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3.  Analysis of purified Wild type and mutant adenovirus particles by SILAC based quantitative proteomics 
The Journal of General Virology  2014;95(Pt 11):2504-2511.
We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120.
PMCID: PMC4202269  PMID: 25096814
4.  Immune Biomarkers Predictive of Respiratory Viral Infection in Elderly Nursing Home Residents 
PLoS ONE  2014;9(10):e108481.
To determine if immune phenotypes associated with immunosenescence predict risk of respiratory viral infection in elderly nursing home residents.
Residents ≥65 years from 32 nursing homes in 4 Canadian cities were enrolled in Fall 2009, 2010 and 2011, and followed for one influenza season. Following influenza vaccination, peripheral blood mononuclear cells (PBMCs) were obtained and analysed by flow cytometry for T-regs, CD4+ and CD8+ T-cell subsets (CCR7+CD45RA+, CCR7-CD45RA+ and CD28-CD57+) and CMV-reactive CD4+ and CD8+ T-cells. Nasopharyngeal swabs were obtained and tested for viruses in symptomatic residents. A Cox proportional hazards model adjusted for age, sex and frailty, determined the relationship between immune phenotypes and time to viral infection.
1072 residents were enrolled; median age 86 years and 72% female. 269 swabs were obtained, 87 were positive for virus: influenza (24%), RSV (14%), coronavirus (32%), rhinovirus (17%), human metapneumovirus (9%) and parainfluenza (5%). In multivariable analysis, high T-reg% (HR 0.41, 95% CI 0.20–0.81) and high CMV-reactive CD4+ T-cell% (HR 1.69, 95% CI 1.03–2.78) were predictive of respiratory viral infection.
In elderly nursing home residents, high CMV-reactive CD4+ T-cells were associated with an increased risk and high T-regs were associated with a reduced risk of respiratory viral infection.
PMCID: PMC4183538  PMID: 25275464
5.  Alterations to the Frequency and Function of Peripheral Blood Monocytes and Associations with Chronic Disease in the Advanced-Age, Frail Elderly 
PLoS ONE  2014;9(8):e104522.
Circulating myeloid cells are important mediators of the inflammatory response, acting as a major source of resident tissue antigen presenting cells and serum cytokines. They represent a number of distinct subpopulations whose functional capacity and relative concentrations are known to change with age. Little is known of these changes in the very old and physically frail, a rapidly increasing proportion of the North American population.
In the following study the frequency and receptor expression of blood monocytes and dendritic cells (DCs) were characterized in a sample of advanced-age, frail elderly (81–100 yrs), and compared against that of adults (19–59 yrs), and community-dwelling seniors (61–76 yrs). Cytokine responses following TLR stimulation were also investigated, as well as associations between immunophenotyping parameters and chronic diseases.
The advanced-age, frail elderly had significantly fewer CD14(++) and CD14(+)CD16(+), but not CD14(++)CD16(+) monocytes, fewer plasmacytoid and myeloid DCs, and a lower frequency of monocytes expressing the chemokine receptors CCR2 and CX3CR1. At baseline and following stimulation with TLR-2 and -4 agonists, monocytes from the advanced-age, frail elderly produced more TNF than adults, although the overall induction was significantly lower. Finally, monocyte subset frequency and CX3CR1 expression was positively associated with dementia, while negatively associated with anemia and diabetes in the advanced-age, frail elderly.
These data demonstrate that blood monocyte frequency and phenotype are altered in the advanced-age, frail elderly and that these changes correlate with certain chronic diseases. Whether these changes contribute to or are caused by these conditions warrants further investigation.
PMCID: PMC4126708  PMID: 25105870
6.  Distinguishing West Nile virus infection using a recombinant envelope protein with mutations in the conserved fusion-loop 
BMC Infectious Diseases  2014;14:246.
West Nile Virus (WNV) is an emerging mosquito-transmitted flavivirus that continues to spread and cause disease throughout several parts of the world, including Europe and the Americas. Specific diagnosis of WNV infections using current serological testing is complicated by the high degree of cross-reactivity between antibodies against other clinically relevant flaviviruses, including dengue, tick-borne encephalitis (TBEV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. Cross-reactivity is particularly problematic in areas where different flaviviruses co-circulate or in populations that have been immunized with vaccines against TBEV, JEV, or YFV. The majority of cross-reactive antibodies against the immunodominant flavivirus envelope (E) protein target a conserved epitope in the fusion loop at the distal end of domain II.
We tested a loss-of-function bacterially expressed recombinant WNV E protein containing mutations in the fusion loop and an adjacent loop domain as a possible diagnostic reagent. By comparing the binding of sera from humans infected with WNV or other flaviviruses to the wild type and the mutant E proteins, we analyzed the potential of this technology to specifically detect WNV antibodies.
Using this system, we could reliably determine WNV infections. Antibodies from WNV-infected individuals bound equally well to the wild type and the mutant protein. In contrast, sera from persons infected with other flaviviruses showed significantly decreased binding to the mutant protein. By calculating the mean differences between antibody signals detected using the wild type and the mutant proteins, a value could be assigned for each of the flaviviruses, which distinguished their pattern of reactivity.
Recombinant mutant E proteins can be used to discriminate infections with WNV from those with other flaviviruses. The data have important implications for the development of improved, specific serological assays for the detection of WNV antibodies in regions where other flaviviruses co-circulate or in populations that are immunized with other flavivirus vaccines.
PMCID: PMC4028281  PMID: 24884467
West Nile virus; Diagnosis; Antibodies; Envelope protein
7.  Immunosenescence in the nursing home elderly 
BMC Geriatrics  2014;14:50.
To describe T-cell and natural killer (NK) cell phenotypes within nursing home elderly.
Nursing home elderly were recruited from four nursing homes in Hamilton, Ontario between September 2010 and December 2011. Healthy adults were recruited from McMaster University between September 2011 and December 2011. Nursing home elderly ≥65 years were eligible; those on immunosuppressive medications were excluded. Healthy adults ≥18-64 years were eligible. CD8+ and CD4+ T-cells% and their subsets, T-regs% and NK cell subset% were compared between the nursing home elderly and healthy adults.
262 nursing home elderly were enrolled; median age 87 years and 81% were female. 16 healthy adults were enrolled; median age 31 and 50% were female. There was no significant difference between CD8+ T-cell% in nursing home and healthy adults (median 17.1 versus 18.0, p = 0.56), however there were fewer naïve CD8 + T-cell% (median 0.9 versus 5.2, p < 0.001), more terminally differentiated CD8 + T-cell% (median 7.3 versus 4.1, p = 0.004) and more senescent T-cell% (median 5.3 versus 3.1, p = 0.04) in the nursing home elderly. There were more CD4+ T-cell% in the nursing home elderly compared to healthy adults (median 45.5 versus 37.1, p = 0.001). Nursing home elderly had a higher CD4+/CD8+ ratio than healthy adults (2.6 versus 1.9, p = 0.048), higher T-reg% (median 1.8 versus 0.8, p < 0.001) and increased mature NK cell% (median 12.1 versus 5.4, p = 0.001) compared to healthy adults.
Differences in naïve CD8+ T-cells, terminally differentiated and senescent CD8+ T-cells, T-regs and NK cell subsets were similar to studies involving community dwelling elderly. In contrast, the CD4+/CD8+ ratio was higher in nursing home elderly.
PMCID: PMC4013821  PMID: 24742120
Immunosenescence; Aging; Immune phenotypes; Nursing home elderly
8.  A novel computer algorithm improves antibody epitope prediction using affinity-selected mimotopes: A case study using monoclonal antibodies against the West Nile virus E protein 
Molecular immunology  2008;46(1):10.1016/j.molimm.2008.07.020.
Understanding antibody function is often enhanced by knowledge of the specific binding epitope. Here, we describe a computer algorithm that permits epitope prediction based on a collection of random peptide epitopes (mimotopes) isolated by antibody affinity purification. We applied this methodology to the prediction of epitopes for five monoclonal antibodies against the West Nile virus (WNV) E protein, two of which exhibit therapeutic activity in vivo. This strategy was validated by comparison of our results with existing F(ab)-E protein crystal structures and mutational analysis by yeast surface display. We demonstrate that by combining the results of the mimotope method with our data from mutational analysis, epitopes could be predicted with greater certainty. The two methods displayed great complementarity as the mutational analysis facilitated epitope prediction when the results with the mimotope method were equivocal and the mimotope method revealed a broader number of residues within the epitope than the mutational analysis. Our results demonstrate that the combination of these two prediction strategies provides a robust platform for epitope characterization.
PMCID: PMC3856767  PMID: 18760481
Epitope mapping; Monoclonal antibody; Phage display; Neutralization; Flavivirus
9.  Oncolytic vesicular stomatitis virus quantitatively and qualitatively improves primary CD8+ T-cell responses to anticancer vaccines 
Oncoimmunology  2013;2(8):e26013.
The ability of heterologous prime-boost vaccination to elicit robust CD8+ T cell responses has been well documented. In contrast, relatively little is known about how this immunotherapeutic strategy impacts the functional qualities of expanded T cells in the course of effector and memory responses. Using vesicular stomatitis virus (VSV) as a boosting vector in mice, we demonstrate that a massive secondary expansion of CD8+ T cells can be achieved shortly after priming with recombinant adenoviral vectors. Importantly, VSV-boosted CD8+ T cells were more potent than those primed by adenoviruses only, as measured by cytokine production, granzyme B expression, and functional avidity. Upon adoptive transfer, equivalent numbers of VSV-expanded CD8+ T cells were more effective (on a per-cell basis) in mediating antitumor and antiviral immunity than T cells only primed with adenoviruses. Furthermore, VSV boosting accelerated the progression of expanded CD8+ T lymphocytes to a central memory phenotype, thereby altering the effector memory profile typically associated with adenoviral vaccination. Finally, the functional superiority of VSV-expanded T cells remained evident 100 d after boosting, suggesting that VSV-driven immunological responses are of sufficient duration for therapeutic applications. Our data strongly support the choice of VSV as a boosting vector in prime-boost vaccination strategies, enabling a rapid amplification of CD8+ T cells and improving the quality of expanded T cells during both early and late immunological responses.
PMCID: PMC3782525  PMID: 24083086
adenovirus; CD8+ T cells; prime-boost; vaccination; vesicular stomatitis virus
10.  A quantitative systems approach to identify paracrine mechanisms that locally suppress immune response to Interleukin-12 in the B16 melanoma model 
Interleukin-12 (IL12) enhances anti-tumor immunity when delivered to the tumor microenvironment. However, local immunoregulatory elements dampen the efficacy of IL12. The identity of these local mechanisms used by tumors to suppress immunosurveillance represents a key knowledge gap for improving tumor immunotherapy. From a systems perspective, local suppression of anti-tumor immunity is a closed-loop system - where system response is determined by an unknown combination of external inputs and local cellular cross-talk. Here, we recreated this closed-loop system in vitro and combined quantitative high content assays, in silico model-based inference, and a proteomic workflow to identify the biochemical cues responsible for immunosuppression. Following an induction period, the B16 melanoma cell model, a transplantable model for spontaneous malignant melanoma, inhibited the response of a T helper cell model to IL12. This paracrine effect was not explained by induction of apoptosis or creation of a cytokine sink, despite both mechanisms present within the co-culture assay. Tumor-derived Wnt-inducible signaling protein-1 (WISP-1) was identified to exert paracrine action on immune cells by inhibiting their response to IL12. Moreover, WISP-1 was expressed in vivo following intradermal challenge with B16F10 cells and was inferred to be expressed at the tumor periphery. Collectively, the data suggest that (1) biochemical cues associated with epithelial-to-mesenchymal transition can shape anti-tumor immunity through paracrine action and (2) remnants of the immunoselective pressure associated with evolution in cancer include both sculpting of tumor antigens and expression of proteins that proactively shape anti-tumor immunity.
PMCID: PMC3428131  PMID: 22777646
11.  Blood CD33(+)HLA-DR(−) myeloid-derived suppressor cells are increased with age and a history of cancer 
Journal of Leukocyte Biology  2013;93(4):633-637.
Myeloid-derived suppressor cells are increased with age and elevated in donors with a history of cancer; an age-related effect has never been shown in humans.
As we age, the composition of our peripheral leukocytes changes dramatically. Many of these alterations contribute to the general immune dysfunction that burdens the elderly, which in turn, contributes to increased susceptibility to disease. MDSCs represent a heterogeneous population of immunosuppressive leukocytes that are elevated in the peripheral blood of cancer patients. Given the relation between cancer incidence and age, this study examined the frequency of peripheral blood CD33(+)HLA-DR(−) MDSCs across three cohorts: healthy adults (19–59 years old), community-dwelling seniors (61–76 years old), and frail elderly (67–99 years old). This analysis is the first to demonstrate that MDSCs and specifically the CD11b(+)CD15(+) MDSC subset are increased with age. Proinflammatory cytokines that are required for the differentiation of MDSCs (e.g., TNF-α, IL-6, and IL-1β) were similarly found to be increased in the serum of the frail elderly. Furthermore, the proportion of MDSCs and the CD11b(+)CD15(+) subset were found to be elevated significantly in elderly donors with a history of cancer. This age-related elevation in the frequency of MDSCs may contribute to the increased cancer incidence that occurs with age. Further investigation into the functional consequences of elevated MDSCs will provide valuable insight into the progression of age-related pathologies.
PMCID: PMC3701116  PMID: 23341539
aging; elderly; inflammaging; mortality
12.  The Polyfunctionality of Human Memory CD8+ T Cells Elicited by Acute and Chronic Virus Infections Is Not Influenced by Age 
PLoS Pathogens  2012;8(12):e1003076.
As humans age, they experience a progressive loss of thymic function and a corresponding shift in the makeup of the circulating CD8+ T cell population from naïve to memory phenotype. These alterations are believed to result in impaired CD8+ T cell responses in older individuals; however, evidence that these global changes impact virus-specific CD8+ T cell immunity in the elderly is lacking. To gain further insight into the functionality of virus-specific CD8+ T cells in older individuals, we interrogated a cohort of individuals who were acutely infected with West Nile virus (WNV) and chronically infected with Epstein Barr virus (EBV) and Cytomegalovirus (CMV). The cohort was stratified into young (<40 yrs), middle-aged (41–59 yrs) and aged (>60 yrs) groups. In the aged cohort, the CD8+ T cell compartment displayed a marked reduction in the frequency of naïve CD8+ T cells and increased frequencies of CD8+ T cells that expressed CD57 and lacked CD28, as previously described. However, we did not observe an influence of age on either the frequency of virus-specific CD8+ T cells within the circulating pool nor their functionality (based on the production of IFNγ, TNFα, IL2, Granzyme B, Perforin and mobilization of CD107a). We did note that CD8+ T cells specific for WNV, CMV or EBV displayed distinct functional profiles, but these differences were unrelated to age. Collectively, these data fail to support the hypothesis that immunosenescence leads to defective CD8+ T cell immunity and suggest that it should be possible to develop CD8+ T cell vaccines to protect aged individuals from infections with novel emerging viruses.
Author Summary
The prevalence and severity of viral infections increases with advanced age, a phenomenon associated with a defective immune system. The thymic output of naïve T cells declines as we age and it is this lack of naïve T cells that is believed to contribute to the inability of the aged to respond to novel infections and develop subsequent memory T cell responses. Here we show that individuals aged 60+ are capable of developing memory CD8+ T cells to West Nile virus, novel pathogen, indistinguishable in terms of polyfunctionality to those of subjects <60 years of age. Furthermore, we show that chronic and life-long infections with CMV and EBV result in similar polyfunctional virus-specific memory CD8+ T cell responses in subjects of all age groups. Our work demonstrates that aged individuals can elicit functional memory CD8+ T cell responses to a new pathogen while maintaining polyfunctional CD8+ T cells against recurrent chronic virus infections. Current vaccine platforms, which rely upon inactivated pathogens or recombinant subunits, are poorly effective in the aged. Our data suggest that live viruses may be more effective vaccine platforms in older humans.
PMCID: PMC3521721  PMID: 23271970
13.  Genetic Variants and Susceptibility to Neurological Complications Following West Nile Virus Infection 
The Journal of Infectious Diseases  2011;204(7):1031-1037.
To determine genetic factors predisposing to neurological complications following West Nile virus infection, we analyzed a cohort of 560 neuroinvasive case patients and 950 control patients for 13 371 mostly nonsynonymous single-nucleotide polymorphisms (SNPs). The top 3 SNPs on the basis of statistical significance were also in genes of biological plausibility: rs2066786 in RFC1 (replication factor C1) (P = 1.88 × 10−5; odds ratio [OR], 0.68 [95% confidence interval {CI}, .56–.81]); rs2298771 in SCN1A (sodium channel, neuronal type I α subunit) (P = 5.87 × 10−5; OR, 1.47 [95% CI, 1.21–1.77]); and rs25651 in ANPEP (ananyl aminopeptidase) (P = 1.44 × 10−4; OR, 0.69 [95% CI, .56–.83]). Additional genotyping of these SNPs in a separate sample of 264 case patients and 296 control patients resulted in a lack of significance in the replication cohort; joint significance was as follows: rs2066786, P = .0022; rs2298771, P = .005; rs25651, P = .042. Using mostly nonsynonymous variants, we therefore did not identify genetic variants associated with neuroinvasive disease.
PMCID: PMC3203390  PMID: 21881118
14.  Combined vaccination and immunostimulatory antibodies provides durable cure of murine melanoma and induces transcriptional changes associated with positive outcome in human melanoma patients 
Oncoimmunology  2012;1(4):419-431.
We have developed a recombinant adenovirus vaccine encoding dopachrome tautomerase (rHuAd5-hDCT) that produces robust DCT-specific immunity, but only provides modest suppression of murine melanoma. In the current study, an agonist antibody against 4-1BB was shown to enhance rHuAd5-hDCT efficacy and evoke tumor regression, but most tumors ultimately relapsed. The vaccine triggered upregulation of the immune inhibitory PD-1 signaling pathway and PD-1 blockade dramatically enhanced the rHuAd5-hDCT + anti-4-1BB strategy, resulting in complete regression of growing tumors in > 70% of recipients. The impact of the combined anti-4-1BB/anti-PD-1 treatment did not manifest as a dramatic enhancement in either the magnitude or functionality of DCT-specific tumor infiltrating lymphocytes relative to either treatment alone. Rather, a synergistic enhancement in intratumoral cytokine expression was observed, suggesting that the benefit of the combined therapy was a local event within the tumor. Global transcriptional analysis revealed immunological changes within the tumor following the curative vaccination, which extended beyond the T cell compartment. We identified an immune signature of 85 genes associated with clearance of murine melanoma that correlated with improved survival outcome in two independent cohorts of human melanoma patients. Our data reinforce the concept that successful vaccination must overcome local hurdles in the tumor microenvironment that are not manifest within the periphery. Further, tumor rejection following vaccination involves more than simply T cells. Finally, the association of our immune signature with positive survival outcome in human melanoma patients suggests that similar vaccination strategies may be promising for melanoma treatment.
PMCID: PMC3382903  PMID: 22754760
4-1BB; PD-1; T lymphocyte; gene profiling; immune suppression; vaccine
15.  Defining the critical hurdles in cancer immunotherapy 
Fox, Bernard A | Schendel, Dolores J | Butterfield, Lisa H | Aamdal, Steinar | Allison, James P | Ascierto, Paolo Antonio | Atkins, Michael B | Bartunkova, Jirina | Bergmann, Lothar | Berinstein, Neil | Bonorino, Cristina C | Borden, Ernest | Bramson, Jonathan L | Britten, Cedrik M | Cao, Xuetao | Carson, William E | Chang, Alfred E | Characiejus, Dainius | Choudhury, A Raja | Coukos, George | de Gruijl, Tanja | Dillman, Robert O | Dolstra, Harry | Dranoff, Glenn | Durrant, Lindy G | Finke, James H | Galon, Jerome | Gollob, Jared A | Gouttefangeas, Cécile | Grizzi, Fabio | Guida, Michele | Håkansson, Leif | Hege, Kristen | Herberman, Ronald B | Hodi, F Stephen | Hoos, Axel | Huber, Christoph | Hwu, Patrick | Imai, Kohzoh | Jaffee, Elizabeth M | Janetzki, Sylvia | June, Carl H | Kalinski, Pawel | Kaufman, Howard L | Kawakami, Koji | Kawakami, Yutaka | Keilholtz, Ulrich | Khleif, Samir N | Kiessling, Rolf | Kotlan, Beatrix | Kroemer, Guido | Lapointe, Rejean | Levitsky, Hyam I | Lotze, Michael T | Maccalli, Cristina | Maio, Michele | Marschner, Jens-Peter | Mastrangelo, Michael J | Masucci, Giuseppe | Melero, Ignacio | Melief, Cornelius | Murphy, William J | Nelson, Brad | Nicolini, Andrea | Nishimura, Michael I | Odunsi, Kunle | Ohashi, Pamela S | O'Donnell-Tormey, Jill | Old, Lloyd J | Ottensmeier, Christian | Papamichail, Michael | Parmiani, Giorgio | Pawelec, Graham | Proietti, Enrico | Qin, Shukui | Rees, Robert | Ribas, Antoni | Ridolfi, Ruggero | Ritter, Gerd | Rivoltini, Licia | Romero, Pedro J | Salem, Mohamed L | Scheper, Rik J | Seliger, Barbara | Sharma, Padmanee | Shiku, Hiroshi | Singh-Jasuja, Harpreet | Song, Wenru | Straten, Per Thor | Tahara, Hideaki | Tian, Zhigang | van Der Burg, Sjoerd H | von Hoegen, Paul | Wang, Ena | Welters, Marij JP | Winter, Hauke | Withington, Tara | Wolchok, Jedd D | Xiao, Weihua | Zitvogel, Laurence | Zwierzina, Heinz | Marincola, Francesco M | Gajewski, Thomas F | Wigginton, Jon M | Disis, Mary L
Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.
PMCID: PMC3338100  PMID: 22168571
16.  IL-1α/IL-1R1 Expression in Chronic Obstructive Pulmonary Disease and Mechanistic Relevance to Smoke-Induced Neutrophilia in Mice 
PLoS ONE  2011;6(12):e28457.
Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood.
Methodology and Principal Findings
The objective of this study was to assess IL-1 α and β expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and β. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and β levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1β- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation.
Conclusions and Significance
This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD.
PMCID: PMC3232226  PMID: 22163019
17.  Association between HLA Class I and Class II Alleles and the Outcome of West Nile Virus Infection: An Exploratory Study 
PLoS ONE  2011;6(8):e22948.
West Nile virus (WNV) infection is asymptomatic in most individuals, with a minority developing symptoms ranging from WNV fever to serious neuroinvasive disease. This study investigated the impact of host HLA on the outcome of WNV disease.
A cohort of 210 non-Hispanic mostly white WNV+ subjects from Canada and the U.S. were typed for HLA-A, B, C, DP, DQ, and DR. The study subjects were divided into three WNV infection outcome groups: asymptomatic (AS), symptomatic (S), and neuroinvasive disease (ND). Allele frequency distribution was compared pair-wise between the AS, S, and ND groups using χ2 and Fisher's exact tests and P values were corrected for multiple comparisons (Pc). Allele frequencies were compared between the groups and the North American population (NA) used as a control group. Logistic regression analysis was used to evaluate the potential synergistic effect of age and HLA allele phenotype on disease outcome.
The alleles HLA-A*68, C*08 and DQB*05 were more frequently associated with severe outcomes (ND vs. AS, PA*68 = 0.013/Pc = 0.26, PC*08 = 0.0075/Pc = 0.064, and PDQB1*05 = 0.029/Pc = 0.68), However the apparent DQB1*05 association was driven by age. The alleles HLA-B*40 and C*03 were more frequently associated with asymptomatic outcome (AS vs. S, PB*40 = 0.021/Pc = 0.58 and AS vs. ND PC*03 = 0.039/Pc = 0.64) and their frequencies were lower within WNV+ subjects with neuroinvasive disease than within the North American population (NA vs. S, PB*40 = 0.029 and NA vs. ND, PC*03 = 0.032).
Host HLA may be associated with the outcome of WNV disease; HLA-A*68 and C*08 might function as “susceptible” alleles, whereas HLA-B*40 and C*03 might function as “protective” alleles.
PMCID: PMC3148246  PMID: 21829673
18.  Surface Phenotype and Functionality of WNV Specific T Cells Differ with Age and Disease Severity 
PLoS ONE  2010;5(12):e15343.
West Nile virus (WNV) infection can result in severe neuroinvasive disease, particularly in persons with advanced age. As rodent models demonstrate that T cells play an important role in limiting WNV infection, and strong T cell responses to WNV have been observed in humans, we postulated that inadequate antiviral T cell immunity was involved in neurologic sequelae and the more severe outcomes associated with age. We previously reported the discovery of six HLA-A*0201 restricted WNV peptide epitopes, with the dominant T cell targets in naturally infected individuals being SVG9 (Env) and SLF9 (NS4b). Here, memory phenotype and polyfunctional CD8+ T cell responses to these dominant epitopes were assessed in 40 WNV seropositive patients displaying diverse clinical symptoms. The patients' PBMC were stained with HLA-I multimers loaded with the SVG9 and SLF9 epitopes and analyzed by multicolor flow cytometry. WNV-specific CD8+ T cells were found in peripheral blood several months post infection. The number of WNV-specific T cells in older individuals was the same, if not greater, than in younger members of the cohort. WNV-specific T cells were predominantly monofunctional for CD107a, MIP-1β, TNFα, IL-2, or IFNγ. When CD8+ T cell responses were stratified by disease severity, an increased number of terminally differentiated, memory phenotype (CD45RA+ CD27− CCR7− CD57+) T cells were detected in patients suffering from viral neuroinvasion. In conclusion, T cells of a terminally differentiated/cytolytic profile are associated with neuroinvasion and, regardless of age, monofunctional T cells persist following infection. These data provide the first indication that particular CD8+ T cell phenotypes are associated with disease outcome following WNV infection.
PMCID: PMC3001480  PMID: 21179445
19.  Applying bioinformatics for antibody epitope prediction using affinity-selected mimotopes – relevance for vaccine design 
Immunome Research  2010;6(Suppl 2):S6.
To properly characterize protective polyclonal antibody responses, it is necessary to examine epitope specificity. Most antibody epitopes are conformational in nature and, thus, cannot be identified using synthetic linear peptides. Cyclic peptides can function as mimetics of conformational epitopes (termed mimotopes), thereby providing targets, which can be selected by immunoaffinity purification. However, the management of large collections of random cyclic peptides is cumbersome. Filamentous bacteriophage provides a useful scaffold for the expression of random peptides (termed phage display) facilitating both the production and manipulation of complex peptide libraries. Immunoaffinity selection of phage displaying random cyclic peptides is an effective strategy for isolating mimotopes with specificity for a given antiserum. Further epitope prediction based on mimotope sequence is not trivial since mimotopes generally display only small homologies with the target protein. Large numbers of unique mimotopes are required to provide sufficient sequence coverage to elucidate the target epitope. We have developed a method based on pattern recognition theory to deal with the complexity of large collections of conformational mimotopes. The analysis consists of two phases: 1) The learning phase where a large collection of epitope-specific mimotopes is analyzed to identify epitope specific “signs” and 2) The identification phase where immunoaffinity-selected mimotopes are interrogated for the presence of the epitope specific “signs” and assigned to specific epitopes. We are currently using computational methods to define epitope “signs” without the need for prior knowledge of specific mimotopes. This technology provides an important tool for characterizing the breadth of antibody specificities within polyclonal antisera.
PMCID: PMC2981875  PMID: 21067548
20.  Identification of CD8+ T Cell Epitopes in the West Nile Virus Polyprotein by Reverse-Immunology Using NetCTL 
PLoS ONE  2010;5(9):e12697.
West Nile virus (WNV) is a growing threat to public health and a greater understanding of the immune response raised against WNV is important for the development of prophylactic and therapeutic strategies.
Methodology/Principal Findings
In a reverse-immunology approach, we used bioinformatics methods to predict WNV-specific CD8+ T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8+ T cell epitopes, which we propose are restricted by 11 different HLA class I alleles. Aiming for optimal coverage of human populations, we suggest that 11 of these new WNV epitopes would be sufficient to cover from 48% to 93% of ethnic populations in various areas of the World.
The 26 identified CD8+ T cell epitopes contribute to our knowledge of the immune response against WNV infection and greatly extend the list of known WNV CD8+ T cell epitopes. A polytope incorporating these and other epitopes could possibly serve as the basis for a WNV vaccine.
PMCID: PMC2939062  PMID: 20856867
21.  Persistence of Transgene Expression Influences CD8+ T-Cell Expansion and Maintenance following Immunization with Recombinant Adenovirus▿  
Journal of Virology  2009;83(23):12027-12036.
Previous studies determined that the CD8+ T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8+ T-cell maintenance following immunization with a recombinant adenovirus.
PMCID: PMC2786755  PMID: 19759135
22.  Characterizing Complex Polysera Produced by Antigen-Specific Immunization through the Use of Affinity-Selected Mimotopes 
PLoS ONE  2009;4(4):e5309.
Antigen-based (as opposed to whole organism) vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity.
Methodology/Principal Findings
Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes) can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets.
By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines.
PMCID: PMC2668798  PMID: 19390580
23.  Induction of Epitope-Specific Neutralizing Antibodies against West Nile Virus▿  
Journal of Virology  2007;81(21):11828-11839.
Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.
PMCID: PMC2168772  PMID: 17715236
24.  TNF-α is a critical negative regulator of type 1 immune activation during intracellular bacterial infection 
Journal of Clinical Investigation  2004;113(3):401-413.
TNF-α has long been regarded as a proimmune cytokine involved in antimicrobial type 1 immunity. However, the precise role of TNF-α in antimicrobial type 1 immunity remains poorly understood. We found that TNF-α–deficient (TNF–/–) mice quickly succumbed to respiratory failure following lung infection with replication-competent mycobacteria, because of apoptosis and necrosis of granuloma and lung structure. Tissue destruction was a result of an uncontrolled type 1 immune syndrome characterized by expansion of activated CD4 and CD8 T cells, increased frequency of antigen-specific T cells, and overproduction of IFN-γ and IL-12. Depletion of CD4 and CD8 T cells decreased IFN-γ levels, prevented granuloma and tissue necrosis, and prolonged the survival of TNF–/– hosts. Early reconstitution of TNF-α by gene transfer reduced the frequency of antigen-specific T cells and improved survival. TNF-α controlled type 1 immune activation at least in part by suppressing T cell proliferation, and this suppression involved both TNF receptor p55 and TNF receptor p75. Heightened type 1 immune activation also occurred in TNF–/– mice treated with dead mycobacteria, live replication-deficient mycobacteria, or mycobacterial cell wall components. Our study thus identifies TNF-α as a type 1 immunoregulatory cytokine whose primary role, different from those of other type 1 cytokines, is to keep an otherwise detrimental type 1 immune response in check.
PMCID: PMC324534  PMID: 14755337
25.  Detailed Analysis of the CD8+ T-Cell Response following Adenovirus Vaccination 
Journal of Virology  2003;77(24):13407-13411.
We examined CD8+ T-cell expansion and function following intramuscular immunization with a recombinant adenovirus. This study has identified a number of properties which may explain the strong immunogenicity of adenovirus vectors: (i) the ability to deliver large amounts of antigen into the lymphoid tissues, (ii) the ability to induce rapid expansion and migration of CD8+ T cells throughout the lymphatics, and (iii) the ability to produce a sustained, high-level CD8+ T-cell response.
PMCID: PMC296052  PMID: 14645597

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