Multiple T-cell epitopes overlap positively-selected residues in the p1 spacer protein of HIV-1 gag.

OBJECTIVES: The p1 region of HIV-1 gag contains the frameshift stem-loop, gag-pol transframe and a protease cleavage site that are crucial for viral assembly, replication and infectivity. Identifying and characterizing CD8+ epitopes that are under host immune selection in this region will help in designing effective vaccines for HIV-1. DESIGN: An approach combining bioinformatical analysis and interferon gamma enzyme-linked immunosorbent spot (ELISPOT) assays is used to identify and characterize the epitopes. Potential human leukocyte antigen (HLA)-restricted epitopes were identified by correlating the positively-selected mutations with host HLA alleles. METHODS: ELISPOT analysis with overlapping peptides was used to confirm and characterize the epitopes. RESULTS: Four positively-selected residues were significantly associated with HLA class I alleles, including HLA B*1302 (K4R, P = 0.0008 and I5L, P = 0.0108), A*7401 (S9N, P = 0.0002) and A*30 genotypes (P7S, P = 0.009), suggesting epitopes restricted by these alleles are present in this region. ELISPOT analysis with patient peripheral blood mononuclear cells (PBMCs) identified seven novel epitopes restricted by the 3 alleles. Two types of epitopes were observed in this region based on the ELISPOT responses, Type I: the positively-selected variation does not affect CD8+ T-cell responses; and Type II: the CD8+ T-cell responses are determined by the epitope variants. CONCLUSION: We identified and characterized seven novel CD8+ epitopes in the p1 spacer protein region. Classifying the effects of positively-selected variants on CD8+ T-cell responses will help in designing effective vaccines for HIV-1.

An integrative bioinformatic approach for studying escape mutations in human immunodeficiency virus type 1 gag in the Pumwani Sex Worker Cohort.

Human immunodeficiency virus type 1 (HIV-1) is able to evade the host cytotoxic T-lymphocyte (CTL) response through a variety of escape avenues. Epitopes that are presented to CTLs are first processed in the presenting cell in several steps, including proteasomal cleavage, transport to the endoplasmic reticulum, binding by the HLA molecule, and finally presentation to the T-cell receptor. An understanding of the potential of the virus to escape CTL responses can aid in designing an effective vaccine. To investigate such a potential, we analyzed HIV-1 gag from 468 HIV-1-positive Kenyan women by using several bioinformatic approaches that allowed the identification of positively selected amino acids in the HIV-1 gag region and study of the effects that these mutations could have on the various stages of antigen processing. Correlations between positively selected residues and mean CD4 counts also allowed study of the effect of mutation on HIV disease progression. A number of mutations that could create or destroy proteasomal cleavage sites or reduce binding affinity of the transport antigen processing protein, effectively hindering epitope presentation, were identified. Many mutations correlated with the presence of specific HLA alleles and with lower or higher CD4 counts. For instance, the mutation V190I in subtype A1-infected individuals is associated with HLA-B*5802 (P = 4.73 x 10(-4)), a rapid-progression allele according to other studies, and also to a decreased mean CD4 count (P = 0.019). Thus, V190I is a possible HLA escape mutant. This method classifies many positively selected mutations across the entire gag region according to their potential for immune escape and their effect on disease progression.

Development of a competitive enzyme linked immunosorbent assay to identify epitope specific antibodies in recipients of the U.S. licensed anthrax vaccine.

Vaccination with anthrax vaccine adsorbed (AVA) results in the production of protective antigen (PA) specific antibodies, which play an important protective role against anthrax toxins. Analyzing the specificity of serum antibodies generated in response to AVA vaccination can provide insight into the mechanisms of protective immunity against this important pathogen. The goal of this study was to develop a competitive enzyme linked immunosorbent assay (cELISA) to test human immune serum for antibodies specific for a known lethal toxin neutralizing epitope in PA. PA-specific antibodies in sera from individuals who received the six-dose AVA vaccine series competed for binding to immobilized PA with monoclonal antibody F20G75, which binds to a linear epitope in domain 2 of PA and neutralizes lethal toxin activity in vitro. These results suggest that antibodies in human AVA vaccinee serum recognize the same epitope as F20G75, or one in close proximity to it, and may serve a protective role against anthrax lethal toxin. This assay may be used for serological confirmation of successful immunization against anthrax and for the identification of antibodies in human vaccinee serum that recognize protective epitopes on PA.

Production and characterization of a monoclonal antibody to Francisella tularensis lipopolysaccharide.

Having the capacity to detect and identify pathogens that can be employed in a bioterror attack is critical from both a public health and defence perspective. Immunodiagnostic assays are useful tools for enhancing such detection capabilities. In order to develop an immunodiagnostic assay for the detection of Francisella tularensis, a murine monoclonal antibody (MAb) was developed, using the live vaccine strain (LVS) of F. tularensis as the inoculating antigen. A single MAb, F94G2-1, which is specific for the lipopolysaccharide (LPS) of this bacterium was developed and characterized. An indirect ELISA using purified LPS was effective in determining reactivity of the MAb against its target. An immunodotblot and a manually printed antigen microarray were also tested as suitable detection methods. Both assays showed that MAb F94G2-1 has excellent specificity for F. tularensis LPS and demonstrate the utility of using the same MAb in a variety of immunodiagnostic applications.

Production and characterization of neutralizing monoclonal antibodies that recognize an epitope in domain 2 of Bacillus anthracis protective antigen.

Antibodies against the protective antigen (PA) of Bacillus anthracis play a key role in response to infection by this important pathogen. The aim of this study was to produce and characterize monoclonal antibodies (mAbs) specific for PA and to identify novel neutralizing epitopes. Three murine mAbs with high specificity and nanomolar affinity for B. anthracis recombinant protective antigen (rPA) were produced and characterized. Western immunoblot analysis, coupled with epitope mapping using overlapping synthetic peptides, revealed that these mAbs recognize a linear epitope within domain 2 of rPA. Neutralization assays demonstrate that these mAbs effectively neutralize lethal toxin in vitro.

Molecular characterization of a panel of murine monoclonal antibodies specific for the SARS-coronavirus.

The availability of monoclonal antibodies (mAbs) specific for the SARS-coronavirus (SARS-CoV) is important for the development of both diagnostic tools and treatment of infection. A molecular characterization of nine monoclonal antibodies raised in immune mice, using highly purified, inactivated SARS-CoV as the inoculating antigen, is presented in this report. These antibodies are specific for numerous viral protein targets, and six of them are able to effectively neutralize SARS-CoV in vitro, including one with a neutralizing titre of 0.075 nM. A phylogenetic analysis of the heavy and light chain sequences reveals that the mAbs share considerable homology. The majority of the heavy chains belong to a single Ig germline V-gene family, while considerably more sequence variation is evident in the light chain sequences. These analyses demonstrate that neutralization ability can be correlated with specific murine V(H)-gene alleles. For instance, one evident trend is high sequence conservation in the V(H) chains of the neutralizing mAbs, particularly in CDR-1 and CDR-2. The results suggest that optimization of murine mAbs for neutralization of SARS-CoV infection will likely be possible, and will aid in the development of diagnostic tools and passive treatments for SARS-CoV infection.

A simple and rapid protocol for the sequence determination of functional kappa light chain cDNAs from aberrant-chain-positive murine hybridomas.

This protocol describes the application of a polymerase chain reaction to allow the cloning and sequencing of new functional kappa light chain cDNAs from murine hybridomas co-expressing aberrant endogenous kappa chain mRNAs. The presence of kappa light chain aberrant mRNAs can hinder or even prevent determination of the sequence of functional murine kappa light chain cDNAs amplified by PCR from hybridomas. The method described here employs a panel of kappa primers in the presence of molar excess of a primer complementary to the complementary determining region (CDR) 3 of the known aberrant chain sequence. Analysis of the PCR products reveals two bands for some reactions: one the functional, full-length kappa chain cDNA (approximately 400 bp) and another shorter (approximately 100 bp) band corresponding to short aberrant chain kappa CDR3-constant region. The full-length product is gel purified and cloned prior to sequencing and aligned with V-region germline sequences available in NCBI and GenBank databases. This method is used routinely in our laboratory and demonstrates consistency and reliability for sequence determination of kappa light chain V-gene cDNA of mAbs to diverse antigens. This protocol is a rapid and convenient method for determining the sequence of murine V kappa region genes from hybridomas expressing aberrant kappa chain mRNAs.

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus.

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.

FinO is an RNA chaperone that facilitates sense-antisense RNA interactions.

The protein FinO represses F-plasmid conjugative transfer by facilitating interactions between the mRNA of the major F-plasmid transcriptional activator, TraJ, and an antisense RNA, FinP. FinO is known to bind stem-loop structures in both FinP and traJ RNAs; however, the mechanism by which FinO facilitates sense-antisense pairing is poorly understood. Here we show that FinO acts as an RNA chaperone to promote strand exchange and duplexing between minimal RNA targets derived from FinP. This strongly suggests that FinO may function to destabilize internal secondary structures within FinP and traJ RNAs that would otherwise act as a kinetic trap to sense-antisense pairing. The energy for FinO-catalyzed base-pair destabilization does not arise from ATP hydrolysis but appears to be supplied directly from FinO RNA binding free energy. An analysis of the activities of mutants that are specifically deficient in strand exchange but not RNA-binding activity demonstrates that strand exchange is essential to the ability of FinO to mediate sense-antisense RNA recognition, and that this function also plays a role in repression of conjugation in vivo.

Characterizing the structural features of RNA/RNA interactions of the F-plasmid FinOP fertility inhibition system.

F-like plasmid transfer is mediated by the FinOP fertility inhibition system. Expression of the F positive regulatory protein, TraJ, is controlled by the action of the antisense RNA, FinP, and the RNA-binding protein FinO. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA, leading to repression of both traJ expression and conjugative F transfer. FinP antisense RNA secondary structure is composed of two stem-loops separated by a 4-base single-stranded spacer and flanked on each side by single-stranded tails. Here we show that disruption of the expected Watson-Crick base pairing between the loops of FinP stem-loop I and its cognate RNA binding partner, traJ mRNA stem-loop Ic, led to a moderate reduction in the rate of duplex formation in vitro. In vivo, alterations of the anti-ribosome binding site region in the loop of FinP stem-loop I reduced the ability of the mutant FinP to mediate fertility inhibition and to inhibit TraJ expression when expressed in trans at an elevated copy number. Alterations of intermolecular complementarity between the stems of these RNAs reduced the rate of duplex formation. Our results suggest that successful interaction between stem-loop I of FinP and stem-loop Ic of traJ mRNA requires that base pairing must proceed from an initial loop-loop interaction through the top portion of the stems for stable duplex formation to occur.

The positive regulator, TraJ, of the Escherichia coli F plasmid is unstable in a cpxA* background.

The Cpx (conjugative plasmid expression) stress response of Escherichia coli is induced in response to extracytoplasmic signals generated in the cell envelope, such as misfolded proteins in the periplasm. Detection of stress is mediated by the membrane-bound histidine kinase, CpxA. Signaling of the response regulator CpxR by activated CpxA results in the expression of several factors required for responding to cell envelope stress. CpxA was originally thought to be required for the expression of the positive regulator of the F plasmid transfer (tra) operon, TraJ. It was later determined that constitutive gain-of-function mutations in cpxA led to activation of the Cpx envelope stress response and decreased TraJ expression. In order to determine the nature of the downregulation of TraJ, the level of expression of TraJ, TraM, and TraY, the F-encoded regulatory proteins of the F tra region, was determined both in a cpxA* background and in a wild-type background in which the Cpx stress response was induced by overexpression of the outer membrane lipoprotein, NlpE. Our results suggest that TraJ downregulation is controlled by a posttranscriptional mechanism that operates in the cytoplasm in response to upregulation of the Cpx stress response by both the cpxA* gain-of-function mutation and the overexpression of NlpE.