Cost-effectiveness of pembrolizumab for previously treated MSI-H/dMMR solid tumours in the UK.

OBJECTIVES: Patients with previously treated microsatellite instability-high (MSI-H)/mismatch repair deficient (dMMR) tumours have limited chemotherapeutic treatment options. Pembrolizumab received approval from the EMA in 2022 for the treatment of colorectal, endometrial, gastric, small intestine, and biliary MSI-H/dMMR tumour types. This approval was supported by data from the KEYNOTE-164 and KEYNOTE-158 clinical trials. This study evaluated the cost-effectiveness of pembrolizumab compared with standard of care (SoC) for previously treated MSI-H/dMMR solid tumours in line with the approved EMA label from a UK healthcare payer perspective. METHODS: A multi-tumour partitioned survival model was built consisting of pre-progression, progressed disease, and dead health states. Pembrolizumab survival outcomes were extrapolated using Bayesian hierarchical models (BHMs) fitted to pooled data from KEYNOTE-164 and KEYNOTE-158. Comparator outcomes were informed by published sources. Tumour sites were modelled independently and then combined, weighted by tumour site distribution. A SoC comparator was used to formulate the overall cost-effectiveness result with pembrolizumab as the intervention. SoC comprised a weighted average of the comparators by tumour site based on market share. Drug acquisition, administration, adverse events, monitoring, subsequent treatment, end-of-life costs, and testing costs were included. Sensitivity and scenario analyses were performed, including modelling pembrolizumab efficacy using standard parametric survival models. RESULTS: Pembrolizumab, at list price, was associated with £129,469 in total costs, 8.30 LYs, and 3.88 QALYs across the pooled tumour sites. SoC was associated with £28,222 in total costs, 1.14 LYs, and 0.72 QALYs across the pooled tumour sites. This yields an incremental cost-effectiveness ratio (ICER) of £32,085 per QALY. Results were robust to sensitivity and scenario analyses. CONCLUSIONS: This model demonstrates pembrolizumab provides a valuable new alternative therapy for UK patients with MSH-H/dMMR cancer at the cost of £32,085 per QALY, with confidential discounts anticipated to improve cost-effectiveness further.

Policy Options for Infliximab Biosimilars in Inflammatory Bowel Disease Given Emerging Evidence for Switching.

Biosimilars are becoming increasingly available internationally as patents expire on the originator biologic drugs they are intended to copy. Although substitution policies seen with generic drugs are being considered as a means to reduce expenditures on biologics, some biosimilars pose particular challenges in that the act of substitution may eventually lead to increased rates of therapeutic failure. As evidence requirements from regulators do not directly address this challenge, switch trials of biosimilars have emerged that may provide further answers. Using infliximab in inflammatory bowel disease as an example, we critically examine emerging evidence from two key switch trials (NOR-SWITCH and NCT020968610) and discuss the clinical and economic implications of these and what policy options may be most reasonable for payers. Options include reimbursing biosimilars for only newly diagnosed patients, using product-listing agreements to manage uncertainty, or using tiered co-payments or other incentives to promote biosimilar use.

Development of an enhanced health-economic model and cost-effectiveness analysis of tiotropium + olodaterol Respimat® fixed-dose combination for chronic obstructive pulmonary disease patients in Italy.

BACKGROUND: The objective of this study was to compare the cost-effectiveness of the fixed-dose combination (FDC) of tiotropium + olodaterol Respimat(®) FDC with tiotropium alone for patients with chronic obstructive pulmonary disease (COPD) in the Italian health care setting using a newly developed patient-level Markov model that reflects the current understanding of the disease. METHODS: While previously published models have largely been based around a cohort approach using a Markov structure and GOLD stage stratification, an individual-level Markov approach was selected for the new model. Using patient-level data from the twin TOnado trials assessing Tiotropium + olodaterol Respimat(®) FDC versus tiotropium, outcomes were modelled based on the trough forced expiratory volume (tFEV1) of over 1000 patients in each treatment arm, tracked individually at trial visits through the 52-week trial period, and after the trial period it was assumed to decline at a constant rate based on disease stage. Exacerbation risk was estimated based on a random-effects logistic regression analysis of exacerbations in UPLIFT. Mortality by age and disease stage was estimated from an analysis of TIOSPIR trial data. Cost of bronchodilators and other medications, routine management, and costs of treatment for moderate and severe exacerbations for the Italian setting were included. A cost-effectiveness analysis was conducted over a 15-year time horizon from the perspective of the Italian National Health Service. RESULTS: Aggregating total costs and quality-adjusted life years (QALYs) for each treatment cohort over 15 years and comparing tiotropium + olodaterol Respimat(®) FDC with tiotropium alone, resulted in mean incremental costs per patient of €1167 and an incremental cost-effectiveness ratio (ICER) of €7518 per additional QALY with tiotropium + olodaterol Respimat(®) FDC. The lung function outcomes observed for tiotropium + olodaterol Respimat(®) FDC in TOnado drove the results in terms of slightly higher mean life-years (12.24 versus 12.07) exacerbation-free months (11.36 versus 11.32) per patient and slightly fewer moderate and severe exacerbations per patient-year (0.411 versus 0.415; 0.21 versus 0.24) versus tiotropium. Probabilistic sensitivity analyses showed tiotropium + olodaterol Respimat(®) FDC to be the more cost-effective treatment in 95.2% and 98.4% of 500 simulations at thresholds of €20,000 and €30,000 per QALY respectively. CONCLUSION: Tiotropium + olodaterol Respimat(®) FDC is a cost-effective bronchodilator in the maintenance treatment of COPD for the Italian health care system.

Identification of Outer Membrane and Exoproteins of Carbapenem-Resistant Multilocus Sequence Type 258 Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae strains have emerged as a cause of life-threatening infections in susceptible individuals (e.g., transplant recipients and critically ill patients). Strains classified as multilocus sequence type (ST) 258 are among the most prominent causes of carbapenem-resistant K. pneumoniae infections worldwide, but the basis for the success of this lineage remains incompletely determined. To gain a more comprehensive view of the molecules potentially involved in the success of ST258, we used a proteomics approach to identify surface-associated and culture supernatant proteins produced by ST258. Protein samples were prepared from varied culture conditions in vitro, and were analyzed by a combination of two-dimensional electrophoresis and liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). We identified a total of 193 proteins in outer membrane preparations from bacteria cultured in Luria-Bertani broth (LB) or RPMI 1640 tissue culture media (RPMI). Compared with LB, several iron-acquisition proteins, including IutA, HmuR, HmuS, CirA, FepA, FitA, FoxA, FhuD, and YfeX, were more highly expressed in RPMI. Of the 177 proteins identified in spent media, only the fimbrial subunit, MrkA, was predicted to be extracellular, a finding that suggests few proteins (or a limited quantity) are freely secreted by ST258. Notably, we discovered 203 proteins not reported in previous K. pneumoniae proteome studies. In silico modeling of proteins with unknown function revealed several proteins with beta-barrel transmembrane structures typical of porins, as well as possible host-interacting proteins. Taken together, these findings contribute several new targets for the mechanistic study of drug-resistance and pathogenesis by ST258 K. pneumoniae isolates.

Interactions of the vaccinia virus A19 protein.

The A19 protein of vaccinia virus (VACV) is conserved among chordopoxviruses, expressed late in infection, packaged in the virus core, and required for a late step in morphogenesis. Multiple-sequence alignments of A19 homologs indicated conservation of a series of lysines and arginines, which could represent a nuclear localization or nucleic acid binding motif, and a pair of CXXC motifs that suggested a zinc finger or redox active sites. The importance of the CXXC motif was confirmed by cysteine-to-serine substitutions, which rendered the altered protein unable to trans-complement infectivity of a null mutant. Nevertheless, the cysteines were not required for function of the poxvirus-specific redox pathway. Epitope-tagged A19 proteins were detected in the nucleus and cytoplasm in both infected and uninfected cells, but this distribution was unaffected by alanine substitutions of the arginine residues, which only partially reduced the ability of the mutated protein to trans-complement infectivity. Viral proteins specifically associated with affinity-purified A19 were identified by mass spectrometry as components of the transcription complex, including RNA polymerase subunits, RAP94 (RNA polymerase-associated protein 94), early transcription factors, capping enzyme, and nucleoside triphosphate phosphohydrolase I, and two core proteins required for morphogenesis. Further studies suggested that the interaction of A19 with the RNA polymerase did not require RAP94 or other intermediate or late viral proteins but was reduced by mutation of cysteines in the putative zinc finger domain. Although A19 was not required for incorporation of the transcription complex in virus particles, the transcriptional activity of A19-deficient virus particles was severely reduced.

Serologic reactivity to the emerging pathogen Granulibacter bethesdensis.

BACKGROUND: Granulibacter bethesdensis is a recently described member of the Acetobacteraceae family that has been isolated from patients with chronic granulomatous disease (CGD). Its pathogenesis, environmental reservoir(s), and incidence of infection among CGD patients and the general population are unknown. METHODS: Detected antigens were identified by mass spectroscopy after 2-dimensional electrophoresis and immunoaffinity chromatography. The prevalence of Granulibacter immunoreactivity was assessed through immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: Methanol dehydrogenase (MDH) and formaldehyde-activating enzyme were recognized during analysis of sera from infected patients. Unique patterns of immunoreactive bands were identified in Granulibacter extracts, compared with extracts of other Acetobacteraceae species. By use of criteria based on these specific bands, specimens from 79 of 175 CGD patients (45.1%) and 23 of 93 healthy donors (24.7%) reacted to all 11 bands. An ELISA that used native MDH to capture and detect immunoglobulin G was developed and revealed high-titer MDH seroreactivity in culture-confirmed cases and 5 additional CGD patients. Testing of samples collected prior to culture-confirmed infection demonstrated instances of recent seroconversion, as well as sustained seropositivity. Infection of CGD mice with G. bethesdensis confirmed acquisition of high-titer antibody-recognizing MDH. CONCLUSIONS: These serologic tests suggest that Granulibacter immunoreactivity is more common among CGD patients and, perhaps, among healthy donors than was previously suspected. This finding raises the possibility that clinical presentations of Granulibacter infection may be underappreciated.

Identification of a novel Staphylococcus aureus two-component leukotoxin using cell surface proteomics.

Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin-herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.

Identification of conserved antigens for early serodiagnosis of relapsing fever Borrelia.

Borrelia hermsii is a blood-borne pathogen transmitted by the argasid tick Ornithodoros hermsi. Since spirochaete clearance in mice is associated with an IgM-mediated response, an immunoproteomic analysis was used to identify proteins reactive with IgM. We report that IgM from both mice and human patients infected with B. hermsii not only reacted with the previously identified variable membrane proteins but also identified candidate antigens including heat-shock proteins, an adhesin protein, ABC transporter proteins, flagellar proteins, housekeeping proteins, an immune evasion protein, and proteins with unknown function. Furthermore, IgM reactivity to recombinant glycerophosphodiester phosphodiesterase was detected during early spirochaete infection and prior to a detectable IgG response. Lastly, a conserved hypothetical protein was produced in Escherichia coli and tested with immune serum against B. hermsii and Borrelia recurrentis. These results identify a much larger set of immunoreactive proteins, and could help in the early serodiagnosis of this tick-borne infection.

Towards a rational approach for heavy-atom derivative screening in protein crystallography.

Heavy-atom derivatization is routinely used in protein structure determination and is thus of critical importance in structural biology. In order to replace the current trial-and-error heavy-atom derivative screening with a knowledge-based rational derivative-selection method, the reactivity of more than 40 heavy-atom compounds over a wide range of buffer and pH values was systematically examined using peptides which contained a single reactive amino-acid residue. Met-, Cys- and His-containing peptides were derivatized against Hg, Au and Pt compounds, while Tyr-, Glu-, Asp-, Asn- and Gln-containing peptides were assessed against Pb compounds. A total of 1668 reactive conditions were examined using mass spectrometry and were compiled into heavy-atom reactivity tables (http://sis.niaid.nih.gov/cgi-bin/heavyatom_reactivity.cgi). The results showed that heavy-atom derivatization reactions are highly linked to buffer and pH, with the most accommodating buffer being MES at pH 6. A group of 21 compounds were identified as most successful irrespective of ligand or buffer/pH conditions. To assess the applicability of the peptide heavy-atom reactivity to proteins, lysozyme crystals were derivatized with a list of peptide-reactive compounds that included both known and new compounds for lysozyme derivatization. The results showed highly consistent heavy-atom reactivities between the peptides and lysozyme.

Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior.

Ticks evolved various mechanisms to modulate their host's hemostatic and immune defenses. Differences in the anti-hemostatic repertoires suggest that hard and soft ticks evolved anti-hemostatic mechanisms independently, but raise questions on the conservation of salivary gland proteins in the ancestral tick lineage. To address this issue, the sialome (salivary gland secretory proteome) from the soft tick, Argas monolakensis, was determined by proteomic analysis and cDNA library construction of salivary glands from fed and unfed adult female ticks. The sialome is composed of approximately 130 secretory proteins of which the most abundant protein folds are the lipocalin, BTSP, BPTI and metalloprotease families which also comprise the most abundant proteins found in the salivary glands. Comparative analysis indicates that the major protein families are conserved in hard and soft ticks. Phylogenetic analysis shows, however, that most gene duplications are lineage specific, indicating that the protein families analyzed possibly evolved most of their functions after divergence of the two major tick families. In conclusion, the ancestral tick may have possessed a simple (few members for each family), but diverse (many different protein families) salivary gland protein domain repertoire.

Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA-MRSA pathogenesis are poorly understood and a comprehensive analysis of CA-MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2-dimensional gel electrophoresis coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS) analysis. Eleven known virulence-related exoproteins differed in abundance between the strains, including alpha-haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1-phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host-pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA-MRSA as a human pathogen.

Characterization of a lysyl aminopeptidase activity associated with phosphoglucose isomerase of Vibrio vulnificus.

Phosphoglucose isomerase (PGI) is a multifunctional enzyme involved in glycolysis and gluconeogenesis and, in mammalian cells, functions as neuroleukin, autocrine motility factor (AMF), and differentiation and maturation factor (MF). We isolated and characterized PGI with a novel lysyl aminopeptidase (LysAP) activity (PGI-LysAP) from Vibrio vulnificus. Mass spectrometry revealed that PGI-LysAP is a heterodimer consisting of 23.4- and 60.8-kDa subunits. Only the heterodimer displayed LysAP activity. PGI-LysAP has a pI around 6.0 and high specificity toward the synthetic, fluorogenic substrate l-lysyl-7-amino-4-methylcoumarin. LysAP activity is optimal at pH 8.0, is 64% higher at 37 degrees C than at 21 degrees C, does not directly correlate with virulence, and is strongly inhibited by serine protease and metalloprotease inhibitors. PGI-LysAP was also identified in Vibrio parahaemolyticus and V. cholerae, but was absent from non-Vibrio human pathogens. Sequencing of the pgi gene revealed 1653 bp coding for a 550-amino-acid protein. Cloned and expressed PGI formed a homodimer with isomerase activity, but not LysAP activity. The finding of LysAP activity associated with heterodimeric PGI should foster a broad search for putative substrates in an effort to elucidate the role of PGI-LysAP in bacteria and its roles in the pathophysiology of diseases.

In vitro proteolytic processing of the MD145 norovirus ORF1 nonstructural polyprotein yields stable precursors and products similar to those detected in calicivirus-infected cells.

The MD145-12 strain (GII/4) is a member of the genus Norovirus in the Caliciviridae and was detected in a patient with acute gastroenteritis in a Maryland nursing home. The open reading frame 1 (ORF1) (encoding the nonstructural polyprotein) was cloned as a consensus sequence into various expression vectors, and a proteolytic cleavage map was determined. The virus-encoded cysteine proteinase mediated at least five cleavages (Q(330)/G(331), Q(696)/G(697), E(875)/G(876), E(1008)/A(1009), and E(1189)/G(1190)) in the ORF1 polyprotein in the following order: N-terminal protein; nucleoside triphosphatase; 20-kDa protein (p20); virus protein, genome linked (VPg); proteinase (Pro); polymerase (Pol). A time course analysis of proteolytic processing of the MD145-12 ORF1 polyprotein in an in vitro coupled transcription and translation assay allowed the identification of stable precursors and final mapped cleavage products. Stable precursors included p20VPg (analogous to the 3AB of the picornaviruses) and ProPol (analogous to the 3CD of the picornaviruses). Less stable processing intermediates were identified as p20VPgProPol, p20VPgPro, and VPgPro. The MD145-12 Pro and ProPol proteins were expressed in bacteria as active forms of the proteinase and used to further characterize their substrate specificities in trans cleavage assays. The MD145-12 Pro was able to cleave its five mapped cleavage sites in trans and, in addition, could mediate trans cleavage of the Norwalk virus (GI/I) ORF1 polyprotein into a similar proteolytic processing profile. Taken together, our data establish a model for proteolytic processing in the noroviruses that is consistent with nonstructural precursors and products identified in studies of caliciviruses that replicate in cell culture systems.