RESUMO
Collapsing focal segmental glomerulosclerosis (FSGS), also known as collapsing glomerulopathy (CG), is the most aggressive variant of FSGS and is characterized by a rapid progression to kidney failure. Understanding CG pathogenesis represents a key step for the development of targeted therapies. Previous work implicated the telomerase protein component TERT in CG pathogenesis, as transgenic TERT expression in adult mice resulted in a CG resembling that seen in human primary CG and HIV-associated nephropathy (HIVAN). Here, we used the telomerase-induced mouse model of CG (i-TERTci mice) to identify mechanisms to inhibit CG pathogenesis. Inactivation of WIP1 phosphatase, a p53 target acting in a negative feedback loop, blocked disease initiation in i-TERTci mice. Repression of disease initiation upon WIP1 deficiency was associated with senescence enhancement and required transforming growth factor-ß functions. The efficacy of a pharmacologic treatment to reduce disease severity in both i-TERTci mice and in a mouse model of HIVAN (Tg26 mice) was then assessed. Pharmacologic inhibition of WIP1 enzymatic activity in either the telomerase mice with CG or in the Tg26 mice promoted partial remission of proteinuria and ameliorated kidney histopathologic features. Histological as well as high-throughput sequencing methods further showed that selective inhibition of WIP1 does not promote kidney fibrosis or inflammation. Thus, our findings suggest that targeting WIP1 may be an effective therapeutic strategy for patients with CG.
Assuntos
Nefropatia Associada a AIDS , Glomerulosclerose Segmentar e Focal , Insuficiência Renal , Telomerase , Adulto , Humanos , Camundongos , Animais , Glomerulosclerose Segmentar e Focal/patologia , Telomerase/uso terapêutico , Nefropatia Associada a AIDS/patologia , Proteinúria , Insuficiência Renal/complicações , Modelos Animais de DoençasRESUMO
Like most epithelial organs, the bladder and kidney can be directly accessed by bacteria evolved for invasion. Epithelia and immune cells attempt to stymie this infection with biophysical and chemical mechanisms. Goldspink et al. connected the Na+ gradient in the kidney medulla with an immune defense mounted by dead cells (namely, the explosive death of neutrophils and macrophages), resulting in extracellular DNA traps. The pathway from Na+ concentration to immune death is depicted.
Assuntos
Armadilhas Extracelulares , Imunidade Inata , Macrófagos , Neutrófilos , Sistema Urinário , Sistema Urinário/imunologia , Neutrófilos/imunologia , Macrófagos/imunologia , Rim , Sódio , Morte Celular , Proteína-Arginina Desiminase do Tipo 4 , Humanos , Animais , Camundongos , Infecções Urinárias/imunologia , Infecções Bacterianas/imunologiaRESUMO
The murine serous cavities contain a rare and enigmatic population of short-lived F4/80lo MHCII+ macrophages but what regulates their development, survival, and fate is unclear. Here, we show that mature F4/80lo MHCII+ peritoneal macrophages arise after birth, but that this occurs largely independently of colonization by microbiota. Rather, microbiota specifically regulate development of a subpopulation of CD11c+ cells that express the immunoregulatory cytokine RELM-α, are reliant on the transcription factor EGR2, and develop independently of the growth factor CSF1. Furthermore, we demonstrate that intrinsic expression of RELM-α, a signature marker shared by CD11c+ and CD11c- F4/80lo MHCII+ cavity macrophages, regulates survival and differentiation of these cells in the peritoneal cavity in a sex-specific manner. Thus, we identify a previously unappreciated diversity in serous cavity F4/80lo MHCII+ macrophages that is regulated by microbiota, and describe a novel sex and site-specific function for RELM-α in regulating macrophage endurance that reveals the unique survival challenge presented to monocyte-derived macrophages by the female peritoneal environment.
Assuntos
Antígeno CD11c , Proteína 2 de Resposta de Crescimento Precoce , Macrófagos Peritoneais , Microbiota , Animais , Antígeno CD11c/metabolismo , Diferenciação Celular , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Feminino , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Caracteres SexuaisRESUMO
BACKGROUND: To gain insight into the pathogenesis of collapsing glomerulopathy, a rare form of FSGS that often arises in the setting of viral infections, we performed a genome-wide association study (GWAS) among inbred mouse strains using a murine model of HIV-1 associated nephropathy (HIVAN). METHODS: We first generated F1 hybrids between HIV-1 transgenic mice on the FVB/NJ background and 20 inbred laboratory strains. Analysis of histology, BUN, and urinary NGAL demonstrated marked phenotypic variation among the transgenic F1 hybrids, providing strong evidence for host genetic factors in the predisposition to nephropathy. A GWAS in 365 transgenic F1 hybrids generated from these 20 inbred strains was performed. RESULTS: We identified a genome-wide significant locus on chromosome 13-C3 and multiple additional suggestive loci. Crossannotation of the Chr. 13 locus, including single-cell transcriptomic analysis of wildtype and HIV-1 transgenic mouse kidneys, nominated Ssbp2 as the most likely candidate gene. Ssbp2 is highly expressed in podocytes, encodes a transcriptional cofactor that interacts with LDB1 and LMX1B, which are both previously implicated in FSGS. Consistent with these data, older Ssbp2 null mice spontaneously develop glomerulosclerosis, tubular casts, interstitial fibrosis, and inflammation, similar to the HIVAN mouse model. CONCLUSIONS: These findings demonstrate the utility of GWAS in mice to uncover host genetic factors for rare kidney traits and suggest Ssbp2 as susceptibility gene for HIVAN, potentially acting via the LDB1-LMX1B transcriptional network.
Assuntos
Nefropatia Associada a AIDS/genética , Proteínas de Ligação a DNA/genética , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Glomerulosclerose Segmentar e Focal/genética , Animais , Modelos Animais de Doenças , Feminino , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: In the context of kidney transplantation, genomic incompatibilities between donor and recipient may lead to allosensitization against new antigens. We hypothesized that recessive inheritance of gene-disrupting variants may represent a risk factor for allograft rejection. METHODS: We performed a two-stage genetic association study of kidney allograft rejection. In the first stage, we performed a recessive association screen of 50 common gene-intersecting deletion polymorphisms in a cohort of kidney transplant recipients. In the second stage, we replicated our findings in three independent cohorts of donor-recipient pairs. We defined genomic collision as a specific donor-recipient genotype combination in which a recipient who was homozygous for a gene-intersecting deletion received a transplant from a nonhomozygous donor. Identification of alloantibodies was performed with the use of protein arrays, enzyme-linked immunosorbent assays, and Western blot analyses. RESULTS: In the discovery cohort, which included 705 recipients, we found a significant association with allograft rejection at the LIMS1 locus represented by rs893403 (hazard ratio with the risk genotype vs. nonrisk genotypes, 1.84; 95% confidence interval [CI], 1.35 to 2.50; P = 9.8×10-5). This effect was replicated under the genomic-collision model in three independent cohorts involving a total of 2004 donor-recipient pairs (hazard ratio, 1.55; 95% CI, 1.25 to 1.93; P = 6.5×10-5). In the combined analysis (discovery cohort plus replication cohorts), the risk genotype was associated with a higher risk of rejection than the nonrisk genotype (hazard ratio, 1.63; 95% CI, 1.37 to 1.95; P = 4.7×10-8). We identified a specific antibody response against LIMS1, a kidney-expressed protein encoded within the collision locus. The response involved predominantly IgG2 and IgG3 antibody subclasses. CONCLUSIONS: We found that the LIMS1 locus appeared to encode a minor histocompatibility antigen. Genomic collision at this locus was associated with rejection of the kidney allograft and with production of anti-LIMS1 IgG2 and IgG3. (Funded by the Columbia University Transplant Center and others.).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Variações do Número de Cópias de DNA , Rejeição de Enxerto/genética , Transplante de Rim , Proteínas com Domínio LIM/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Estudos de Coortes , Estudos de Associação Genética , Genótipo , Antígenos HLA/genética , Teste de Histocompatibilidade , Humanos , Imunoglobulina G/sangue , Proteínas com Domínio LIM/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Polimorfismo de Nucleotídeo Único , Doadores de TecidosRESUMO
BACKGROUND: The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. METHODS: We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. RESULTS: We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10-14). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. CONCLUSIONS: We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Deleção Cromossômica , Síndrome de DiGeorge/genética , Haploinsuficiência , Rim/anormalidades , Proteínas Nucleares/genética , Sistema Urinário/anormalidades , Adolescente , Animais , Criança , Cromossomos Humanos Par 22 , Exoma , Feminino , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Modelos Animais , Análise de Sequência de DNA , Adulto Jovem , Peixe-ZebraRESUMO
Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4(+) T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4(+) T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Células Dendríticas/enzimologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Proteína do Núcleo p24 do HIV/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Proteína do Núcleo p24 do HIV/química , Proteína do Núcleo p24 do HIV/imunologia , HIV-1/imunologia , Antígeno HLA-B27/imunologia , Antígeno HLA-B35/imunologia , Humanos , Ativação Linfocitária , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologiaRESUMO
The bladder supports a diversity of macrophage populations with functional roles related to homeostasis and host defense, including clearance of cell debris from tissue, immune surveillance, and inflammatory responses. This review examines these roles with particular attention given to macrophage origins, differentiation, recruitment, and engagement in host defense against urinary tract infections (UTIs), where these cells recognize uropathogens through a combination of receptor-mediated responses. Time is an important variable that is often overlooked in many clinical and biological studies, including in relation to macrophages and UTIs. Given that ageing is a significant factor in urinary tract infection pathogenesis and macrophages have been shown to harbor their own circadian system, this review also explores the influence of age on macrophage functions and the role of diurnal variations in macrophage functions in host defense and inflammation during UTIs. We provide a conceptual framework for future studies that address these key knowledge gaps.
Assuntos
Ritmo Circadiano , Infecções Urinárias , Humanos , Macrófagos , Inflamação/etiologia , EnvelhecimentoRESUMO
Immunoglobulin A (IgA) mediates mucosal responses to food antigens and the intestinal microbiome and is involved in susceptibility to mucosal pathogens, celiac disease, inflammatory bowel disease, and IgA nephropathy. We performed a genome-wide association study of serum IgA levels in 41,263 individuals of diverse ancestries and identified 20 genome-wide significant loci, including 9 known and 11 novel loci. Co-localization analyses with expression QTLs prioritized candidate genes for 14 of 20 significant loci. Most loci encoded genes that produced immune defects and IgA abnormalities when genetically manipulated in mice. We also observed positive genetic correlations of serum IgA levels with IgA nephropathy, type 2 diabetes, and body mass index, and negative correlations with celiac disease, inflammatory bowel disease, and several infections. Mendelian randomization supported elevated serum IgA as a causal factor in IgA nephropathy. African ancestry was consistently associated with higher serum IgA levels and greater frequency of IgA-increasing alleles compared to other ancestries. Our findings provide novel insights into the genetic regulation of IgA levels and its potential role in human disease.
Assuntos
Doença Celíaca , Diabetes Mellitus Tipo 2 , Glomerulonefrite por IGA , Doenças Inflamatórias Intestinais , Humanos , Camundongos , Animais , Glomerulonefrite por IGA/genética , Glomerulonefrite por IGA/complicações , Estudo de Associação Genômica Ampla , Doença Celíaca/genética , Predisposição Genética para Doença , Diabetes Mellitus Tipo 2/complicações , Imunoglobulina A/genética , Rim/metabolismoRESUMO
Proteasomes are the major source of proteases responsible for the generation of peptides bound to major histocompatibility complex class I molecules. Antigens, adjuvants, and cytokines can modulate the composition and enzymatic activity of proteasomes and thus alter the epitopes generated. In the present study, we examined the effect of human immunodeficiency virus type 1 (HIV-1) p24 on proteasomes from a dendritic cell line (JAWS II), from a macrophage cell line (C2.3), and from murine primary bone marrow-derived macrophages and dendritic cells. HIV-1 p24 downregulated PA28beta and the beta2i subunit of the immunoproteasome complex in JAWS II cells but did not decrease the immunoproteasome subunits in macrophages, whereas in primary dendritic cells, PA28alpha, beta2i, and beta5i were downregulated. Exposure of JAWS II cells and primary dendritic cells to HIV-1 p24 for 90 min significantly decreased the presentation of ovalbumin to a SIINFEKL-specific CD8(+) T-cell hybridoma. The decrease in antigen presentation and the downmodulation of the immunoproteasome subunits in JAWS II cells and primary dendritic cells could be overcome by pretreating the cells with gamma interferon for 6 h or by exposing the cells to HIV-1 p24 encapsulated in liposomes containing lipid A. These results suggest that early antigen processing kinetics could influence the immunogenicity of CD8(+) T-cell epitopes generated.
Assuntos
Apresentação de Antígeno , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Animais , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/virologia , Regulação para Baixo , Feminino , Infecções por HIV/virologia , Humanos , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by several neutralizing antibodies (NAbs) and is of interest for vaccine design. In this study, we identified novel MPER peptide mimotopes and evaluated their reactivity with HIV + plasma antibodies to characterize the diversity of the immune responses to MPER during natural infection. We utilized phage display technology to generate novel mimotopes that fit antigen-binding sites of MPER NAbs 4E10, 2F5 and Z13. Plasma antibodies from 10 HIV + patients were mapped by phage immunoprecipitation, to identify unique patient MPER binding profiles that were distinct from, and overlapping with, those of MPER NAbs. 4E10 mimotope binding profiles correlated with plasma neutralization of HIV-2/HIV-1 MPER chimeric virus, and with overall plasma neutralization breadth and potency. When administered as vaccines, 4E10 mimotopes elicited low titer NAb responses in mice. HIV mimotopes may be useful for detailed analysis of plasma antibody specificity.
Assuntos
Epitopos/química , Anticorpos Anti-HIV/biossíntese , Proteína gp41 do Envelope de HIV/química , Infecções por HIV/prevenção & controle , HIV-1/imunologia , HIV-2/imunologia , Vacinas contra a AIDS/administração & dosagem , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Especificidade de Anticorpos , Antígenos Virais/química , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Proteína gp41 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-2/efeitos dos fármacos , HIV-2/genética , Humanos , Imunidade Humoral/efeitos dos fármacos , Imunização/métodos , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação ProteicaRESUMO
Natural killer (NK) cells limit viral replication by direct recognition of infected cells, antibody-dependent cellular cytotoxicity (ADCC), and releasing cytokines. Although growing evidence supports NK cell antiviral immunity in HIV-1 infection, further knowledge of their response is necessary. Here we show that NK cells responding to models of direct cell recognition, ADCC, and cytokine activation have unique transcriptional fingerprints. Compared with healthy volunteers, individuals with chronic HIV-1 infection have higher expression of genes commonly associated with activation, and lower expression of genes associated with direct cell recognition and cytokine stimulation in their NK cells. By contrast, NK cell transcriptional profiles of individuals receiving a modified vaccinia Ankara (MVA) vectored HIV-1 vaccine show upregulation of genes associated with direct cell recognition. These findings demonstrate that targeted transcriptional profiling provides a sensitive assessment of NK cell activity, which helps understand how NK cells respond to viral infections and vaccination.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/metabolismo , Células Matadoras Naturais/metabolismo , Transcriptoma , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular , Citocinas/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Anticorpos Anti-HIV/sangue , HIV-1 , Humanos , Transcrição Gênica , Vacinação , Vaccinia virusRESUMO
Peritoneal macrophages are one of the most studied macrophage populations in the body, yet the composition, developmental origin and mechanisms governing the maintenance of this compartment are controversial. Here we show resident F4/80(hi)GATA6(+) macrophages are long-lived, undergo non-stochastic self-renewal and retain cells of embryonic origin for at least 4 months in mice. However, Ly6C(+) monocytes constitutively enter the peritoneal cavity in a CCR2-dependent manner, where they mature into short-lived F4/80(lo)MHCII(+) cells that act, in part, as precursors of F4/80(hi)GATA6(+) macrophages. Notably, monocyte-derived F4/80(hi) macrophages eventually displace the embryonic population with age in a process that is highly gender dependent and not due to proliferative exhaustion of the incumbent embryonic population, despite the greater proliferative activity of newly recruited cells. Furthermore, although monocyte-derived cells acquire key characteristics of the embryonic population, expression of Tim4 was impaired, leading to cumulative changes in the population with age.
Assuntos
Envelhecimento/fisiologia , Autorrenovação Celular , Embrião de Mamíferos/citologia , Macrófagos/citologia , Animais , Proliferação de Células , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células Mieloides/metabolismo , Peritônio/citologia , Fenótipo , Receptores CCR2/metabolismo , Fatores de TempoRESUMO
Proteasomes play an important role in cell homeostasis and in orchestrating the immune response by systematically degrading foreign proteins and misfolded or damaged host cell proteins. We describe a protocol to purify functionally active proteasomes from human CD4(+) T cells and dendritic cells derived from peripheral blood mononuclear cells. The purification is a three-step process involving ion-exchange chromatography, ammonium sulfate precipitation, and sucrose density gradient ultracentrifugation. This method can be easily adapted to purify proteasomes from cell lines or from organs. Methods to characterize and visualize the purified proteasomes are also described.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Células Dendríticas/enzimologia , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Complexo de Endopeptidases do Proteassoma/imunologiaRESUMO
The flanking amino acids that surround epitopes are critical for effective antigen processing and maintenance of epitope integrity. In the present study, the frequency and characteristics of each amino acid that flanked the peptides generated from the proteasomal degradation of three different subtypes of HIV-1 Gag-p24 were determined. Synthetic flanking regions were designed based on the highest and the lowest frequencies of amino acid with the ideal characteristics at positions upstream and downstream of the proteasomal cleavage site. Peptides were synthesized that contained known CD8+ CTL-epitopes from HIV-1 Gag, CMV pp65, and vaccinia proteins HRP-2, and C16, flanked by amino acid sequences specifically designed to either generate or inhibit the known CD8+ CTL-epitopes. As predicted, the known CD8+ CTL-epitopes were effectively generated from the peptides with synthetic flanking regions specifically designed to promote epitope generation in a proteasome-dependent manner. The majority of the proteasome-generated epitopes were cleaved immediately after the C-terminal amino acid of the specific CTL-epitope. The synthetic peptide sequences containing known CD8+ CTL-epitopes with the flanking regions that promote epitope generation were effectively processed and presented to epitope specific CD8+ T-cells resulting in the production of IFN-γ. These results highlight the importance of flanking regions in promoting efficient antigen processing and presentation. This concept can have important implications in vaccine design and development strategies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Proteína do Núcleo p24 do HIV/imunologia , Sequência de Aminoácidos , Humanos , Interferon gama/imunologia , Dados de Sequência Molecular , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologiaRESUMO
Vaccine development efforts will be guided by algorithms that predict immunogenic epitopes. Such prediction methods rely on classification-based algorithms that are trained against curated data sets of known B and T cell epitopes. It is unclear whether this empirical approach can be applied prospectively to predict epitopes associated with protective immunity for novel antigens. We present a comprehensive comparison of in silico B and T cell epitope predictions with in vivo validation using an previously uncharacterized malaria antigen, CelTOS. CelTOS has no known conserved structural elements with any known proteins, and thus is not represented in any epitope databases used to train prediction algorithms. This analysis represents a blind assessment of this approach in the context of a novel, immunologically relevant antigen. The limited accuracy of the tested algorithms to predict the in vivo immune responses emphasizes the need to improve their predictive capabilities for use as tools in vaccine design.