Regulation of antigen processing and presentation molecules in West Nile virus-infected human skin fibroblasts

Infection of humans with the West Nile flavivirus principally occurs via tick and mosquito bites. Here, we document the expression of antigen processing and presentation molecules in West Nile virus (WNV)-infected human skin fibroblast (HFF) cells. Using a new Flavivirus-specific antibody, 4G4, we have analyzed cell surface human leukocyte antigen (HLA) expression on virus-infected cells at a single cell level. Using this approach, we show that West Nile Virus infection alters surface HLA expression on both infected HFF and neighboring uninfected HFF cells. Interestingly, increased surface HLA evident on infected HFF cultures is almost entirely due to virus-induced interferon (IFN)alpha/beta because IFNalpha/beta-neutralizing antibodies completely prevent increased surface HLA expression. In contrast, RT-PCR analysis indicates that WNV infection results in increased mRNAs for HLA-A, -B, and -C genes, and HLA-associated molecules low molecular weight polypeptide-2 (LMP-2) and transporter associated with antigen presentation-1 (TAP-1), but induction of these mRNAs is not diminished in HFF cells cultured with IFNalpha/beta-neutralizing antibodies. Taken together, these data support the idea that that both cytokine-dependent and cytokine-independent mechanisms account for WNV-induced HLA expression in human skin fibroblasts. (C) 2004 Elsevier Inc. All rights reserved.

Potent T cell response to a class I-binding 13-mer viral epitope and the influence of HLA micropolymorphism in controlling epitope length

The BZLF1 antigen of Epstein-Barr virus includes three overlapping sequences of different lengths that conform to the binding motif of human leukocyte antigen (HLA) B*3501. These 9-mer ((56)LPOGQLTAy(64)), 11-mer ((54)EPLPQGQLTAy(64)), and 13-mer ((52)LPEPLPQGQLTAY(64)) peptides all bound well to B*3501; however, the CTL response in individuals expressing this HILA allele was directed strongly and exclusively towards the 11-mer peptide. In contrast, EBV-exposed donors expressing HLA B*3503 showed no significant CTL response to these peptides because the single amino acid difference between B*3501 and B*3503 within the F pocket inhibited HLA binding by these peptides. The extraordinarily long 13-mer peptide was the target for the CTL response in individuals expressing B*3508, which differs from B*3501 at a single position within the D pocket (B*3501, 156 Leucine; B*3508, 156 Arginine). This minor difference was shown to enhance binding of the 13-mer peptide, presumably through a stabilizing interaction between the negatively charged glutamate at position 3 of the peptide and the positively charged arginine at HLA position 156. The 13-mer epitope defined in this study represents the longest class I-binding viral epitope identified to date as a minimal determinant. Furthermore, the potency of the response indicates that peptides of this length do not present a major structural barrier to CTL recognition.

Predictive vaccinology: Optimisation of predictions using support vector machine classifiers

Promiscuous human leukocyte antigen (HLA) binding peptides are ideal targets for vaccine development. Existing computational models for prediction of promiscuous peptides used hidden Markov models and artificial neural networks as prediction algorithms. We report a system based on support vector machines that outperforms previously published methods. Preliminary testing showed that it can predict peptides binding to HLA-A2 and -A3 super-type molecules with excellent accuracy, even for molecules where no binding data are currently available.

MULTIPRED: A computational system for prediction of promiscuous HLA binding peptides

MULTIPRED is a web-based computational system for the prediction of peptide binding to multiple molecules ( proteins) belonging to human leukocyte antigens (HLA) class I A2, A3 and class II DR supertypes. It uses hidden Markov models and artificial neural network methods as predictive engines. A novel data representation method enables MULTIPRED to predict peptides that promiscuously bind multiple HLA alleles within one HLA supertype. Extensive testing was performed for validation of the prediction models. Testing results show that MULTIPRED is both sensitive and specific and it has good predictive ability ( area under the receiver operating characteristic curve A(ROC) > 0.80). MULTIPRED can be used for the mapping of promiscuous T-cell epitopes as well as the regions of high concentration of these targets termed T-cell epitope hotspots. MULTIPRED is available at http:// antigen.i2r.a-star.edu.sg/ multipred/.

TCR{alpha} Genes Direct MHC Restriction in the Potent Human T Cell Response to a Class I-Bound Viral Epitope

The underlying generic properties of {alpha}β TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR β-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ β-chain was associated with distinct TCR {alpha}-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR {alpha}-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR {alpha}-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.

Association between chronic fatigue syndrome and the corticosteroid-binding globulin gene ALA SER224 polymorphism

Chronic fatigue syndrome (CFS) is characterized by idiopathic fatigue of greater than 6 months' duration with postexertional exacerbation and many other symptoms. A trend toward relative hypocortisolism is described in CFS. Twin and family studies indicate a substantial genetic etiologic component to CFS. Recently, severe corticosteroid-binding globulin (CBG) gene mutations have been associated with CFS in isolated kindreds. Human leukocyte elastase, an enzyme important in CBG catabolism at inflammatory sites, is reported to be elevated in CFS. We hypothesized that CBG gene polymorphisms may act as a genetic risk factor for CFS. A total of 248 patients with CFS defined by Centers for Disease Control criteria, and 248 controls were recruited. Sequencing and restriction enzyme testing of the CBG gene coding region allowed detection of severe CBG gene mutations and a common exon 3 polymorphism (c.825G --> T, Ala-Ser(224)). Plasma CBG levels were measured in 125 CFS patients and 198 controls by radioimmunoassay. Total and free (calculated and measured) cortisol levels were ascertained in single samples between 8-10 a.m. The age of onset (mid 30s) and gender ratio (2.2:1, female:male) of the patients were similar to those reported in U.S. epidemiologic studies. A trend toward a preponderance of serine(224) homozygosity among the CFS patients was noted, compared with controls (chi(2) = 5.31, P = 0.07). Immunoreactive-CBG (IR-CBG) levels were higher in Serine/Alanine (Ser/Ala) than Ala/Ala subjects and higher again in Ser/Ser subjects, this effect was strongest in controls; Ser/Ser: 46.1 +/- 1.8 (n = 31, P = 0.03) vs. Ser/Ala: 42.4 +/- 1.0 (n = 56, P = 0.05) vs. Ala/Ala: 40.8 +/- 1.7 mug/mL (n = 21). Despite higher CBG levels, there was a nonsignificant trend toward lower total and free plasma cortisol in serine allele positive patients, total cortisol: Ser/Ser: 13.3 +/- 1.4 (n = 34) vs. Ser/Ala: 14.0 +/- 0.7 (n = 66) vs. Ala/Ala: 15.4 +/- 1.0 (n = 23). Homozygosity for the serine allele of the CBG gene may predispose to CFS, perhaps due to an effect on hypothalamic-pituitary-adrenal axis function related to altered CBG-cortisol transport function or immune-cortisol interactions.

Extreme learning machine for predicting HLA-peptide binding

Machine learning techniques have been recognized as powerful tools for learning from data. One of the most popular learning techniques, the Back-Propagation (BP) Artificial Neural Networks, can be used as a computer model to predict peptides binding to the Human Leukocyte Antigens (HLA). The major advantage of computational screening is that it reduces the number of wet-lab experiments that need to be performed, significantly reducing the cost and time. A recently developed method, Extreme Learning Machine (ELM), which has superior properties over BP has been investigated to accomplish such tasks. In our work, we found that the ELM is as good as, if not better than, the BP in term of time complexity, accuracy deviations across experiments, and most importantly - prevention from over-fitting for prediction of peptide binding to HLA.

Investigation of oxidative stress defenses of Neisseria gonorrhoeae by using a human polymorphonuclear leukocyte survival assay

Neisseria gonorrhoeae has well-characterized oxidative stress defense systems that protect against oxidative killing in in vitro assays. In contrast, mutant strains of N. gonorrhoeae lacking oxidative stress defenses are identical to the wild type when tested in an ex vivo survival assay using human polymorphonuclear leukocytes.

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single similar to 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')(2) also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to Fc gamma R. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-a, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

The CD1d natural killer T-cell antigen presentation pathway is highly conserved between humans and rhesus macaques

Natural killer T (NKT) cells play an important role in controlling cancers, infectious diseases and autoimmune diseases. Although the rhesus macaque is a useful primate model for many human diseases such as infectious and autoimmune diseases, little is known about their NKT cells. We analyzed Valpha24TCR+ T cells from rhesus macaque peripheral blood mononuclear cells stimulated with aalpha-galactosylceramide (a-GalCer) and interleukin-2. We found that rhesus macaques possess Va24TCR+ T cells, suggesting that recognition of alpha-GalCer is highly conserved between rhesus macaques and humans. The amino acid sequences of the V-J junction for the Valpha24TCR of rhesus macaque and human NKT cells are highly conserved (93% similarity), and the CD1d alpha1-alpha2 domains of both species are highly homologous (95.6%). These findings indicate that the rhesus macaque is a useful primate model for understanding the contribution of NKT cells to the control of human diseases.