International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

BACKGROUND: Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. METHODS: Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. FINDINGS: PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. CONCLUSIONS: The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation

MHC-I peptides get out of the groove and enable a novel mechanism of HIV-1 escape

Major histocompatibility complex class I (MHC-I) molecules play a crucial role in immunity by capturing peptides for presentation to T cells and natural killer (NK) cells. The peptide termini are tethered within the MHC-I antigen-binding groove, but it is unknown whether other presentation modes occur. Here we show that 20% of the HLA-B*57:01 peptide repertoire comprises N-terminally extended sets characterized by a common motif at position 1 (P1) to P2. Structures of HLA-B*57:01 presenting N-terminally extended peptides, including the immunodominant HIV-1 Gag epitope TW10 (TSTLQEQIGW), showed that the N terminus protrudes from the peptide-binding groove. The common escape mutant TSNLQEQIGW bound HLA-B*57:01 canonically, adopting a dramatically different conformation than the TW10 peptide. This affected recognition by killer cell immunoglobulin-like receptor (KIR) 3DL1 expressed on NK cells. We thus define a previously uncharacterized feature of the human leukocyte antigen class I (HLA-I) immunopeptidome that has implications for viral immune escape. We further suggest that recognition of the HLA-B*57:01-TW10 epitope is governed by a 'molecular tension' between the adaptive and innate immune systems

Reduction of incidence and severity of Septoria lycopersici leaf spot of tomato with bacteria and yeasts Redução da incidência e severidade da mancha foliar do tomateiro causada por Septoria lycopersici com bacteria e leveduras

Septoria leaf spot, caused by Septoria lycopersici, is an important disease of tomato (Lycopersicon esculentum) which is mainly controlled by fungicide sprays. One of the alternatives to reduce fungicide applications is the use of leaf antagonists such as yeast and bacterium. This study was conducted from 1994 through 1995 in Auburn, AL, USA. The pathogen and one antagonist were isolated from field plants. In greenhouse, six yeast and one bacterial isolates were tested, in a set of seven experiments. The experiments were conducted in a completely randomized design with four to eight treatments and six replications. The antagonists (1-3 × 10(8) colony forming units ml-1) were inoculated 48h before the inoculation of the pathogen (1-2 × 10(5) conidia ml-1), under conditions of intermittent misting. The yeast isolate Y236 (Cryptococcus laurentii) and the bacterial isolate BTL (Pseudomonas putida) significantly (P <= 0.05) reduced the incidence or the severity of the disease in most experiments.<br>A mancha foliar causada pelo fungo Septoria lycopersici é uma doença no tomateiro (Lycopersicon esculentum), controlada basicamente pela aplicação de fungicidas. Uma das alternativas ao controle químico dessa enfermidade é a utilização de bactérias e leveduras antagonistas. Este estudo foi conduzido de 1994 a 1995 em Auburn, Alabama, EUA. O patógeno e um dos antagonistas foram isolados do filoplano de plantas de tomateiro infectadas pela doença em questão. Sete antagonistas (um isolado de bacteria e seis de leveduras) foram testados em uma série de sete experimentos conduzidos em casa de vegetação. Os experimentos foram conduzidos em um delineamento completamente casualizado com quatro a oito tratamentos e seis repetições. Em todos os experimentos, os antagonistas (1-3 × 10(8) unidades formadoras de colonia ml-1) foram inoculados 48h antes da inoculação com o patógeno (1-2 × 10(5) conidios ml-1), sob condições de nebulosidade intermitente. Entre os antagonistas testados, destacaram-se o isolado de levedura Y236 (Cryptococcus laurentii) e o isolado bacteriano BTL (Pseudomonas putida). Ambos os isolados reduziram significativamente (P <= 0.05) a incidência e a severidade da doença na maioria dos experimentos

Behcet disease-associated MHC class I residues implicate antigen binding and regulation of cell-mediated cytotoxicity

The HLA protein, HLA-B*51, encoded by HLA-B in MHC, is the strongest known genetic risk factor for Behçet disease (BD). Associations between BD and other factors within the MHC have been reported also, although strong regional linkage disequilibrium complicates their confident disentanglement from HLA-B*51. In the current study, we examined a combination of directly obtained and imputed MHC-region SNPs, directly obtained HLA-B locus types, and imputed classical HLA types with their corresponding polymorphic amino acid residues for association with BD in 1,190 cases and 1,257 controls. SNP mapping with logistic regression of the MHC identified the HLA-B/MICA region and the region between HLA-F and HLA-A as independently associated with BD (P < 1.7 × 10(−8)). HLA-B*51, -A*03, -B*15, -B*27, -B*49, -B*57, and -A*26 each contributed independently to BD risk. We directly examined rs116799036, a noncoding SNP upstream of HLA-B that was recently suggested to underlie the association of HLA-B*51 with BD, but we were unable to replicate that finding in our collection. Instead, we mapped the BD association to seven MHC class I (MHC-I) amino acid residues, including anchor residues that critically define the selection and binding of peptides to MHC-I molecules, residues known to influence MHC-I–killer immunoglobulin-like receptor interactions, and a residue located in the signal peptide of HLA-B. The locations of these variants collectively implicate MHC-I peptide binding in the pathophysiology of BD. Furthermore, several lines of evidence suggest a role for altered regulation of cellular cytotoxicity in BD pathogenesis

Human herpesvirus 6A accelerates AIDS progression in macaques

Although HIV is the necessary and sufficient causative agent of AIDS, genetic and environmental factors markedly influence the pace of disease progression. Clinical and experimental evidence suggests that human herpesvirus 6A (HHV-6A), a cytopathic T-lymphotropic DNA virus, fosters the progression to AIDS in synergy with HIV-1. In this study, we investigated the effect of coinfection with HHV-6A on the progression of simian immunodeficiency virus (SIV) disease in pig-tailed macaques (Macaca nemestrina). Inoculation of HHV-6A resulted in a rapid appearance of plasma viremia associated with transient clinical manifestations and followed by antibody seroconversion, indicating that this primate species is susceptible to HHV-6A infection. Whereas animals infected with HHV-6A alone did not show any long-term clinical and immunological sequelae, a progressive loss of CD4(+) T cells was observed in all of the macaques inoculated with SIV. However, progression to full-blown AIDS was dramatically accelerated by coinfection with HHV-6A. Rapid disease development in dually infected animals was heralded by an early depletion of both CD4(+) and CD8(+) T cells. These results provide in vivo evidence that HHV-6A may act as a promoting factor in AIDS progression