemicity. Using data from an age stratified seroprevalence study conducted in Recife, we estimated the force of infection (a measure of transmission intensity) between 1986–2006 and used these estimates to simulate the accumulation of immunity since the re-emergence. As the length of time that dengue has circulated increases, adults have a lower probability of remaining susceptible to primary or secondary infection and thus, cases become on average younger. If in fact the shift represents the transition from re-emergence to hyperendemicity, similar shifts are likely to be observed in the rest of Brazil, the American continent and other regions where transmission emerges

A Phase I Double Blind, Placebo-Controlled, Randomized Study of a Multigenic HIV-1 Adenovirus Subtype 35 Vector Vaccine in Healthy Uninfected Adults

<div><h3>Background</h3><p>We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35) vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN) and env (Ad35-ENV), both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults.</p> <h3>Methods</h3><p>Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions) or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively) within one of four dosage groups: Ad35-GRIN/ENV 2×10⁹ (A), 2×10¹⁰ (B), 2×10¹¹ (C), or Ad35-GRIN 1×10¹⁰ (D) viral particles.</p> <h3>Results</h3><p>No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A–D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC) per 10⁶ PBMC to any antigen was 78–139 across Groups A–C and 158–174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A–C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination.</p> <h3>Conclusion/Significance</h3><p>Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination. T-cell responses were broad and polyfunctional.</p> <h3>Trial Registration</h3><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/results?term=NCT00851383">NCT00851383</a></p> </div

Clinical evaluation of dengue and identification of risk factors for severe disease: protocol for a multicentre study in 8 countries

Background: The burden of dengue continues to increase globally, with an estimated 100 million clinically apparent infections occurring each year. Although most dengue infections are asymptomatic, patients can present with a wide spectrum of clinical symptoms ranging from mild febrile illness through to severe manifestations of bleeding, organ impairment, and hypovolaemic shock due to a systemic vascular leak syndrome. Clinical diagnosis of dengue and identification of which patients are likely to develop severe disease remain challenging. This study aims to improve diagnosis and clinical management through approaches designed a) to differentiate between dengue and other common febrile illness within 72 h of fever onset, and b) among patients with dengue to identify markers that are predictive of the likelihood of evolving to a more severe disease course. Method/Design: This is a prospective multi-centre observational study aiming to enrol 7–8000 participants aged ≥ 5 years presenting with a febrile illness consistent with dengue to outpatient health facilities in 8 countries across Asia and Latin America. Patients presenting within 72 h of fever onset who do not exhibit signs of severe disease are eligible for the study. A broad range of clinical and laboratory parameters are assessed daily for up to 6 days during the acute illness, and also at a follow up visit 1 week later. Discussion: Data from this large cohort of patients, enrolled early with undifferentiated fever, will be used to develop a practical diagnostic algorithm and a robust clinical case definition for dengue. Additionally, among patients with confirmed dengue we aim to identify simple clinical and laboratory parameters associated with progression to a more severe disease course. We will also investigate early virological and serological correlates of severe disease, and examine genetic associations in this large heterogeneous cohort. In addition the results will be used to assess the new World Health Organization classification scheme for dengue in practice, and to update the guidelines for “Integrated Management of Childhood Illness” used in dengue-endemic countries. Trial registration: NCT01550016. Registration Date: March 7, 201

Reliable Classifier to Differentiate Primary and Secondary Acute Dengue Infection Based on IgG ELISA

Dengue virus infection causes a wide spectrum of illness, ranging from sub-clinical to severe disease. Severe dengue is associated with sequential viral infections. A strict definition of primary versus secondary dengue infections requires a combination of several tests performed at different stages of the disease, which is not practical.We developed a simple method to classify dengue infections as primary or secondary based on the levels of dengue-specific IgG. A group of 109 dengue infection patients were classified as having primary or secondary dengue infection on the basis of a strict combination of results from assays of antigen-specific IgM and IgG, isolation of virus and detection of the viral genome by PCR tests performed on multiple samples, collected from each patient over a period of 30 days. The dengue-specific IgG levels of all samples from 59 of the patients were analyzed by linear discriminant analysis (LDA), and one- and two-dimensional classifiers were designed. The one-dimensional classifier was estimated by bolstered resubstitution error estimation to have 75.1% sensitivity and 92.5% specificity. The two-dimensional classifier was designed by taking also into consideration the number of days after the onset of symptoms, with an estimated sensitivity and specificity of 91.64% and 92.46%. The performance of the two-dimensional classifier was validated using an independent test set of standard samples from the remaining 50 patients. The classifications of the independent set of samples determined by the two-dimensional classifiers were further validated by comparing with two other dengue classification methods: hemagglutination inhibition (HI) assay and an in-house anti-dengue IgG-capture ELISA method. The decisions made with the two-dimensional classifier were in 100% accordance with the HI assay and 96% with the in-house ELISA.Once acute dengue infection has been determined, a 2-D classifier based on common dengue virus IgG kits can reliably distinguish primary and secondary dengue infections. Software for calculation and validation of the 2-D classifier is made available for download