Immunogenicity of the outer domain of a HIV-1 clade C gp120

Background: The possibility that a sub domain of a C clade HIV-1 gp120 could act as an effective immunogen was investigated. To do this, the outer domain ( OD) of gp120(CN54) was expressed and characterized in a construct marked by a re-introduced conformational epitope for MAb 2G12. The expressed sequence showed efficient epitope retention on the isolated ODCN54 suggesting authentic folding. To facilitate purification and subsequent immunogenicity ODCN54 was fused to the Fc domain of human IgGl. Mice were immunised with the resulting fusion proteins and also with gp120(CN54)-Fc and gp120 alone. Results: Fusion to Fc was found to stimulate antibody titre and Fc tagged ODCN54 was substantially more immunogenic than non-tagged gp120. Immunogenicity appeared the result of Fc facilitated antigen processing as immunisation with an Fc domain mutant that reduced binding to the FcR lead to a reduction in antibody titre when compared to the parental sequence. The breadth of the antibody response was assessed by serum reaction with five overlapping fragments of gp120(CN54) expressed as GST fusion proteins in bacteria. A predominant anti-inner domain and anti-V3C3 response was observed following immunisation with gp120(CN54)-Fc and an anti-V3C3 response to the ODCN54-Fc fusion. Conclusion: The outer domain of gp120(CN54) is correctly folded following expression as a C terminal fusion protein. Immunogenicity is substantial when targeted to antigen presenting cells but shows V3 dominance in the polyvalent response. The gp120 outer domain has potential as a candidate vaccine component.

Vaccine glycoproteins tagged with the human Fc domain as second generation vaccine candidates

Traditional vaccines such as inactivated or live attenuated vaccines, are gradually giving way to more biochemically defined vaccines that are most often based on a recombinant antigen known to possess neutralizing epitopes. Such vaccines can offer improvements in speed, safety and manufacturing process but an inevitable consequence of their high degree of purification is that immunogenicity is reduced through the lack of the innate triggering molecules present in more complex preparations. Targeting recombinant vaccines to antigen presenting cells (APCs) such as dendritic cells however can improve immunogenicity by ensuring that antigen processing is as efficient as possible. Immune complexes, one of a number of routes of APC targeting, are mimicked by a recombinant approach, crystallizable fragment (Fc) fusion proteins, in which the target immunogen is linked directly to an antibody effector domain capable of interaction with receptors, FcR, on the APC cell surface. A number of virus Fc fusion proteins have been expressed in insect cells using the baculovirus expression system and shown to be efficiently produced and purified. Their use for immunization next to non-Fc tagged equivalents shows that they are powerfully immunogenic in the absence of added adjuvant and that immune stimulation is the result of the Fc-FcR interaction.

Expression of the receptor tyrosine kinase ephb2 on dendritic cells is modulated by toll-like receptor ligation but is not required for t cell activation

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.

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Intra-individual variation of serum prostate specific antigen levels in men with benign prostate biopsies

To determine the intra-individual (physiological) variation of prostate-specific antigen (PSA) measurements in men after a benign prostatic biopsy. Sixty-four men were prospectively assessed, all of whom had a benign prostatic biopsy within the preceding 13 months. The degree of intra-individual variability was established by calculating the coefficient of variation on four PSA levels obtained from each patient weekly over a month. Six patients were subsequently diagnosed with prostate cancer and their data are presented separately. In the remaining 58 patients the median (range) individual mean PSA value was 6.3 (0.5-34.1) ng/mL. The median (range) coefficient of variation within the group was 9.5 (2.4-76.1)%. There was a clear linear relationship between mean PSA level and the standard deviation. In 48 of the 63 patients analysed, the coefficient of variation for serum PSA values in the group as a whole was greater than the variation claimed for the assay technique. The significance of the linear relationship between PSA and the standard deviation is discussed, with particular reference to those men who had a benign prostate biopsy.

Analysis of the antigen combining site: Correlations between length and sequence composition of the hypervariable loops and the nature of the antigen

It has long been suggested that the overall shape of the antigen combining site (ACS) of antibodies is correlated with the nature of the antigen. For example, deep pockets are characteristic of antibodies that bind haptens, grooves indicate peptide binders, while antibodies that bind to proteins have relatively flat combining sites. In. 1996, MacCallum, Martin and Thornton used a fractal shape descriptor and showed a strong correlation of the shape of the binding region with the general nature of the antigen. However, the shape of the ACS is determined primarily by the lengths of the six complementarity-determining regions (CDRs). Here, we make a direct correlation between the lengths of the CDRs and the nature of the antigen. In addition, we show significant differences in the residue composition of the CDRs of antibodies that bind to different antigen classes. As well as helping us to understand the process of antigen recognition, autoimmune disease and cross-reactivity these results are of direct application in the design of antibody phage libraries and modification of affinity. (C) 2003 Elsevier Science Ltd. All rights reserved.

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: Preserved folding energy drives fiber formation

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.

Childhood emotional abuse and neglect as predictors of psychological and physical symptoms in women presenting to a primary care practice

Objective: There were two aims to this study: first to examine whether emotional abuse and neglect are significant predictors of psychological and somatic symptoms, and lifetime trauma exposure in women presenting to a primary care practice, and second to examine the strength of these relationships after controlling for the effects of other types of childhood abuse and trauma. Method: Two-hundred and five women completed the Childhood Trauma Questionnaire (Bernstein et al., 1994), Trauma History Questionnaire (Green, 1996), the Symptom Checklist-revised (Derogatis, 1997), and the Revised Civilian Mississippi Scale for posttraumatic stress disorder (Norris & Perilla, 1996) when presenting to their primary care physician for a visit. Hierarchical multiple regression analyses were conducted to examine unique contributions of emotional abuse and neglect variables on symptom measures while controlling for childhood sexual and physical abuse and lifetime trauma exposure. Results: A history of emotional abuse and neglect was associated with increased anxiety, depression, posttraumatic stress and physical symptoms, as well as lifetime trauma exposure. Physical and sexual abuse and lifetime trauma were also significant predictors of physical and psychological symptoms. Hierarchical multiple regressions demonstrated that emotional abuse and neglect predicted symptomatology in these women even when controlling for other types of abuse and lifetime trauma exposure. Conclusions: Long-standing behavioral consequences may arise as a result of childhood emotional abuse and neglect, specifically, poorer emotional and physical functioning, and vulnerability to further trauma exposure. (C) 2003 Elsevier Ltd. All rights reserved.

Effects of presenting the baseline risk when communicating absolute and relative risk reductions

This study compares relative and absolute forms of presenting risk information about influenza and the need for vaccination. It investigates whether differences in people's risk estimates and their evaluations of risk information, as a result of the different presentation formats, are still apparent when they are provided with information about the baseline level of risk. The results showed that, in the absence of baseline information, the relative risk format resulted in higher ratings of satisfaction, perceived effectiveness of vaccination, and likelihood of being vaccinated. However, these differences were not apparent when baseline information was presented. Overall, provision of baseline information resulted in more accurate risk estimates and more positive evaluations of the risk messages. It is recommended that, in order to facilitate shared and fully informed decision making, information about baseline level of risk should be included in all health communications specifying risk reductions, irrespective of the particular format adopted.

Comparison of two methods of presenting risk information to patients about the side effects of medicines

Objective: To determine whether the use of verbal descriptors suggested by the European Union (EU) such as "common" (1-10% frequency) and "rare" (0.01-0.1%) effectively conveys the level of risk of side effects to people taking a medicine. Design: Randomised controlled study with unconcealed allocation. Participants: 120 adults taking simvastatin or atorvastatin after cardiac surgery or myocardial infarction. Setting: Cardiac rehabilitation clinics at two hospitals in Leeds, UK. Intervention: A written statement about one of the side effects of the medicine (either constipation or pancreatitis). Within each side effect condition half the patients were given the information in verbal form and half in numerical form (for constipation, "common" or 2.5%; for pancreatitis, "rare" or 0.04%). Main outcome measure: The estimated likelihood of the side effect occurring. Other outcome measures related to the perceived severity of the side effect, its risk to health, and its effect on decisions about whether to take the medicine. Results: The mean likelihood estimate given for the constipation side effect was 34.2% in the verbal group and 8.1% in the numerical group; for pancreatitis it was 18% in the verbal group and 2.1% in the numerical group. The verbal descriptors were associated with more negative perceptions of the medicine than their equivalent numerical descriptors. Conclusions: Patients want and need understandable information about medicines and their risks and benefits. This is essential if they are to become partners in medicine taking. The use of verbal descriptors to improve the level of information about side effect risk leads to overestimation of the level of harm and may lead patients to make inappropriate decisions about whether or not they take the medicine.

Non-promoting diterpene esters can induce Epstein-Barr Virus early antigen expression in the Raji cell line

A range of diterpene ester ligands with selective biological activity (e.g., irritant but not tumour promoting) were tested for their ability to induce Epstein-Barr virus (EBV) early antigen expression in the lymphoblastoid Raji cell line. All substituted compounds were found to be capable of inducing some antigen expression at nM−μM levels, including desacetyl-α-sapinine, a compound largely devoid of biological activity. The non-promoting, fluorescent compound, sapintoxin A, was virtually equipotent with promoting compounds. It was concluded that, although the assay has relevance to the specific condition of chronic diterpene ester exposure occurring in conjunction with high EBV infection rates, there was relatively poor correlation with mouse skin tumour promoting potential.

Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen

Induction of humoral responses to HIV at mucosal compartments without inflammation is important for vaccine design. We developed charged wax nanoparticles that efficiently adsorb protein antigens and are internalized by DC in the absence of inflammation. HIV-gp140-adsorbed nanoparticles induced stronger in vitro T-cell proliferation responses than antigen alone. Such responses were greatly enhanced when antigen was co-adsorbed with TLR ligands. Immunogenicity studies in mice showed that intradermal vaccination with HIV-gp140 antigen-adsorbed nanoparticles induced high levels of specific IgG. Importantly, intranasal immunization with HIV-gp140-adsorbed nanoparticles greatly enhanced serum and vaginal IgG and IgA responses. Our results show that HIV-gp140-carrying wax nanoparticles can induce strong cellular/humoral immune responses without inflammation and may be of potential use as effective mucosal adjuvants for HIV vaccine candidates.

Conserved hydrophobic clusters on the surface of the Caf1A usher C-terminal domain are important for F1 antigen assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A(C)). Caf1A(C) is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A(C) is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A(C) were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.