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Background: There is an urgent need for effective treatments against glioblastoma (GBM). In this trial, we investigated the efficacy and safety of an adoptive cell-based immunotherapy. Methods: Patients with newly diagnosed GBM were recruited at 4 study sites in Sweden. The patients were randomized 1:2 to receive either radiotherapy (RT), 60 Gy/30 fractions, with concomitant and adjuvant temozolomide (TMZ) only, or RT and TMZ with the addition of Autologous Lymphoid Effector Cells Specific Against Tumor (ALECSAT) in an open-label phase II trial. The primary endpoint was investigator-assessed progression-free survival (PFS). The secondary endpoints were survival and safety of ALECSAT. Results: Sixty-two patients were randomized to either standard of care (SOC) with RT and TMZ alone (n = 22) or SOC with ALECSAT (n = 40). Median age was 57 years (range 38-69), 95% of the patients were in good performance status (WHO 0-1). There was no significant difference between the study arms (SOC vs ALECSAT + SOC) in PFS (7.9 vs 7.8 months; hazard ratio [HR] 1.28; 95% confidence interval [CI] 0.70-2.36; P = .42) or in median overall survival (OS) (18.3 vs 19.2 months; HR 1.16, 95% CI 0.58-2.31; P = .67). The treatment groups were balanced in terms of serious adverse events (52.4% vs 52.5%), but adverse events ≥grade 3 were more common in the experimental arm (81.0% vs 92.5%). Conclusion: Addition of ALECSAT immunotherapy to standard treatment with radiochemotherapy was well tolerated but did not improve PFS or OS for patients with newly diagnosed GBM.

The comparative virulence of Yersinia ruckeri serotype O1 biotype 2 strain 07111224 from a Danish outbreak of enteric redmouth disease in rainbow trout (Oncorhynchus mykiss) was investigated in different sizes of rainbow trout (5, 19, 25 and 52 g) using a panel of challenge methods (bath, intraperitoneal (IP) injection, anal intubation and cohabitation). The results show that Y. ruckeri 07111224 is virulent enough to successfully cause mortality in 5–52 g rainbow trout through three different infection routes (bath, IP injection and anal intubation), and strongly suggests that susceptibility to Y. ruckeri infection is dependent on fish size. Bath challenge (108 CFU/ml) caused 79% mortality in 5 g fish, while the mortality in 19–52 g fish was approximately 26%. IP injection challenges (105–106 CFU/individual) resulted in high mortalites in fish of all sizes. Anal intubation challenges (109 CFU/individual) induced 82% mortality in 5 g fish and decreased with increasing fish weight. Finally, cohabitation challenges induced 45% mortality in 5 g test fish when test fish were cohabitated 1:1 with shedder fish, while mortalities in test fish of five other challenge groups were lower (5–25%). Statistical models were fit to the data from each challenge model. The model parameter predictions suggests statistically significant effects of fish weight for bath, IP and anal intubation challenges. Furthermore, they highlight the importance of the ratio of shedders to test fish in cohabitation challenges and of the challenge doses, in particular for the IP challenge model.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) in rainbow trout, and the first commercially available fish vaccine was an immersion vaccine against ERM consisting of Y. ruckeri bacterin. The ERM immersion vaccine has been successfully used in aquaculture farming of salmonids for more than 35 years. The gills and the gastrointestinal (GI) tract are believed to be the portals of antigen uptake during waterborne vaccination against ERM; however, the actual sites of bacterin uptake are only partly understood. In order to obtain insight into bacterin uptake during waterborne vaccination, optical projection tomography (OPT) together with immunohistochemistry (IHC) was applied to visualize bacterin uptake and processing in whole rainbow trout fry. Visualization by OPT revealed that the bacterin was initially taken up via gill lamellae from within 30 seconds post vaccination. Later, bacterin uptake was detected on other mucosal surfaces such as skin and olfactory bulb from 5 to 30 minutes post vaccination. The GI tract was found to be filled with a complex of bacterin and mucus at 3 hours post vaccination and the bacterin remained in the GI tract for at least 24 hours. Large amounts of bacterin were present in the blood, and an accumulation of bacterin was found in filtering lymphoid organs such as spleen and trunk kidney where the bacterin accumulates 24 hours post vaccination as demonstrated by OPT and IHC. These results suggest that bacterin is taken up via the gill epithelium in the earliest phases of the bath exposure and from the GI tract in the later phase. The bacterin then enters the blood circulatory system, after which it is filtered by spleen and trunk kidney, before finally accumulating in lymphoid organs where adaptive immunity against ERM is likely to develop.

The effect of oral vaccines against bacterial fish diseases has been a topic for debate for decades. Recently both M-like cells and dendritic cells have been discovered in the intestine of rainbow trout. It is therefore likely that antigens reaching the intestine can be taken up and thereby induce immunity in orally vaccinated fish. The objective of this project was to investigate whether oral and anal vaccination of rainbow trout induces protection against an experimental waterborne infection with the pathogenic enterobacteria Yersinia ruckeri O1 biotype 1 the causative agent of enteric redmouth disease (ERM). Rainbow trout were orally vaccinated with AquaVac ERM Oral (MERCK Animal Health) or an experimental vaccine bacterin of Y. ruckeri O1. Both vaccines were tested with and without a booster vaccination four months post the primary vaccination. Furthermore, two groups of positive controls were included, one group receiving the experimental oral vaccine in a 50 times higher dose, and the other group receiving a single dose administered anally in order to bypass the stomach. Each group was bath challenged with 6.3×108 CFU/ml Y. ruckeri, six months post the primary vaccination. The challenge induced significant mortality in all the infected groups except for the groups vaccinated anally with a single dose or orally with the high dose of bacterin. Both of these groups had 100% survival. These results show that a low dose of Y. ruckeri bacterin induces full protection when the bacterin is administered anally. Oral vaccination also induces full protection, however, at a dose 50 times higher than if the fish were to be vaccinated anally. This indicates that much of the orally fed antigen is digested in the stomach before it reaches the second segment of the intestine where it can be taken up as immunogenic antigens and presented to lymphocytes.

Despite the fact that enteric redmouth disease (ERM) in farmed rainbow trout is one of the most devastating disease problems, little is known about the initial route of infection and pathogenicity of the aetiological agent, Yersinia ruckeri. In order to determine the initially infected organs, optical projection tomography (OPT), a novel three-dimensional (3D) bio-imaging technique, was applied. OPT not only enables the visualization of Y. ruckeri on mucosal surfaces but also the 3D spatial distribution in whole organs, without sectioning. Rainbow trout were infected by bath challenge exposure to 1 × 10(8) CFU/ml of Y. ruckeri O1 for 1 hour. Three fish were sampled for OPT and immunohistochemistry (IHC) 1, 10 and 30 minutes, 1, 3, 6, 12 and 24 hours, as well as 2, 3, 7 and 21 days after the start of the infection period. Y. ruckeri was re-isolated from the blood of infected fish as early as 1 minute post infection. Both OPT and IHC analysis confirmed that the secondary gill lamellae were the only tissues infected at this early time point, indicating that Y. ruckeri initially infects gill epithelial cells. The experimentally induced infection caused septicemia, and Y. ruckeri was found in all examined organs 7 days post infection including the brain, which correlated with the peak in mortality. To the best of our knowledge this is the first description of Y. ruckeri infection in the brain, which is likely to cause encephalitis. This in part could explain the lethality of ERM in rainbow trout. Using OPT scanning it was possible to visualize the initial route of entry, as well as secondary infection routes along with the proliferation and spread of Y. ruckeri, ultimately causing significant mortality in the exposed rainbow trout. These results demonstrate that OPT is a state-of-the-art technique capable of visualizing pathogenesis at high resolution.

Yersinia ruckeri (Y.r.) O1 biotype (bt) 2 causes outbreaks of Enteric Redmouth disease (ERM) in vaccinated, farmed rainbow trout around the world. Y.r. O1 bt 1 has been known since first described from the Hagerman Valley in Idaho in the 1950’s and outbreaks have now been controlled by vaccination for more than 35 years. Recent years have shown ERM outbreaks caused by Y.r. O1 bt 2 reported from ERM vaccinated farmed rainbow trout stocks in both Europe and the USA. It has been suggested that Y.r. O1 bt 2 is more virulent than bt 1. In order to test this hypothesis we have collected Y.r. O1 isolates from ERM outbreaks in Danish rainbow trout farms during 2011. Some of the isolates were obtained from vaccinated stocks, which subsequently developed ERM disease outbreaks. The Y.r. O1 isolates have been characterized and biotyped according to mobility and hydrolysis of Tween-80, and almost all isolates obtained in 2011 were double negative, which characterizes them as bt 2. Only one Y.r. O1 bt 1 strain was isolated, the rest of all the Y.r. O1 strains belonged to bt 2. Y.r. O1 bt 1 has been isolated from Danish rainbow trout farms for 30 years, but Y.r. O1 bt 2 has first been detected 12 years later and has caused many outbreaks of ERM which has led to an increase in the use of antibiotics. In order to determine the virulence of the newly isolated Y.r. O1 strains, experimental infections were carried out. The mortalities of the newly isolated strains were compared to well characterized isolates of both bt 1 and 2. In order to mimic a natural Y.r. infection, a bath infection model was applied. All Y.r. O1 isolates caused significant mortality compared to the un-infected control group. The Y.r. O1 isolates could be divided into two groups, regarding their virulence: A low virulence group causing less than 50 % mortality, and a high virulence group causing more than 50 % mortality. Interestingly, all tested bt 1 isolates grouped in the low virulence group together

In recent years, there has been an increase in reported outbreaks of enteric redmouth disease (ERM) in ERM vaccinated farmed rainbow trout in a number of countries, including the United States and a range of European countries such as Denmark, Spain and the UK, which both affect animal welfare and cause considerable economic losses. These reported disease outbreaks have been associated with non-motile, virulent serovar O1 strains of Yersinia ruckeri, classified as biotype 2. A standardised challenge model has been developed based on a newly isolated and highly virulent Y. ruckeri O1 biotype 2 strain obtained from an ERM disease outbreak in a Danish trout farm. This waterborne infection model gives us the opportunity to test and evaluate the effect of commercial and experimental vaccines against Y. ruckeri O1 biotype 2. An experimental vaccine containing equal amounts of Y. ruckeri O1 biotype 1 and biotype 2 (whole cell bacterin) was developed, based on the most immunogenic strains from our collection. Rainbow trout were vaccinated by immersion (30sec) or bath (5min) in 1•10^9 CFU/ml, or given an intraperitoneal (i.p.) injection (5•10^8 CFU/fish). The effect of the experimental vaccine has been compared to a state-of-the-art commercial ERM immersion vaccine (AquaVac® ReleraTM). Un-vaccinated and sham vaccinated rainbow trout were included as controls. Two months post vaccination the rainbow trout were challenged in duplicate with Y. ruckeri O1 biotype 2 by bath. No effect of the experimental immersion or bath vaccine was observed in the present study. However, full protection was achieved with i.p. injection of the experimental vaccine (p<0.0001, RPS=100%). Bath vaccination with AquaVac® ReleraTM induced a significant, partial protection relative to the control groups (p=0.002, RPS=58.5%). It is suggested that the immunity induced by immersion and bath vaccination is inadequate for protection of the rainbow trout fry, since mortalitie

Optical projection tomography (OPT) is a new tool for three-dimensional (3D) imaging of small tissues or embryos, based on multi-angle recording of internal fluorescent signals using intact whole mount tissue or fish. To understand the route of infection, gills of Y. ruckeri infected rainbow trout were labeled with fluorescent antibody and visualized in 3D by the OPT scanner. Rainbow trout were infected with Y. ruckeri O1 biotype 1 (1 x 109 cells/ml) for 1 hour at 18 °C, and then transferred to clean water. Three fish were sampled at 12 different time points and fixed in 4% PFA. The gills were incubated whole with rabbit anti-Y. ruckeri polyclonal antibody and Alexa Fluor®594 conjugated goat anti-rabbit IgG. After embedding in 1% low melting point agarose, specimens were dehydrated in 100% methanol and cleared in BABB (benzyl alcohol: benzyl benzoate) for OPT scanning. 3D imaging results showed that Y. ruckeri were observed in connection with the primary gill filaments, at the base of gill arches. In contrast, no bacteria were found in control fish gills. More detailed analyses are being processed.

Vaccination is a keystone in prophylactic strategies preventing outbreaks of fish pathogenic bacterial diseases in aquaculture. The first commercial fish vaccine consisted of a bacterin of Yersinia ruckeri serotype O1 biotype 1. The vaccine has been very successful and has been used for more than 35 years. A vast experience has been gained concerning the applications of the vaccine, which can be utilized through several mucosal immunization routes such as bath, oral and anal application, all resulting in significantly increased survival compared to un-vaccinated control groups during bath challenge experiments. Little is known regarding the nature of the protective immune response. We have shown that even low titers of Y. ruckeri specific antibodies confer protective immunity in vivo, and shown that is possible to pass immunity to naïve fish by passing serum containing Y. ruckeri specific antibodies. Further, plasma from bath vaccinated fish kills significantly more Y. ruckeri in vitro than plasma from un-vaccinated control fish. Increased plasma antibody titer against Y. ruckeri seems to be an important part of the protective immune response obtained post bath vaccination. These results all point towards an important role of specific antibodies as part of a vaccine-induced protective mechanism. However, we have shown full protection in both orally and anally immunized rainbow trout without detecting any increase in circulating levels of Y. ruckeri specific antibodies. Since both dendritic cells and M-like cells have been found in fish, is it suggested that gut-associated lymphoid tissue (GALT) associated with the gastrointestinal tract are involved in antigen uptake and generation of a local protective immune response against Y. ruckeri.

Yersinia ruckeri O1 biotype 2 (Y.r. bt2) has recently caused several outbreaks of Enteric Redmouth disease (ERM) in ERM-vaccinated farmed rainbow trout in Denmark, Finland, Spain, the UK and the USA. Recently, we have isolated a very virulent Y.r. bt2 strain from an ERM outbreak in a Danish rainbow trout stock which had been vaccinated against both Y.r. bt 1 and 2 with a commercial “state of the art” immersion vaccine and boosted with a commercial ERM oral vaccine 4 month later. The newly isolated Y.r. bt2 strain has been used to develop a standardized challenge model which gives us the opportunity to test and evaluate the effect of both commercial and experimental vaccines against Y.r. bt2 in a waterborne infection experiment, mimicking a natural infection. We have developed an experimental immersion and bath vaccine containing equal amounts of the most immunogenic Y.r. bt 1 and 2 strains among the isolates obtained from ERM outbreaks in Denmark as well as other countries. The effect of the experimental vaccine is compared with a “state of the art” commercial vaccine which also contains Y.r. bt 1 and 2 in equal amounts. Groups of rainbow trout were immersion vaccinated in a 1:10 dilution of the vaccine for 30 seconds as recommended by the manufacturer. As a positive control groups of trout were bath vaccinated for 5 minutes. Two months later was all groups bath challenged with Y.r. bt 2 in duplicate. The challenge data obtained in the present study have indicated that neither a “state of the art” commercial nor the experimental immersion ERM vaccine provided significant protective immunity against the virulent Y.r. bt2 infections. IP. injection of the experimental vaccine without adjuvant induced full protection. Significant mortality were seen in all immersion and bath vaccinated groups. The insufficient immunity induced by immersion and bath vaccination explains why ERM disease outbreaks are observed in fish farms where all the rainbow trout are

Furunculosis caused by infection with Aeromonas salmonicida subsp. salmonicida has been a known threat to aquaculture for more than a century. Efficient prophylactic approaches against this disease are essential for continued growth of salmonid aquaculture. Since the introduction of successful oil-adjuvanted vaccines in the early 1990's, a number of studies have been published on the protective as well as adverse effects of these vaccines. Most studies focus on vaccination of salmon (Salmo salar). However, rainbow trout (Oncorhynchus mykiss) are also very susceptible to infection and are vaccinated accordingly. In this study we have examined the protection against infection with a Danish strain of A. salmonicida in both vaccinated and non-vaccinated rainbow trout. A commercial and an experimental auto-vaccine were tested. The protective effects of the vaccines were evaluated through an A. salmonicida challenge 18 weeks post vaccination. Both vaccines resulted in a significantly increased survival in the vaccinated fish during a 28 day challenge period relative to non-vaccinated fish (P = 0.01 and P = 0.001 for the commercial and experimental vaccine, respectively). Throughout the entire experiment, the presence of specific antibodies in plasma was monitored using ELISA. A significant increase in specific antibody levels was seen in fish vaccinated with both vaccines during the 18 weeks between vaccination and challenge. Within 3 days post challenge, a significant decrease in specific antibodies occurred in vaccinated fish. A positive correlation was found between mean levels of specific antibodies pre challenge and overall survival. This correlation, along with the observed depletion of antibodies during the initial phase of infection, suggests that specific antibodies play an essential role in vaccine mediated protection against A. salmonicida in rainbow trout.

Innate immune factors play a crucial role in survival of young fish especially during early stages of life when adaptive immunity is not fully developed. In the present study, we investigated the immune response of rainbow trout (Oncorhynchus mykiss) larvae and fry at an early stage of development. We exposed 17 and 87 days post hatch larvae and fry (152 and 1118 degree days post hatch; avg. wt. 70 and 770 mg, respectively) to the bacterial pathogen, Yersinia ruckeri for 4 h by bath challenge. Samples were taken at 4, 24, 72 and 96 h post exposure for qPCR and immunohistochemical analyses to elucidate the immune response mounted by these young fish. Larvae showed no mortality although infected larvae at 48 h post exposure showed hyperaemia in the mouth region and inflammation on the dorsal side of the body. Gene expression studies showed an up-regulation of iNOS and IL-22 in infected larvae 24 h post exposure but most of the investigated genes did not show any difference between infected and uninfected larvae. Immunohistochemical studies demonstrated a high expression of IgT molecules in gills and CD8 positive cells in thymus of both infected and uninfected larvae. Infection of rainbow trout fry with Y. ruckeri, in contrast, induced a cumulative mortality of 74%. A high expression of cytokines (IL-1b, TNF-a, IL-22, IL-8 and IL- 10), acute phase proteins (SAA, hepcidin, transferrin and precerebellin), complement factors (C3, C5 and factor B), antimicrobial peptide (cathelicidin-2) and iNOS was found in infected fry when compared to the uninfected control. IgT molecules and mannose binding lectins in gills of both infected and uninfected fry were detected by immunohistochemistry. The study indicated that early life stages (yolk-sac larvae), merely up-regulate a few genes and suggests a limited capacity of larvae to mount an immune response by gene regulation at the transcriptional level. Based on