Survival update of neoadjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma in the OpACIN and OpACIN-neo trials.

BACKGROUND: Neoadjuvant ipilimumab plus nivolumab has yielded high response rates in patients with macroscopic stage III melanoma. These response rates translated to high short-term survival rates. However, data on long-term survival and disease recurrence are lacking. PATIENTS AND METHODS: In OpACIN, 20 patients with macroscopic stage III melanoma were randomized to ipilimumab 3 mg/kg plus nivolumab 1 mg/kg q3w four cycles of adjuvant or split two cycles of neoadjuvant and two adjuvant. In OpACIN-neo, 86 patients with macroscopic stage III melanoma were randomized to arm A (2× ipilimumab 3 mg/kg plus nivolumab 1 mg/kg q3w; n = 30), arm B (2× ipilimumab 1 mg/kg plus nivolumab 3 mg/kg q3w; n = 30), or arm C (2× ipilimumab 3 mg/kg q3w plus 2× nivolumab 3 mg/kg q2w; n = 26) followed by surgery. RESULTS: The median recurrence-free survival (RFS) and overall survival (OS) were not reached in either trial. After a median follow-up of 69 months for OpACIN, 1/7 patients with a pathologic response to neoadjuvant therapy had disease recurrence. The estimated 5-year RFS and OS rates for the neoadjuvant arm were 70% and 90% versus 60% and 70% for the adjuvant arm. After a median follow-up of 47 months for OpACIN-neo, the estimated 3-year RFS and OS rates were 82% and 92%, respectively. The estimated 3-year RFS rate for OpACIN-neo was 95% for patients with a pathologic response versus 37% for patients without a pathologic response (P < 0.001). In multiple regression analyses, pathologic response was the strongest predictor of disease recurrence. Of the 12 patients with distant disease recurrence after neoadjuvant therapy, 5 responded to subsequent anti-PD-1 and 8 to targeted therapy, although 7 patients showed progression after the initial response. CONCLUSIONS: Updated data confirm the high survival rates after neoadjuvant combination checkpoint inhibition in macroscopic stage III melanoma, especially for patients with a pathologic response. Pathologic response is the strongest surrogate marker for long-term outcome.

Additions to the knowledge of the genus Helvella in Europe. New records and de novo description of five species from the Nordic region.

Helvella is a species-rich genus, forming a large variation of astounding ascocarps in many different habitats. During the last decade, molecular markers and morphological characters have been combined to delimit and identify cryptic species in this genus. We report on a list of 54 species of Helvella s.s. in the Nordic region and describe five new species, i.e. H. bresadolae, H. convexa, H. japonica, H. nordlandica and H. oroarctica. The morphological and molecular characteristics of the new species and the emended / hypocrateriformis, / fibrosa-macropus, and / fallax-pezizoides lineages of Helvella s.s. are shortly commented upon. Further we include a discussion of the distribution of species in the Nordic region based on a large set of studied collections. The ecological versatility and variable geographic patterns of these species indicate that cryptic species may have contrasting ecology in their local habitats. Citation: Skrede I, Løken SB, Mathiesen C, Schumacher T (2023). Additions to the knowledge of the genus Helvella in Europe. New records and de novo description of five species from the Nordic region. Fungal Systematics and Evolution 11: 71-84. doi: 10.3114/fuse.2023.11.06.

Conversion of unresponsiveness to immune checkpoint inhibition by fecal microbiota transplantation in patients with metastatic melanoma: study protocol for a randomized phase Ib/IIa trial.

BACKGROUND: The gut microbiome plays an important role in immune modulation. Specifically, presence or absence of certain gut bacterial taxa has been associated with better antitumor immune responses. Furthermore, in trials using fecal microbiota transplantation (FMT) to treat melanoma patients unresponsive to immune checkpoint inhibitors (ICI), complete responses (CR), partial responses (PR), and durable stable disease (SD) have been observed. However, the underlying mechanism determining which patients will or will not respond and what the optimal FMT composition is, has not been fully elucidated, and a discrepancy in microbial taxa associated with clinical response has been observed between studies. Furthermore, it is unknown whether a change in the microbiome itself, irrespective of its origin, or FMT from ICI responding donors, is required for reversion of ICI-unresponsiveness. To address this, we will transfer microbiota of either ICI responder or nonresponder metastatic melanoma patients via FMT. METHODS: In this randomized, double-blinded phase Ib/IIa trial, 24 anti-PD1-refractory patients with advanced stage cutaneous melanoma will receive an FMT from either an ICI responding or nonresponding donor, while continuing anti-PD-1 treatment. Donors will be selected from patients with metastatic melanoma treated with anti-PD-1 therapy. Two patients with a good response (≥ 30% decrease according to RECIST 1.1 within the past 24 months) and two patients with progression (≥ 20% increase according to RECIST 1.1 within the past 3 months) will be selected as ICI responding or nonresponding donors, respectively. The primary endpoint is clinical benefit (SD, PR or CR) at 12 weeks, confirmed on a CT scan at 16 weeks. The secondary endpoint is safety, defined as the occurrence of grade ≥ 3 toxicity. Exploratory endpoints are progression-free survival and changes in the gut microbiome, metabolome, and immune cells. DISCUSSION: Transplanting fecal microbiota to restore the patients' perturbed microbiome has proven successful in several indications. However, less is known about the potential role of FMT to improve antitumor immune response. In this trial, we aim to investigate whether administration of FMT can reverse resistance to anti-PD-1 treatment in patients with advanced stage melanoma, and whether the ICI-responsiveness of the feces donor is associated with its effectiveness. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05251389 (registered 22-Feb-2022). Protocol V4.0 (08-02-2022).

MART-1 TCR gene-modified peripheral blood T cells for the treatment of metastatic melanoma: a phase I/IIa clinical trial.

Background: Adoptive cell therapy with peripheral blood T cells expressing transgenic T-cell receptors (TCRs) is an innovative therapeutic approach for solid malignancies. We investigated the safety and feasibility of adoptive transfer of autologous T cells expressing melanoma antigen recognized by T cells 1 (MART-1)-specific TCR, cultured to have less differentiated phenotypes, in patients with metastatic melanoma. Materials and methods: In this phase I/IIa trial, peripheral blood T cells from HLA-A2∗02:01-positive patients with unresectable stage IIIC/IV melanoma expressing MART-1 were selected and stimulated with anti-CD3/CD28 beads, transduced with a modified MART-1(26-35)-specific 1D3 TCR (1D3HMCys) and expanded in interleukin (IL)-7 and IL-15. Patients received a single infusion of transgenic T cells in a dose-escalating manner. Feasibility, safety and objective response rate were assessed. Results: Twelve pretreated metastatic cutaneous (n = 7) and uveal (n = 5) melanoma patients were included. Patient 1 received 4.6 × 109 1D3HMCys T cells and experienced grade 5 toxicity after 9 days. Subsequent patients received 5.0 × 107 [n = 3; cohort (c) 2], 2.5 × 108 (n = 2; c3) and 1.0 × 108 (n = 6; c4) 1D3HMCys T cells. The study was prematurely terminated because of dose-dependent toxicity, concerning skin (10/12), eyes (3/12), ears (4/12) and cytokine release syndrome (5/12), with 7 patients experiencing grade 3-5 toxicity. Partial responses were seen in 2/11 (18%) assessable patients and persistence of 1D3HMCys T cells corresponded to infused cell dose. Conclusions: Production of TCR-modified cells as described leads to highly potent T cells. Partial responses were seen in 18% of patients with dose-dependent 'on-target, off-tumor' toxicity and a maximum tolerated dose of 1.0 × 108 cells.

Survival and biomarker analyses from the OpACIN-neo and OpACIN neoadjuvant immunotherapy trials in stage III melanoma.

Neoadjuvant ipilimumab plus nivolumab showed high pathologic response rates (pRRs) in patients with macroscopic stage III melanoma in the phase 1b OpACIN ( NCT02437279 ) and phase 2 OpACIN-neo ( NCT02977052 ) studies1,2. While the results are promising, data on the durability of these pathologic responses and baseline biomarkers for response and survival were lacking. After a median follow-up of 4 years, none of the patients with a pathologic response (n = 7/9 patients) in the OpACIN study had relapsed. In OpACIN-neo (n = 86), the 2-year estimated relapse-free survival was 84% for all patients, 97% for patients achieving a pathologic response and 36% for nonresponders (P < 0.001). High tumor mutational burden (TMB) and high interferon-gamma-related gene expression signature score (IFN-γ score) were associated with pathologic response and low risk of relapse; pRR was 100% in patients with high IFN-γ score/high TMB; patients with high IFN-γ score/low TMB or low IFN-γ score/high TMB had pRRs of 91% and 88%; while patients with low IFN-γ score/low TMB had a pRR of only 39%. These data demonstrate long-term benefit in patients with a pathologic response and show the predictive potential of TMB and IFN-γ score. Our findings provide a strong rationale for a randomized phase 3 study comparing neoadjuvant ipilimumab plus nivolumab versus standard adjuvant therapy with antibodies against the programmed cell death protein-1 (anti-PD-1) in macroscopic stage III melanoma.

The genera Helvella and Dissingia (Ascomycota: Pezizomycetes) in Europe - Notes on species from Spain.

Phylogenetic analyses of 115 newly collected Helvella specimens from Spain using three genetic markers [heat shock protein 90 (hsp), RNA polymerase II second largest subunit (rpb2) and the nuclear large subunit ribosomal DNA (LSU)] confirm the assignment of the Spanish collections to one Dissingia and 30 Helvella species. The analyses were supplemented with an additional sample of 65 Spanish and extralimital Helvella specimens from the fungaria of Oslo (O), Trondheim (TRH), Copenhagen (C), Uppsala (UPS), Stockholm (S) and Venice (MCVE). Nine species are described as new, i.e. Helvella fuscolacunosa, H. hispanica, H. iberica, H. inexpectata, H. neopallescens, H. phlebophoroides, H. poculiformis, H. retinervis, and H. terricola. We present photographs of a selection of fresh specimens and provide descriptions of all species of this diverse South European Mediterranean element of the genera in Europe.

The Helvella corium species aggregate in Nordic countries - phylogeny and species delimitation.

Mycologists have always been curious about the elaborate morphotypes and shapes of species of the genus Helvella. The small, black, cupulate Helvella specimens have mostly been assigned to Helvella corium, a broadly defined morpho-species. Recent phylogenetic analyses, however, have revealed an aggregate of species hidden under this name. We performed a multispecies coalescent analysis to re-assess species limits and evolutionary relationships of the Helvella corium species aggregate in the Nordic countries. To achieve this, we used morphology and phylogenetic evidence from five loci - heat shock protein 90 (hsp), translation elongation factor 1-alpha (tef), RNA polymerase II (rpb2), and the 5.8S and large subunit (LSU) of the nuclear ribosomal DNA. All specimens under the name Helvella corium in the larger university fungaria of Norway, Sweden and Denmark were examined and barcoded, using partial hsp and/or rpb2 as the preferential secondary barcodes in Helvella. Additional fresh specimens were collected in three years (2015-2018) to obtain in vivo morphological data to aid in species discrimination. The H. corium species aggregate consists of seven phylogenetically distinct species, nested in three divergent lineages, i.e. H. corium, H. alpina and H. pseudoalpina sp. nov. in the /alpina-corium lineage, H. alpestris, H. macrosperma and H. nannfeldtii in the /alpestris-nannfeldtii lineage, and H. alpicola as a weakly supported sister to the /alpestris-nannfeldtii lineage. Among the seven species, the ribosomal loci expressed substantial variation in evolutionary rates, suggesting care in the use of these regions alone in delimitation of Helvella species. Altogether, 469 out of 496 available fungarium specimens were successfully barcoded.

The role of socio-economic status and energy-density in Australian women of child-bearing age.

INTRODUCTION: An optimal diet is imperative in preparing women for pregnancy and this may be influenced by socio-economic status (SES). This research aims to investigate the role of SES on the dietary energy density (ED) in Australian women of preconception age. METHODS: A secondary analysis of the Australian National Nutrition and Physical Activity Survey 2011-12 for females aged 18-39 years (n = 1617) was conducted. Dietary intake was assessed by 24-hr recalls and dietary ED by dietary energy per weight (kJ.g-1 ). ED was further categorised as ED of foods and beverages separately. SES was assessed by three variables: Socio-Economic Indexes for Areas (SEIFA), developed by the Australian Bureau of Statistics; income decile; and level of education. Linear mixed model regressions were used to identify associations between ED and SES. RESULTS: The median ED for food, beverages and combined food and beverages was 9.38 kJ g-1 , 1.02 kJ g-1 and 7.11 kJ g-1 , respectively. No significant variation was explained by SES variables when analysing combined ED in the adjusted model or ED from foods. Income decile reduced ED of beverages, although with little effect (coefficient: -0.04, P = 0.002). Significant confounders included inactivity, which increased ED in both combined ED and ED foods (coefficient: 0.51, P = 0.001 and coefficient: 0.78, P < 0.001). CONCLUSIONS: SES explained little variation in dietary ED in women of childbearing age. A large proportion of women had high energy-dense diets regardless of their SES. These findings suggest that a large proportion of women, who may become pregnant, have diets that exceed the international recommendations for dietary energy density.

Pindara revisited - evolution and generic limits in Helvellaceae.

The Helvellaceae encompasses taxa that produce some of the most elaborate apothecial forms, as well as hypogeous ascomata, in the class Pezizomycetes (Ascomycota). While the circumscription of the Helvellaceae is clarified, evolutionary relationships and generic limits within the family are debatable. A robust phylogeny of the Helvellaceae, using an increased number of molecular characters from the LSU rDNA, RPB2 and EF-1α gene regions (4 299 bp) and a wide representative sampling, is presented here. Helvella s.lat. was shown to be polyphyletic, because Helvella aestivalis formed a distant monophyletic group with hypogeous species of Balsamia and Barssia. All other species of Helvella formed a large group with the enigmatic Pindara (/Helvella) terrestris nested within it. The ear-shaped Wynnella constitutes an independent lineage and is recognised with the earlier name Midotis. The clade of the hypogeous Balsamia and Barssia, and H. aestivalis is coherent in the three-gene phylogeny, and considering the lack of phenotypic characters to distinguish Barssia from Balsamia we combine species of Barssia, along with H. aestivalis, in Balsamia. The closed/tuberiform, sparassoid H. astieri is shown to be a synonym of H. lactea; it is merely an incidental folded form of the saddle-shaped H. lactea. Pindara is a sister group to a restricted Helvella, i.e., excluding the /leucomelaena lineage, on a notably long branch. We recognise Pindara as a separate genus and erect a new genus Dissingia for the /leucomelaena lineage, viz. H. confusa, H. crassitunicata, H. leucomelaena and H. oblongispora. Dissingia is supported by asci that arise from simple septa; all other species of Helvellaceae have asci that arise from croziers, with one exception being the /alpina-corium lineage of Helvella s.str. This suggests ascus development from croziers is the ancestral state for the Helvellaceae and that ascus development from simple septa has evolved at least twice in the family. Our phylogeny does not determine the evolutionary relationships within Helvella s.str., but it is most parsimonious to infer that the ancestor of the helvelloids produced subsessile or shortly stipitate, cup-shaped apothecia. This shape has been maintained in some lineages of Helvella s.str. The type species of Underwoodia, Underwoodia columnaris, is a sister lineage to the rest of the Helvellaceae.

Publisher Correction: Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer.

The original version of this Article contained an error in Fig. 4. In the left histogram of the right panel of Fig. 4d, several data points were inadvertently deleted from the histogram during the production process. This error has been corrected in both the PDF and HTML versions of the Article. The original, incorrect version of Fig. 4 is presented in the accompanying Publisher Correction.

Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer.

Primary triple negative breast cancers (TNBC) are prone to dissemination but sub-clonal relationships between tumors and resulting metastases are poorly understood. Here we use cellular barcoding of two treatment-naïve TNBC patient-derived xenografts (PDXs) to track the spatio-temporal fate of thousands of barcoded clones in primary tumors, and their metastases. Tumor resection had a major impact on reducing clonal diversity in secondary sites, indicating that most disseminated tumor cells lacked the capacity to 'seed', hence originated from 'shedders' that did not persist. The few clones that continued to grow after resection i.e. 'seeders', did not correlate in frequency with their parental clones in primary tumors. Cisplatin treatment of one BRCA1-mutated PDX model to non-palpable levels had a surprisingly minor impact on clonal diversity in the relapsed tumor yet purged 50% of distal clones. Therefore, clonal features of shedding, seeding and drug resistance are important factors to consider for the design of therapeutic strategies.

Pregnancy stress, healthy pregnancy and birth outcomes - the need for early preventative approaches in pregnant Australian Indigenous women: a prospective longitudinal cohort study.

Adverse pregnancy outcomes including prematurity and low birth weight (LBW) have been associated with life-long chronic disease risk for the infant. Stress during pregnancy increases the risk of adverse pregnancy outcomes. Many studies have reported the incidence of adverse pregnancy outcomes in Indigenous populations and a smaller number of studies have measured rates of stress and depression in these populations. This study sought to examine the potential association between stress during pregnancy and the rate of adverse pregnancy outcomes in Australian Indigenous women residing in rural and remote communities in New South Wales. This study found a higher rate of post-traumatic stress disorder, depression and anxiety symptoms during pregnancy than the general population. There was also a higher incidence of prematurity and LBW deliveries. Unfortunately, missing post-traumatic stress disorder and depressive symptomatology data impeded the examination of associations of interest. This was largely due to the highly sensitive nature of the issues under investigation, and the need to ensure adequate levels of trust between Indigenous women and research staff before disclosure and recording of sensitive research data. We were unable to demonstrate a significant association between the level of stress and the incidence of adverse pregnancy outcomes at this stage. We recommend this longitudinal study continue until complete data sets are available. Future research in this area should ensure prioritization of building trust in participants and overestimating sample size to ensure no undue pressure is placed upon an already stressed participant.

Disparities exist between the dietary intake of Indigenous Australian women during pregnancy and the Australian dietary guidelines: the Gomeroi gaaynggal study.

BACKGROUND: Little is known about the adequacy of nutrient intakes and the overall diet quality of Indigenous Australian pregnant women. The aim of this cross-sectional study was to assess nutrient sufficiency and diet quality, as measured using the Australian Recommended Food Score (ARFS), in pregnant women from the Gomeroi gaaynggal cohort (n = 58). METHODS: Maternal dietary intake during pregnancy was assessed using the Australian Eating Survey Food Frequency Questionnaire, which was self-administered in the third trimester. Diet quality was determined using the ARFS. Food group servings and nutrient intakes were compared to the Australian Guide to Health Eating (AGHE) and Australian Nutrient Reference Values (NRVs). The current analysis examined the adequacy of usual intakes from food sources only, excluding supplements. RESULTS: None of the women met all AGHE daily food group serving recommendations. The highest alignment rates were for dairy (33%), meat/alternatives (31%) and vegetables (29.3%). Almost 93% of participants exceeded the recommended intake of energy-dense, nutrient-poor foods and percentage energy from saturated fat was high (15%). Of the five key nutrients for optimal reproductive health (folate, iron, calcium, zinc and fibre), the nutrients with the highest percentage of pregnant women achieving the NRVs were zinc (77.6%) and folate (68.9%), whereas iron was the lowest. Only one person achieved all NRVs (folate, iron, calcium, zinc and fibre) important in pregnancy. The median ARFS was 28 points (maximum of 73). CONCLUSIONS: Although the small cohort limits the generalisability of the findings of the present study, the data obtained indicate that the diets of these Indigenous pregnant women are inadequate. Therefore, strategies aiming to optimise nutrient intakes of Indigenous pregnant women are needed urgently.

A synopsis of the saddle fungi (Helvella: Ascomycota) in Europe - species delimitation, taxonomy and typification.

Helvella is a widespread, speciose genus of large apothecial ascomycetes (Pezizomycete: Pezizales) that are found in terrestrial biomes of the Northern and Southern Hemispheres. This study represents a beginning on assessing species limits and applying correct names for Helvella species based on type material and specimens in the university herbaria (fungaria) of Copenhagen (C), Harvard (FH) and Oslo (O). We use morphology and phylogenetic evidence from four loci - heat shock protein 90 (hsp), translation elongation factor alpha (tef), RNA polymerase II (rpb2) and the nuclear large subunit ribosomal DNA (LSU) - to assess species boundaries in an expanded sample of Helvella specimens from Europe. We combine the morphological and phylogenetic information from 55 Helvella species from Europe with a small sample of Helvella species from other regions of the world. Little intraspecific variation was detected within the species using these molecular markers; hsp and rpb2 markers provided useful barcodes for species delimitation in this genus, while LSU provided more variable resolution among the pertinent species. We discuss typification issues and identify molecular characteristics for 55 European Helvella species, designate neo- and epitypes for 30 species, and describe seven Helvella species new to science, i.e., H. alpicola, H. alpina, H. carnosa, H. danica, H. nannfeldtii, H. pubescens and H. scyphoides.

Comparison of fatty acid intakes assessed by a cardiovascular-specific food frequency questionnaire with red blood cell membrane fatty acids in hyperlipidaemic Australian adults: a validation study.

BACKGROUND/OBJECTIVES: Limited dietary intake tools have been validated specifically for hyperlipidaemic adults. The Australian Eating Survey (AES) Food Frequency Questionnaire (FFQ) was adapted to include foods with cardio-protective properties (CVD-AES). The aims were to estimate dietary fatty acid (FA) intakes derived from the CVD-AES and AES and compare them with red blood cell (RBC) membrane FA content. SUBJECTS/METHODS: Dietary intake was measured using the semi-quantitative 120-item AES and 177-item CVD-AES. Nutrient intakes were calculated using AUSNUT 2011-2013. Fasting RBC membrane FAs were assessed using gas chromatography. Extent of agreement between intakes estimated by AES or CVD-AES and RBC membrane composition (% of total FAs) for linoleic acid (LA), alpha-linolenic acid (ALA), eicosapentanoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) were assessed using Spearman's correlation coefficients, adjusted linear regressions and Kappa statistics. RESULTS: Data from 39 participants (72% female, 59.3±11.1 years) indicate stronger positive correlations between RBC membrane FAs and CVD-AES dietary estimates compared with the AES. Significant (P<0.05) moderate-strong correlations were found between CVD-AES FAs and FA proportions in RBC membranes for EPA (r=0.62), DHA (r=0.53) and DPA (r=0.42), with a moderate correlation for LA (r=0.39) and no correlation with ALA. Significant moderate correlations were found with the AES for DHA (r=0.39), but not for LA, ALA, EPA or DPA. CONCLUSIONS: The CVD-AES provides a more accurate estimate of long chain FA intakes in hyperlipidaemic adults, compared with AES estimates. This indicates that a CVD-specific FFQ should be used when evaluating FA intakes in this population.

Enhanced procedural blank control for organic geochemical studies of critical sample material.

Organic contamination of sedimentary rocks can produce artefacts in studies of hydrocarbon composition, and this can have significant negative consequences for interpretations of the geobiological record. False positives - that is cases of non-syngenetic hydrocarbon biomarkers - are common in Precambrian studies, and significant challenges persist despite the intensive effort devoted to these studies. Efforts to standardize the 'burden of proof' for distinguishing between contamination and syngenetic material have to date failed to yield a simple or universal protocol, yet the need remains great, as both bitumen-lean rocks and bitumen-rich samples can be vulnerable to the accumulation of false-positive signals. In an effort to determine the best approach to quality control, we tested the capability of different blank materials to collect ambient contamination by assessing their capacity to adsorb hydrocarbons during storage in plastic bags and found that commonly used Quartz sand does not provide an adequate measure of storage- or laboratory-induced contamination. Brick blanks, having the advantage that they can parallel rock samples even during the sawing process, are characterized by similar poor adsorption properties. Primarily steered by mineralogy, organic carbon content and surface area, model-black shales can adsorb up to 20 times more contaminants than sand blanks and up to 200 times more contaminants than organic-free model-carbonates. This observation provides an explanation for reports and observations of seemingly systematic stratigraphic variation of contaminants, but mostly should raise awareness for the evaluation of procedural blanks, in particular of sample-to-blank ratios, when studying bitumen-lean rock samples of varying lithologies. Additionally, differences between the hydrocarbon profiles in plastic bags and the hydrocarbon signatures transferred to blank materials emphasize difficulties in the unequivocal detection of contamination sources. Artificial black shale pellets can provide enhanced contamination control in biomarker studies - particularly for exceptionally vulnerable samples such as Precambrian rocks, meteorites or extraterrestrial sample-return material.

Antigen design enhances the immunogenicity of Semliki Forest virus-based therapeutic human papillomavirus vaccines.

Cellular immunity against cancer can be achieved with viral vector- and DNA-based immunizations. In preclinical studies, cancer vaccines are very potent, but in clinical trials these potencies are not achieved yet. Thus, a rational approach to improve cancer vaccines is warranted. We previously demonstrated that the relatively low intrinsic immunogenicity of DNA vaccines could be enhanced by inclusion of endoplasmic reticulum (ER) targeting and universal helper epitopes within the vaccine. We now evaluated whether an optimal antigen format, as defined in DNA vaccines, can further enhance the effectiveness of recombinant Semliki Forest virus (rSFV) vaccines. To this purpose, we generated, characterized and evaluated the efficacy of rSFV replicon particles expressing human papillomavirus E6 and/or E7 proteins fused to several helper T-cell epitopes and an ER targeting signal. Here, we show that inclusion of a helper cassette and an ER targeting signal enhanced protein stability and markedly augmented the frequencies of human papillomavirus-specific T cells. Even at an immunization dose of as low as 10(5) replicon particles, this novel vaccine achieved tumor regression and protection. Thus, even highly effective viral vector vaccines can benefit from an improved antigen format, based on the inclusion of defined helper epitopes and ER targeting.

Platelet activation in cats with hypertrophic cardiomyopathy.

BACKGROUND: Cats with hypertrophic cardiomyopathy (HCM) are at risk for development of systemic thromboembolic disease. However, the relationship between platelet activation state and cardiovascular parameters associated with HCM is not well described. OBJECTIVES: To characterize platelet activation by flow cytometric evaluation of platelet P-selectin and semiquantitative Western blot analysis of soluble platelet-endothelial cell adhesion molecule-1 (sPECAM-1). ANIMALS: Eight normal healthy cats (controls) owned by staff and students of the School of Veterinary Medicine and 36 cats from the UC Davis Feline HCM Research Laboratory were studied. METHODS: Platelet-rich plasma (PRP) was used for all flow cytometry studies. Platelet surface CD41 and P-selectin expression were evaluated before and after ADP stimulation. sPECAM-1 expression was evaluated by Western blot analysis of platelet-poor plasma that had been stabilized with aprotinin. Standard echocardiographic studies were performed. RESULTS: Resting platelets from cats with severe HCM had increased P-selectin expression compared to controls, and expressed higher surface density of P-selectin reflected by their increased mean fluorescence intensities (MFI). Stimulation with ADP also resulted in significantly increased P-selectin MFI of platelets from cats with severe HCM. Increased P-selectin expression and MFI correlated with the presence of a heart murmur and end-systolic cavity obliteration (ESCO). sPECAM-1 expression from cats with moderate and severe HCM was significantly increased above those of control cats. CONCLUSIONS AND CLINICAL IMPORTANCE: P-selectin and sPECAM expression may be useful biomarkers indicating increased platelet activation in cats with HCM.

Transposon leads to contamination of clinical pDNA vaccine.

We report an unexpected contamination during clinical manufacture of a Human Papilomavirus (HPV) 16 E6 encoding plasmid DNA (pDNA) vaccine, with a transposon originating from the Escherichia coli DH5 host cell genome. During processing, presence of this transposable element, insertion sequence 2 (IS2) in the plasmid vector was not noticed until quality control of the bulk pDNA vaccine when results of restriction digestion, sequencing, and CGE analysis were clearly indicative for the presence of a contaminant. Due to the very low level of contamination, only an insert-specific PCR method was capable of tracing back the presence of the transposon in the source pDNA and master cell bank (MCB). Based on the presence of an uncontrolled contamination with unknown clinical relevance, the product was rejected for clinical use. In order to prevent costly rejection of clinical material, both in-process controls and quality control methods must be sensitive enough to detect such a contamination as early as possible, i.e. preferably during plasmid DNA source generation, MCB production and ultimately during upstream processing. However, as we have shown that contamination early in the process development pipeline (source pDNA, MCB) can be present below limits of detection of generally applied analytical methods, the introduction of "engineered" or transposon-free host cells seems the only 100% effective solution to avoid contamination with movable elements and should be considered when searching for a suitable host cell-vector combination.

DNA vaccines and intradermal vaccination by DNA tattooing.

Over the past two decades, DNA vaccination has been developed as a method for the induction of immune responses. However, in spite of high expectations based on their efficacy in preclinical models, immunogenicity of first generation DNA vaccines in clinical trials was shown to be poor, and no DNA vaccines have yet been licensed for human use. In recent years significant progress has been made in the development of second generation DNA vaccines and DNA vaccine delivery methods. Here we review the key characteristics of DNA vaccines as compared to other vaccine platforms, and recent insights into the prerequisites for induction of immune responses by DNA vaccines will be discussed. We illustrate the development of second generation DNA vaccines with the description of DNA tattooing as a novel DNA delivery method. This technique has shown great promise both in a small animal model and in non-human primates and is currently under clinical evaluation.