Peer support for people living with hepatitis B virus-A foundation for treatment expansion.

Chronic hepatitis B infection (CHB) affects 300 million people worldwide and is being targeted by the United Nations 2030 Sustainable Development Goals (SDGs) and the World Health Organisation (WHO), working towards elimination of hepatitis B virus (HBV) as a public health threat. In this piece, we explore the evidence and potential impact of peer support to enhance and promote interventions for people living with CHB. Peer support workers (PSWs) are those with lived experience of an infection, condition or situation who work to provide support for others, aiming to improve education, prevention, treatment and other clinical interventions and to reduce the physical, psychological and social impacts of disease. Peer support has been shown to be a valuable tool for improving health outcomes for people living with human immunodeficiency virus (HIV) and hepatitis C virus (HCV), but to date has not been widely available for communities affected by HBV. HBV disproportionately affects vulnerable and marginalised populations, who could benefit from PSWs to help them navigate complicated systems and provide advocacy, tackle stigma, improve education and representation, and optimise access to treatment and continuity of care. The scale up of peer support must provide structured and supportive career pathways for PSWs, account for social and cultural needs of different communities, adapt to differing healthcare systems and provide flexibility in approaches to care. Investment in peer support for people living with CHB could increase diagnosis, improve retention in care, and support design and roll out of interventions that can contribute to global elimination goals.

Multiple risk factors for persistent HBV viraemia in an adult receiving nucleos/tide analogue therapy.

Diagnosing and treating chronic hepatitis B virus (HBV) infection are key interventions to support progress towards elimination of viral hepatitis by 2030. Although nucleos/tide analogue (NA) therapy is typically highly effective, challenges remain for viral load (VL) suppression, including medication access, incomplete adherence and drug resistance. We present a case of a long-term HBV and HIV coinfected adult prescribed with sequential NA therapy regimens, with episodes of breakthrough viraemia. Multiple factors contribute to virological breakthrough, including exposure to old NA agents, initial high HBV VL, therapy interruptions, intercurrent illnesses and potential contribution from resistance mutations. The case underscores the importance of individualised treatment approaches and adherence support in achieving HBV suppression. Furthermore, it emphasises the need for improved clinical pathways addressing education, support and access to care, particularly for marginalised populations. Comprehensive data collection inclusive of under-represented individuals is crucial for maintaining retention in the care cascade and informing effective interventions.

Falling treatment uptake in the hepatitis C care cascade is a growing threat to achieving elimination.

Most high-income countries are not on track to achieve the World Health Organization hepatitis C elimination targets. As elimination programmes assess growing proportions of patients in community-based pathways, rates of treatment uptake may fall. We aimed to identify factors associated with DAA treatment uptake and measure changes in their prevalence over time. We performed a time-to-treatment analysis on 2728 patients approved for hepatitis C Direct-Acting Antiviral treatment in the North Central London region between January 2016 and October 2019. We investigated the association between treatment uptake and factors including assessment/treatment setting (hospital, drug service or prison), patient age, gender, injection drug use, harmful alcohol use, cirrhosis status and previous treatment. The likelihood of treatment uptake was reduced by three independent risk factors. These included assessment setting: prison-based or drug-service pathways (aHR 0.29 or 0.81 vs. hospital outpatient pathway, 95% CI 0.21-0.40 and 0.70-0.94 respectively, p < .001); being UK-born (aHR 0.89 vs. non-UK born, 0.82-0.98, p = .01); and history of harmful alcohol use (aHR 0.84 vs. no history, 0.72-0.99, p = .04). The average number of these risk factors for not starting treatment per patient increased over time (R2  = 0.66 p < .001). Independent of these, there was an additional 5% reduction in rate of treatment initiation in each successive year of the programme (aHR 0.95, 0.91-0.99, p = .02). In conclusion, disengagement from care before treatment uptake was found to be a growing threat to elimination. Despite provision of community-based test-to-cure pathways, there are persistent barriers to treatment uptake and these are increasing over time.

Delivery of biannual ultrasound surveillance for individuals with cirrhosis and cured hepatitis C in the UK.

BACKGROUND: Previous studies show the uptake of biannual ultrasound (US) surveillance in patients with cirrhosis is suboptimal. Here, our goal was to understand in broader terms how surveillance is being delivered to cirrhosis patients with cured hepatitis C in the UK. METHODS: Hepatitis C cirrhosis patients achieving a sustained viral response (SVR) to antiviral therapies were identified from the national Hepatitis-C-Research-UK resource. Data on (i) liver/abdominal US examinations, (ii) HCC diagnoses, and (iii) HCC curative treatment were obtained through record-linkage to national health registries. The rate of US uptake was calculated by dividing the number of US episodes by follow-up time. RESULTS: A total of 1908 cirrhosis patients from 31 liver centres were followed for 3.8 (IQR: 3.4-4.9) years. Overall, 10 396 liver/abdominal USs were identified. The proportion with biannual US was 19% in the first 3 years after SVR and 9% for all follow-up years. Higher uptake of biannual US was associated with attending a liver transplant centre; older age and cirrhosis decompensation. Funnel plot analysis indicated significant inter-centre variability in biannual US uptake, with 6/29 centres outside control limits. Incident HCC occurred in 133 patients, of which 49/133 (37%) were treated with curative intent. The number of US episodes in the two years prior to HCC diagnosis was significantly associated with higher odds of curative-intent treatment (aOR: 1.53; 95% CI: 1.12-2,09; p = .007). CONCLUSIONS: This study provides novel data on the cascade of care for HCC in the UK. Our findings suggest biannual US is poorly targeted, inefficient and is not being delivered equitably to all patients.

Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

The fate of indeterminate liver lesions: What proportion are precursors of hepatocellular carcinoma?

BACKGROUND: The natural history and incidence of hepatocellular carcinoma (HCC) arising from indeterminate liver lesions are not well described. We aimed to define the incidence of HCC in a cohort of patients undergoing surveillance by magnetic resonance imaging (MRI) and estimate any associations with incident HCC. METHODS: We performed a retrospective follow-up study, identifying MRI scans in which indeterminate lesions had been reported between January 2006 and January 2017. Subsequent MRI scan reports were reviewed for incident HCC arising from indeterminate lesions, data were extracted from electronic patient records and survival analysis performed to estimate associations with baseline factors. RESULTS: One hundred and nine patients with indeterminate lesions on MRI were identified. HCC developed in 19 (17%) patients over mean follow up of 4.6 years. Univariate Cox proportional hazards analysis found incident HCC to be significantly associated with baseline low platelet count (hazard ratio (HR) = 7.3 (95% confidence intervals (CI) 2.1-24.9), high serum alpha-fetoprotein level (HR = 2.7 (95% CI 1.0-7.1)) and alcohol consumption above fourteen units weekly (HR = 3.1 (95% CI 1.1-8.7)). Multivariate analysis, however, found that only low platelet count was independently associated with HCC (HR = 5.5 (95% CI 0.6-5.1)). CONCLUSIONS: HCC arises in approximately one fifth of indeterminate liver lesions over 4.6 years and is associated with a low platelet count at the time of first diagnosis of an indeterminate lesion. Incidence of HCC was more common in people with viral hepatitis and in those consuming > 14 units of alcohol per week. Our data may be used to support a strategy of enhanced surveillance in patients with indeterminate lesions.

Identification of distinct conformations associated with monomers and fibril assemblies of mutant huntingtin.

The N-terminal fragments of mutant huntingtin (mHTT) misfold and assemble into oligomers, which ultimately bundle into insoluble fibrils. Conformations unique to various assemblies of mHTT remain unknown. Knowledge on the half-life of various multimeric structures of mHTT is also scarce. Using a panel of four new antibodies named PHP1-4, we have identified new conformations in monomers and assembled structures of mHTT. PHP1 and PHP2 bind to epitopes within the proline-rich domain (PRD), whereas PHP3 and PHP4 interact with motifs formed at the junction of polyglutamine (polyQ) and polyproline (polyP) repeats of HTT. The PHP1- and PHP2-reactive epitopes are exposed in fibrils of mHTT exon1 (mHTTx1) generated from recombinant proteins and mHTT assemblies, which progressively accumulate in the nuclei, cell bodies and neuropils in the brains of HD mouse models. Notably, electron microscopic examination of brain sections of HD mice revealed that PHP1- and PHP2-reactive mHTT assemblies are present in myelin sheath and in vesicle-like structures. Moreover, PHP1 and PHP2 antibodies block seeding and subsequent fibril assembly of mHTTx1 in vitro and in a cell culture model of HD. PHP3 and PHP4 bind to epitopes in full-length and N-terminal fragments of monomeric mHTT and binding diminishes as the mHTTx1 assembles into fibrils. Interestingly, PHP3 and PHP4 also prevent the aggregation of mHTTx1 in vitro highlighting a regulatory function for the polyQ-polyP motifs. These newly detected conformations may affect fibril assembly, stability and intercellular transport of mHTT.

Detection, stratification and treatment of hepatitis C-positive prisoners in the United Kingdom prison estate: Development of a pathway of care to facilitate the elimination of hepatitis C in a London prison.

The United Kingdom is committed to eliminating hepatitis C virus (HCV) infection by 2025. The prison estate provides an opportunity to identify and treat HCV-positive individuals in a high-prevalence environment. We designed and implemented a pathway of care within a London prison to diagnose, stratify and link HCV-positive prisoners into care. This study was a two-phase case study of a HCV care pathway. New arrivals to the prison were offered blood-borne virus screening with dried blood spot testing at their secondary health check. Those with active infection completed disease stratification tests and were reviewed at a weekly hospital-based multidisciplinary team meeting to determine management. In Phase-2, the pathway was redesigned to improve testing and the referral of HCV-positive prisoners into treatment. Over the 30-month evaluation period, 12,946 people were received in the prison. During Phase-1, 19.6% of new arrivals completed blood-borne virus testing, with 7.3% identified as HCV-positive. Just 8.3% of HCV-positive individuals were treated or referred for treatment in Phase-1. During Phase-2, 30% of new receptions completed BBV testing and 3.9% were identified as HCV-positive. Linkage into care was improved, with 38.9% treated or referred during the second phase. Poor access to testing and referral to treatment limit the effectiveness of care provision for prisoners with HCV. Elimination of HCV in prisons requires local service configuration to ensure high uptake of testing, with all HCV-positive cases then offered treatment during custody or referral on to treatment after release.

Liver transplant listing for hepatitis C-associated cirrhosis and hepatocellular carcinoma has fallen in the United Kingdom since the introduction of direct-acting antiviral therapy.

Following the introduction of direct-acting antivirals (DAA), there have been reports of declining incidence of hepatitis C (HCV)-related liver disease as a liver transplantation indication. In this study, we assessed the impact of DAA on liver transplant indications in the UK and waiting list outcomes for patients with HCV. We assessed UK adult elective liver transplant registrants between 2006 and 2017. The aetiology of liver disease at registration was reclassified using an accepted hierarchical system and changes were assessed over time and compared before and after the introduction of DAA. Registration UKELD scores and 1-year waiting list outcomes were also compared. The proportion of waiting list patients registered with HCV-related cirrhosis reduced after the introduction of DAA from 10.5% in 2013 to 4.7% in 2016 (P < 0.001). Alcohol-related liver disease (ARLD) was the leading indication for liver transplantation followed by liver cancer (26.1% and 18.4% in 2016, respectively). The proportion of registrations with Hepatocellular carcinoma (HCC) associated with HCV reduced from 46.4% in 2013 to 33.7% in 2016 (P = 0.002). For patients with HCV-related cirrhosis at one year the outcomes of death, transplantation, delisting due to improvement or deterioration and awaiting a graft at 1 year were similar. For patients with HCV-related HCC, the proportion dying at 1 year reduced significantly from 2.9% to 0.0% (P = 0.04). These data demonstrate an association between DAA and reduced listing rates for HCV-related cirrhosis and HCC, but no significant changes in waiting list outcomes other than reduced mortality in the HCC group.

Exploring health economics course competencies' value for nurse administrators.

BACKGROUND: A survey of nursing administrators in the United States was conducted to solicit opinions about health economics (HE) course competencies for baccalaureate nursing programs. PURPOSE: To provide rationale for intensifying educational efforts to increase nurses' awareness about the value of HE skills in their everyday work. METHODS: Participants were asked to rate six HE competencies using a Likert scale. Paired-samples t tests, bivariate correlations, and standard multiple linear regressions were used to analyze survey results. DISCUSSION: A strong direct relationship exists between the amount of prior knowledge nursing administrators have about HE and the perceived level of usefulness of HE skills. CONCLUSION: Nurses' contributions to creating value in healthcare must be encouraged. Increasing the share and accessibility of the HE content in continuing education offerings could be one way to expand HE knowledge among practicing nurses and promote cost-effective delivery of healthcare in the United States.

A critical reanalysis of the relationship between genomics and well-being.

Fredrickson et al. [Fredrickson BL, et al. (2013) Proc Natl Acad Sci USA 110(33):13684-13689] claimed to have observed significant differences in gene expression related to hedonic and eudaimonic dimensions of well-being. Having closely examined both their claims and their data, we draw substantially different conclusions. After identifying some important conceptual and methodological flaws in their argument, we report the results of a series of reanalyses of their dataset. We first applied a variety of exploratory and confirmatory factor analysis techniques to their self-reported well-being data. A number of plausible factor solutions emerged, but none of these corresponded to Fredrickson et al.'s claimed hedonic and eudaimonic dimensions. We next examined the regression analyses that purportedly yielded distinct differential profiles of gene expression associated with the two well-being dimensions. Using the best-fitting two-factor solution that we identified, we obtained effects almost twice as large as those found by Fredrickson et al. using their questionable hedonic and eudaimonic factors. Next, we conducted regression analyses for all possible two-factor solutions of the psychometric data; we found that 69.2% of these gave statistically significant results for both factors, whereas only 0.25% would be expected to do so if the regression process was really able to identify independent differential gene expression effects. Finally, we replaced Fredrickson et al.'s psychometric data with random numbers and continued to find very large numbers of apparently statistically significant effects. We conclude that Fredrickson et al.'s widely publicized claims about the effects of different dimensions of well-being on health-related gene expression are merely artifacts of dubious analyses and erroneous methodology.

Myostatin deficiency but not anti-myostatin blockade induces marked proteomic changes in mouse skeletal muscle.

Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post-developmental Mstn blockade. Using high-resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn(-/-) ) and mice treated for 2-weeks with REGN1033, an anti-Mstn antibody. Relative to wild-type animals, Mstn(-/-) mice had a two-fold greater muscle mass and a >1.5-fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5-fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn(-/-) mice corroborates the mutiple physiological changes including slow-to-fast fiber type switch. Thus, the proteome-wide protein expression differs between Mstn(-/-) mice and mice subjected to specific Mstn blockade post-developmentally, providing molecular-level insights to inform mechanistic hypotheses to explain the observed functional differences.

In-silico discovery of cancer-specific peptide-HLA complexes for targeted therapy.

BACKGROUND: Major Histocompatibility Complex (MHC) or Human Leukocyte Antigen (HLA) Class I molecules bind to peptide fragments of proteins degraded inside the cell and display them on the cell surface. We are interested in peptide-HLA complexes involving peptides that are derived from proteins specifically expressed in cancer cells. Such complexes have been shown to provide an effective means of precisely targeting cancer cells by engineered T-cells and antibodies, which would be an improvement over current chemotherapeutic agents that indiscriminately kill proliferating cells. An important concern with the targeting of peptide-HLA complexes is off-target toxicity that could occur due to the presence of complexes similar to the target complex in cells from essential, normal tissues. RESULTS: We developed a novel computational strategy for identifying potential peptide-HLA cancer targets and evaluating the likelihood of off-target toxicity associated with these targets. Our strategy combines sequence-based and structure-based approaches in a unique way to predict potential off-targets. The focus of our work is on the complexes involving the most frequent HLA class I allele HLA-A*02:01. Using our strategy, we predicted the off-target toxicity observed in past clinical trials. We employed it to perform a first-ever comprehensive exploration of the human peptidome to identify cancer-specific targets utilizing gene expression data from TCGA (The Cancer Genome Atlas) and GTEx (Gene Tissue Expression), and structural data from PDB (Protein Data Bank). We have thus identified a list of 627 peptide-HLA complexes across various TCGA cancer types. CONCLUSION: Peptide-HLA complexes identified using our novel strategy could enable discovery of cancer-specific targets for engineered T-cells or antibody based therapy with minimal off-target toxicity.

Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes.

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.

Impact of direct acting antiviral therapy in patients with chronic hepatitis C and decompensated cirrhosis.

BACKGROUND & AIMS: All oral direct acting antivirals (DAAs) effectively treat chronic hepatitis C virus (HCV) infection, but the benefits in advanced liver disease are unclear. We compared outcomes in treated and untreated patients with decompensated cirrhosis. METHODS: Patients with HCV and decompensated cirrhosis or at risk of irreversible disease were treated in an expanded access programme (EAP) in 2014. Treatment, by clinician choice, was with sofosbuvir, ledipasvir or daclatasvir, with or without ribavirin. For functional outcome comparison, untreated patients with HCV and decompensated cirrhosis who were registered on a database 6months before treatment was available were retrospectively studied. Primary endpoint was sustained virological response 12weeks post antiviral treatment (treated cohort) and the secondary endpoint (both cohorts) was adverse outcomes (worsening in MELD score or serious adverse event) within 6months. RESULTS: 467 patients received treatment (409 decompensated cirrhosis). Viral clearance was achieved in 381 patients (81.6%) - 209 from 231 (90.5%) with genotype 1 and 132 from 192 (68.8%) with genotype 3. MELD scores improved in treated patients (mean change -0.85) but worsened in untreated patients (mean+0.75) (p<0.0001). Patients with initial serum albumin <35g/L, aged >65 or with low (<135mmol/L) baseline serum sodium concentrations were least likely to benefit from therapy. CONCLUSIONS: All oral DAAs effectively cured HCV in patients with advanced liver disease. Viral clearance was associated with improvement in liver function within 6months compared to untreated patients. The longer term impact of HCV treatment in patients with decompensated cirrhosis remains to be determined.

Outcomes after successful direct-acting antiviral therapy for patients with chronic hepatitis C and decompensated cirrhosis.

BACKGROUND & AIMS: Direct-acting antivirals have become widely used for patients with chronic hepatitis C virus infection with decompensated cirrhosis. Virological responses are excellent and early improvements in liver function, at least in a proportion of patients, have been observed but the longer term impact of viral clearance on end-stage liver disease complications is unclear. METHODS: Prospective study of patients with decompensated cirrhosis who received 12weeks of all-oral direct-acting antivirals through the English Expanded Access Programme. Endpoints were deaths, liver transplantation, hepatocellular carcinoma, serious decompensation events, sepsis or hospitalisations, and MELD scores between start of therapy to 15months post-treatment start. An untreated cohort of patients was retrospectively studied over 6months for comparison. RESULTS: Amongst 317/406 patients who achieved sustained virological response at 24weeks post-treatment, there were 9 deaths (3%), 17 new liver cancers (5%), 39 transplantations (12%) and 52 with serious decompensations (16%), over 15months. When compared to the first six months from treatment start and to untreated patients, there was a reduction in incidence of decompensations [30/406 (7%) in months 6-15 and 72/406 (18%) in months 0-6 for treated patients vs. 73/261 (28%) in untreated patients]. There was no significant difference in liver cancer incidence (10/406 (2.5%) in months 6-15 and 17/406 (4%) in months 0-6 for treated patients vs. 11/261 (4%) in untreated patients). CONCLUSIONS: This study suggests that antiviral therapy in patients with decompensated cirrhosis led to prolonged improvement in liver function, with no evidence of paradoxical adverse impact nor increase in liver malignancy. LAY SUMMARY: This is a report of a large group of patients in England who have hepatitis C virus (HCV) infection with advanced liver disease. They have been treated with new anti-HCV drugs, which cured the infection in the majority. This study looks at their outcomes a year following treatment, in terms of deaths, cancers and other complications of advanced liver disease. We conclude that in most patients anti-HCV treatment is beneficial even in advanced liver disease.

Lentiviral expression of GAD67 and CCK promoter-driven opsins to target interneurons in vitro and in vivo.

BACKGROUND: The ability to manipulate the activity of interneurons with optogenetic tools offers the possibility of interfering with diseases caused by altered neuronal inhibition and synchrony, including epilepsy and schizophrenia. To develop vectors for therapeutic approaches, targeting optogenetic constructs to interneurons is therefore a key requirement. We investigated whether the interneuron-specific promoters glutamic acid decarboxylase (GAD)67 and cholecystokinin (CCK) allowed targeted lentiviral delivery of opsins to interneurons as a whole, or specifically CCK+ interneurons. METHODS: We generated lentiviral (LV) plasmids encoding channelrhodopsin (ChR2) and halorhodopsin (NpHR) tagged with fluorophores and driven by GAD67 or CCK promoters. Adeno-associated virus (AAV) and LV vectors carrying opsins driven by pyramidal cell promoters were used as controls. We transduced neuronal cultures and rodent brain in vivo, immunostained specimens 6-8 weeks after in vivo injection and 7-14 days after in vitro transduction, and evaluated volume and specificity of expression by confocal microscopy. RESULTS: In vitro, 90% (19/21) of LV-CCK-NpHR2.0-EYFP expressing neurons were CCK+. In vivo, LV-GAD67-ChR2-mCherry was expressed in 2.6% (5/193), LV-GAD67-NpHR2.0-EYFP in approximately 15% (43/279) and LV-CCK-NpHR2.0-EYFP in 47% (9/19) of hippocampal GABA+ interneurons. GAD67 vectors expressed in larger volumes than CCK-driven constructs. AAV vector controls achieved the largest expression volumes. CONCLUSIONS: LV-CCK-NpHR2.0-EYFP may be useful for targeting CCK+ interneurons in culture. GAD67/CCK-driven lentiviral constructs are expressed in vivo, although expression is not specific for interneurons. Overall, expression levels are low compared to opsins driven by pyramidal cell promoters. A better understanding of GAD67 and CCK promoter structure or alternative techniques is required to reliably target opsins to interneurons using viral vectors.

Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins.

We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders.

4-1BB ligand activates bystander dendritic cells to enhance immunization in trans.

Expression of the costimulatory receptor 4-1BB is induced by TCR recognition of Ag, whereas 4-1BB ligand (4-1BBL) is highly expressed on activated APC. 4-1BB signaling is particularly important for survival of activated and memory CD8(+) T cells. We wished to test whether coexpression of Ag and 4-1BBL by dendritic cells (DC) would be an effective vaccine strategy. Therefore, we constructed lentiviral vectors (LV) coexpressing 4-1BBL and influenza nucleoprotein (NP). Following s.c. immunization of mice, which targets DC, we found superior CD8(+) T cell responses against NP and protection from influenza when 4-1BBL was expressed. However, functionally superior CD8(+) T cell responses were obtained when two LV were coinjected: one expressing 4-1BBL and the other expressing NP. This surprising result suggested that 4-1BBL is more effective when expressed in trans, acting on adjacent DC. Therefore, we investigated the effect of LV expression of 4-1BBL in mouse DC cultures and observed induced maturation of bystander, untransduced cells. Maturation was blocked by anti-4-1BBL Ab, required cell-cell contact, and did not require the cytoplasmic signaling domain of 4-1BBL. Greater maturation of untransduced cells could be explained by LV expression of 4-1BBL, causing downregulation of 4-1BB. These data suggest that coexpression of 4-1BBL and Ag by vaccine vectors that target DC may not be an optimal strategy. However, 4-1BBL LV immunization activates significant numbers of bystander DC in the draining lymph nodes. Therefore, transactivation by 4-1BBL/4-1BB interaction following DC-DC contact may play a role in the immune response to infection or vaccination.

Using a prime and pull approach, lentivector vaccines expressing Ag85A induce immunogenicity but fail to induce protection against Mycobacterium bovis bacillus Calmette-Guérin challenge in mice.

Although bacillus Calmette-Guérin (BCG) is an established vaccine with excellent efficacy against disseminated Mycobacterium tuberculosis infection in young children, efficacy in adults suffering from respiratory tuberculosis (TB) is suboptimal. Prime-boost viral vectored vaccines have been shown to induce effective immune responses and lentivectors (LV) have been shown to improve mucosal immunity in the lung. A mucosal boost to induce local immunogenicity is also referred to as a 'pull' in a prime and pull approach, which has been found to be a promising vaccine strategy. The majority of infants worldwide receive BCG immunization through current vaccine protocols. We therefore aimed to investigate the role of a boost (or pull) immunization with an LV vaccine expressing the promising TB antigen (Ag85A). We immunized BALB/c mice subcutaneously with BCG or an LV vaccine expressing a nuclear factor-κB activator vFLIP together with Ag85A (LV vF/85A), then boosted with intranasal LV vF/85A. Prime and pull immunization with LV85A induced significantly enhanced CD8(+) and CD4(+) T-cell responses in the lung, but did not protect against intranasal BCG challenge. In contrast, little T-cell response in the lung was seen when the prime vaccine was BCG, and intranasal vF/85A provided no additional protection against mucosal BCG infection. Our study demonstrates that not all LV prime and pull approaches may be successful against TB in man and careful antigen and immune activator selection is therefore required.