Reproducibility and Optimal Arterial Input Function Selection in Dynamic Contrast-Enhanced Perfusion MRI in the Healthy Brain.

BACKGROUND: Dynamic contrast-enhanced MRI (DCE-MRI) has seen increasing use for quantification of low level of blood-brain barrier (BBB) leakage in various pathological disease states and correlations with clinical outcomes. However, currently there exists limited studies on reproducibility in healthy controls, which is important for the establishment of a normality threshold for future research. PURPOSE: To investigate the reproducibility of DCE-MRI and to evaluate the effect of arterial input function (AIF) selection and manual region of interests (ROI) delineation vs. automated global segmentation. STUDY TYPE: Prospective. POPULATION: A total of 16 healthy controls; 11 females; mean age 28.7 years (SD 10.1). FIELD STRENGTH/SEQUENCE: A 3T; GE DCE; 3D TFE T1WI. 2D TSE T2. ASSESSMENT: The influx constant Ki , a measure of BBB permeability, and Vp , the blood plasma volume, was calculated using the Patlak model. Cerebral blood flow (CBF) was calculated using Tikhonov model free deconvolution. Manual tissue ROIs, drawn by H.J.S. (30+ years of experience), were compared to automatic tissue segmentation. STATISTICAL TESTS: Intraclass correlation coefficient (ICC) and repeatability coefficient (RC) was used to assess reproducibility. Bland-Altman plots were used to evaluate agreement between measurements day 1 vs. day 2, and manual vs. segmentation method. RESULTS: Ki showed excellent reproducibility in both white and gray matter with an ICC between 0.79 and 0.82 and excellent agreement between manual ROI and automatic segmentation, with an ICC of 0.89 for Ki in WM. Furthermore, Ki values in gray and white matter conforms with histological tissue characteristics, where gray matter generally has a 2-fold higher vessel density. The highest reproducibility measures of Ki (ICC = 0.83), CBF (ICC = 0.77) and Vd (ICC = 0.83) was obtained with the AIF sampled in the internal carotid artery (ICA). DATA CONCLUSION: DCE-MRI shows excellent reproducibility of pharmacokinetic variables derived from healthy controls. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

Long COVID symptoms and duration in SARS-CoV-2 positive children - a nationwide cohort study.

Most children have a mild course of acute COVID-19. Only few mainly non-controlled studies with small sample size have evaluated long-term recovery from SARS-CoV-2 infection in children. The aim of this study was to evaluate symptoms and duration of 'long COVID' in children. A nationwide cohort study of 37,522 children aged 0-17 years with RT-PCR verified SARS-CoV-2 infection (response rate 44.9%) and a control group of 78,037 children (response rate 21.3%). An electronic questionnaire was sent to all children from March 24th until May 9th, 2021. Symptoms lasting > 4 weeks were common among both SARS-CoV-2 children and controls. However, SARS-CoV-2 children aged 6-17 years reported symptoms more frequently than the control group (percent difference 0.8%). The most reported symptoms among pre-school children were fatigue Risk Difference (RD) 0.05 (CI 0.04-0.06), loss of smell RD 0.01 (CI 0.01-0.01), loss of taste RD 0.01 (CI 0.01-0.02) and muscle weakness RD 0.01 (CI 0.00-0.01). Among school children the most significant symptoms were loss of smell RD 0.12 (CI 0.12-0.13), loss of taste RD 0.10 (CI 0.09-0.10), fatigue RD 0.05 (CI 0.05-0.06), respiratory problems RD 0.03 (CI 0.03-0.04), dizziness RD 0.02 (CI 0.02-0.03), muscle weakness RD 0.02 (CI 0.01-0.02) and chest pain RD 0.01 (CI 0.01-0.01). Children in the control group experienced significantly more concentration difficulties, headache, muscle and joint pain, cough, nausea, diarrhea and fever than SARS-CoV-2 infected. In most children 'long COVID' symptoms resolved within 1-5 months. CONCLUSIONS: Long COVID in children is rare and mainly of short duration. WHAT IS KNOWN: • There are increasing reports on 'long COVID' in adults. • Only few studies have evaluated the long-term recovery from COVID-19 in children, and common for all studies is a small sample size (median number of children included 330), and most lack a control group. WHAT IS NEW: • 0.8% of SARS-CoV-2 positive children reported symptoms lasting >4 weeks ('long COVID'), when compared to a control group. • The most common 'long COVID' symptoms were fatigue, loss of smell and loss of taste, dizziness, muscle weakness, chest pain and respiratory problems. • These 'long COVID' symptoms cannot be assigned to psychological sequelae of social restrictions. • Symptoms such as concentration difficulties, headache, muscle- and joint pain as well as nausea are not 'long COVID' symptoms. • In most cases 'long COVID' symptoms resolve within 1-5 months.

Kinetic and phenotypic analysis of CD8+ T cell responses after priming with alphavirus replicons and homologous or heterologous booster immunizations.

UNLABELLED: Alphavirus replicons are potent inducers of CD8(+) T cell responses and thus constitute an attractive vaccine vector platform for developing novel vaccines. However, the kinetics and memory phenotype of CD8(+) T cell responses induced by alphavirus replicons are not well characterized. Furthermore, little is known how priming with alphavirus replicons affects booster immune responses induced by other vaccine modalities. We demonstrate here that a single immunization with an alphavirus replicon, administered as viral particles or naked DNA, induced an antigen-specific CD8(+) T cell response that had a sharp peak, followed by a rapid contraction. Administering a homologous boost before contraction had occurred did not further increase the response. In contrast, boosting after contraction when CD8(+) T cells had obtained a memory phenotype (based on CD127/CD62L expression), resulted in maintenance of CD8(+) T cells with a high recall capacity (based on CD27/CD43 expression). Increasing the dose of replicon particles promoted T effector memory (Tem) and inhibited T central memory development. Moreover, infection with a replicating alphavirus induced a similar distribution of CD8(+) T cells as the replicon vector. Lastly, the distribution of T cell subpopulations induced by a DNA-launched alphavirus replicon could be altered by heterologous boosts. For instance, boosting with a poxvirus vector (MVA) favored expansion of the Tem compartment. In summary, we have characterized the antigen-specific CD8(+) T cell response induced by alphavirus replicon vectors and demonstrated how it can be altered by homologous and heterologous boost immunizations. IMPORTANCE: Alphavirus replicons are promising vaccine candidates against a number of diseases and are by themselves developed as vaccines against, for example, Chikungunya virus infection. Replicons are also considered to be used for priming, followed by booster immunization using different vaccine modalities. In order to rationally design prime-boost immunization schedules with these vectors, characterization of the magnitude and phenotype of CD8(+) T cell responses induced by alphavirus replicons is needed. Here, we demonstrate how factors such as timing and dose affect the phenotypes of memory T cell populations induced by immunization with alphavirus replicons. These findings are important for designing future clinical trials with alphaviruses, since they can be used to tailor vaccination regimens in order to induce a CD8(+) T cell response that is optimal for control and/or clearance of a specific pathogen.

Immune responses to mRNA-based vaccines given as a third COVID-19 vaccine dose in people living with HIV-a literature review.

People living with HIV (PLWH) were not included in the first efficacy studies of mRNA vaccines against SARS-CoV-2. In this literature review, we investigate evidence of humoral and cellular immunity after a third dose of an mRNA vaccine in PLWH. We performed a literature search in PubMed, Embase, Web of Science and SCOPUS published between 1 January 2020 and 31 December 2022. Selection criteria were studies on immunological responses in PLWH, who were given an mRNA-based vaccine as a third vaccine dose against SARS-CoV-2. Eight articles complied with our selection criteria. All studies found a strong humoral response after the third dose. Five studies investigated cellular immunity and found an increased cellular response after the third vaccine dose in PLWH. No difference in humoral response was observed between PLWH and controls after three doses. However, some of the studies suggested a weaker cellular response among PLWH than in controls, which was associated with lower nadir or current CD4+ T-cell counts. In conclusion, we found evidence of strong humoral immunity in PLWH after receiving an mRNA-based COVID-19 vaccine as a third dose, while the cellular immunity may be impaired compared to controls.

Correlation between the radial artery resistance index and the systemic vascular resistance index: a cross-sectional study.

INTRODUCTION: Ultrasound measurement of the radial resistance index (RRI) in the anatomical snuffbox has been proposed as a useful method for assessing the systemic vascular resistance index (SVRI). This study aims to establish the correlation between SVRI measured by pulmonary artery catheter (PAC) and RRI. METHODS: A cross-sectional study included all consecutive patients undergoing postoperative (POP) cardiac surgery with hemodynamic monitoring using PAC. Hemodynamic assessment was performed using PAC, and RRI was measured with ultrasound in the anatomical snuffbox. The Pearson correlation test was used to establish the correlation between RRI and SVRI measured using PAC. Hemodynamic behavior concerning RRI with a cutoff point of 1.1 (described to estimate under SVRI) was examined. Additionally, consistency between two evaluators was assessed for RRI using the intraclass correlation coefficient and Bland-Altman analysis. RESULTS: A total of 35 measurements were obtained. The average cardiac index (CI) was 2.73 ± 0.64 L/min/m², and the average SVRI was 1967.47 ± 478.33 dyn·s·m²/cm5. The correlation between RRI and SVRI measured using PAC was 0.37 [95% CI 0.045-0.62]. The average RRI was 0.94 ± 0.11. RRI measurements > 1.1 had a mean SVRI of 2120.79 ± 673.48 dyn·s·m²/cm5, while RRI measurements ≤ 1.1 had a mean SVRI of 1953.1 ± 468.17 dyn·s·m²/cm5 (p = 0.62). The consistency between evaluators showed an intraclass correlation coefficient of 0.88 [95% CI 0.78-0.93], and Bland-Altman analysis illustrated adequate agreement of RRI evaluators. CONCLUSIONS: For patients in cardiac surgery POP, the correlation between the SVRI measured using PAC and the RRI measured in the anatomical snuffbox is low. Using the RRI as a SVRI estimator for patients is not recommended in this clinical scenario.

Age-related decline in cerebral oxygen consumption in multiple sclerosis.

Cerebral oxygen metabolism is altered in relapsing-remitting multiple sclerosis (RRMS), possibly a result of disease related cerebral atrophy with subsequent decreased oxygen demand. However, MS inflammation can also inhibit brain metabolism. Therefore, we measured cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2) using MRI phase contrast mapping and susceptibility-based oximetry in 44 patients with early RRMS and 36 healthy controls. Cerebral atrophy and white matter lesion load were assessed from high-resolution structural MRI. Expanded Disability Status Scale (EDSS) scores were collected from medical records. The CMRO2 was significantly lower in patients (-15%, p = 0.002) and decreased significantly with age in patients relative to the controls (-1.35 µmol/100 g/min/year, p = 0.036). The lower CMRO2 in RRMS was primarily driven by a higher venous oxygen saturation in the sagittal sinus (p = 0.007) and not a reduction in CBF (p = 0.69). There was no difference in cerebral atrophy between the groups, and no correlation between CMRO2 and MS lesion volume or EDSS score. Therefore, the progressive CMRO2 decline observed before the occurrence of significant cerebral atrophy and despite adequate CBF supports emerging evidence of dysfunctional cellular respiration as a potential pathogenic mechanism and therapeutic target in RRMS.

Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine.

Vaccination using "naked" DNA is a highly attractive strategy for induction of pathogen-specific immune responses; however, it has been only weakly immunogenic in humans. Previously, we constructed DNA-launched Semliki Forest virus replicons (DREP), which stimulate pattern recognition receptors and induce augmented immune responses. Also, in vivo electroporation was shown to enhance immune responses induced by conventional DNA vaccines. Here, we combine these two approaches and show that in vivo electroporation increases CD8(+) T cell responses induced by DREP and consequently decreases the DNA dose required to induce a response. The vaccines used in this study encode the multiclade HIV-1 T cell immunogen HIVconsv, which is currently being evaluated in clinical trials. Using intradermal delivery followed by electroporation, the DREP.HIVconsv DNA dose could be reduced to as low as 3.2 ng to elicit frequencies of HIV-1-specific CD8(+) T cells comparable to those induced by 1 µg of a conventional pTH.HIVconsv DNA vaccine, representing a 625-fold molar reduction in dose. Responses induced by both DREP.HIVconsv and pTH.HIVconsv were further increased by heterologous vaccine boosts employing modified vaccinia virus Ankara MVA.HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Using the same HIVconsv vaccines, the mouse observations were supported by an at least 20-fold-lower dose of DNA vaccine in rhesus macaques. These data point toward a strategy for overcoming the low immunogenicity of DNA vaccines in humans and strongly support further development of the DREP vaccine platform for clinical evaluation.

The impact of drug discontinuation in patients treated with temporary pacemaker due to atrioventricular block.

INTRODUCTION: Patients treated with a temporary pacemaker (TPM) due to atrioventricular (AV) block are often monitored after discontinuation of AV node blocking drugs to evaluate the indication for permanent pacing. However, the impact of drug discontinuation is sparsely documented. We investigated to what extent drug discontinuation abolished the need for permanent pacemaker (PPM) implantation. METHODS AND RESULTS: All hospital records of patients who received a TPM at Aalborg Hospital, Denmark, between January 2000 and March 2011 (n = 575) were retrospectively reviewed. Patients with AV block who were treated with a TPM and concomitant cessation of drug therapy were included if there was no other underlying mechanism causing the AV block. AV blocking drugs included antiarrhythmic agents classes II-IV and digoxin. Fifty-five patients fulfilled our inclusion criteria. Forty-seven patients had an indication for a PPM at the initial hospital admission, despite drug discontinuation. Of the remaining 8 patients who were discharged without a PPM, 3 subsequently experienced events: 2 had recurrence of AV block requiring a PPM, and 1 experienced syncope. Thus, in total, 49 (89%) patients had a final indication for a permanent pacemaker (PPM). Of patients receiving beta-blocker monotherapy, 26 (96%) had an indication for a PPM. TPM implantation was complicated by infection or displacement in 11% of cases. CONCLUSIONS: The vast majority of patients treated with a TPM due to AV block and who receive beta-blockers alone or in combination with digoxin have a final indication for a PPM despite cessation of drug treatment. TPM are frequently associated with complications.

Outcomes of emergency surgical interventions in right-sided colonic cancer: nationwide population-based study based on Danish Colorectal Cancer Group register.

AIM: The aim of this study was to investigate the trends in morbidity and mortality of patients with right-sided colonic cancer who had an emergency surgical procedure in Denmark after the introduction of quality index parameters. METHODS: This was a retrospective nationwide study based on a prospectively maintained Danish Colorectal Cancer Group database focused on right-sided colonic cancer in the interval from 1 May 2001 to 30 April 2018, who underwent emergency surgical intervention (within 48 h of hospital admission). The primary objective was to investigate the trends in morbidity and mortality throughout the study years. Multivariable estimates were adjusted for age, sex, smoking status, alcohol consumption, ASA score classification, tumour localization, type of access to abdominal cavity, surgeon's grade of specialization, and metastatic disease. RESULTS: Out of 2839 patients, a total of 2740 patients fulfilled the inclusion criteria, of whom 2464 underwent right or transverse colon resection (89.9 per cent). The 30-day and 90-day postoperative mortality rates were significantly reduced over the time of the study (OR 0.943, 95 per cent c.i. 0.922 to 0.965, P < 0.001 and OR 0.953, 95 per cent c.i. 0.934 to 0.972, P < 0.001 respectively); however, the complication rates did not follow this trend. Older patients (OR 1.032, 95 per cent c.i. 1.009 to 1.055, P = 0.005) and patients with high ASA scores (OR 1.61, 95 per cent c.i. 1.422 to 1.830, P < 0.001) had higher rates of severe grade 3b postoperative complications. A stoma was constructed in 276 patients (10 per cent), whereas a stent was used in only eight patients. Defunctioning procedures, including stoma construction or colonic stenting (without oncological resection), did not reduce the risk of complications compared with that of definitive surgery. CONCLUSION: The 30-day and 90-day postoperative mortality rates were significantly reduced over the time of the study. Age and ASA score were risk factors for severe postoperative complications.

Subclinical cognitive deficits are associated with reduced cerebrovascular response to visual stimulation in mid-sixties men.

Reduced cerebrovascular response to neuronal activation is observed in patients with neurodegenerative disease. In the present study, we examined the correlation between reduced cerebrovascular response to visual activation (ΔCBFVis.Act) and subclinical cognitive deficits in a human population of mid-sixties individuals without neurodegenerative disease. Such a correlation would suggest that impaired cerebrovascular function occurs before overt neurodegenerative disease. A total of 187 subjects (age 64-67 years) of the Metropolit Danish Male Birth Cohort participated in the study. ΔCBFVis.Act was measured using arterial spin labelling (ASL) MRI. ΔCBFVis.Act correlated positively with cognitive performance in: Global cognition (p = 0.046), paired associative memory (p = 0.025), spatial recognition (p = 0.026), planning (p = 0.016), simple processing speed (p < 0.01), and with highly significant correlations with current intelligence (p < 10-5), and more complex processing speed (p < 10-3), the latter two explaining approximately 11-13% of the variance. Reduced ΔCBFVis.Act was independent of brain atrophy. Our findings suggest that inhibited cerebrovascular response to neuronal activation is an early deficit in the ageing brain and associated with subclinical cognitive deficits. Cerebrovascular dysfunction could be an early sign of a trajectory pointing towards the development of neurodegenerative disease. Future efforts should elucidate if maintenance of a healthy cerebrovascular function can protect against the development of dementia.

Blood-brain barrier permeability changes in the first year after alemtuzumab treatment predict 2-year outcomes in relapsing-remitting multiple sclerosis.

BACKGROUND: In relapsing-remitting multiple sclerosis (RRMS), early disease control reduces the risk of permanent disability. The blood-brain barrier (BBB) is compromised in MS, and its permeability is a potential biomarker. OBJECTIVE: To investigate BBB permeability measured by MRI as a marker of alemtuzumab efficacy. METHODS: Patients with RRMS initiating alemtuzumab treatment were recruited prospectively. BBB permeability was assessed as the Patlak-derived influx constant (Ki) by dynamic contrast-enhanced MRI before and 6, 12, and 18 months after the first course of alemtuzumab. No Evidence of Disease Activity-3 (NEDA-3) status was ascertained two years after treatment initiation. RESULTS: Patients who maintained NEDA-3 status at two years (n = 7) had a larger decrease in Ki between baseline and six months (-0.029 ml/100 g/min [CI -0.005 - -0.053]) and between baseline and 12 months in normal appearing white matter (0.043 [CI 0.022 - -0.065]), than those who experienced disease activity (n = 8). ROC curve analysis of the Ki change between baseline and 12 months in NAWM predicted a loss of NEDA status at 2 years with 86% sensitivity and 86% specificity (AUC 0.98, p = 0.002). CONCLUSION: BBB permeability predicted alemtuzumab efficacy at two years, indicating that BBB permeability is a biomarker of treatment response in RRMS.

Adjuvants influence the maturation of VRC01-like antibodies during immunization.

Once naive B cells expressing germline VRC01-class B cell receptors become activated by germline-targeting immunogens, they enter germinal centers and undergo affinity maturation. Booster immunizations with heterologous Envs are required for the full maturation of VRC01-class antibodies. Here, we examined whether and how three adjuvants, Poly(I:C), GLA-LSQ, or Rehydragel, that activate different pathways of the innate immune system, influence the rate and type of somatic mutations accumulated by VRC01-class BCRs that become activated by the germline-targeting 426c.Mod.Core immunogen and the heterologous HxB2.WT.Core booster immunogen. We report that although the adjuvant used had no influence on the durability of plasma antibody responses after the prime, it influenced the plasma VRC01 antibody titers after the boost and the accumulation of somatic mutations on the elicited VRC01 antibodies.

Long peptides induce polyfunctional T cells against conserved regions of HIV-1 with superior breadth to single-gene vaccines in macaques.

A novel T-cell vaccine strategy designed to deal with the enormity of HIV-1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T-cell immunogen HIVconsv, which directs vaccine-induced responses to the most conserved regions of the HIV-1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub-pools and delivered into anatomically separate sites induced T-cell responses that were markedly broader than those elicited by traditional single-open-reading-frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV-1-derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T-cell responses than vaccines previously used in humans.

Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs.

UNLABELLED: Six major hepatitis C virus (HCV) genotypes and numerous subtypes have been described, and recently a seventh major genotype was discovered. Genotypes show significant molecular and clinical differences, such as differential response to combination therapy with interferon-alpha and ribavirin. Recently, HCV research has been accelerated by cell culture systems based on the unique growth capacity of strain JFH1 (genotype 2a). By development of JFH1-based intergenotypic recombinants containing Core, envelope protein 1 and 2 (E1, E2), p7, and nonstructural protein 2 (NS2) of genotype 6a and 7a strains, as well as subtype 1b and 2b strains, we have completed a panel of culture systems for all major HCV genotypes. Efficient growth in Huh7.5 cells depended on adaptive mutations for HK6a/JFH1 (6a/2a, in E1 and E2) and J4/JFH1 (1b/2a, in NS2 and NS3); viability of J8/JFH1 (2b/2a) and QC69/JFH1 (7a/2a) did not require adaptation. To facilitate comparative studies, we generated virus stocks of genotype 1-7 recombinants with infectivity titers of 10(3.7) to 10(5.2) 50% tissue culture infectious dose/mL and HCV RNA titers of 10(7.0) to 10(7.9) IU/mL. Huh7.5 cultures infected with genotype 1-6 viruses had similar spread kinetics, intracellular Core, NS5A, and lipid amounts, and colocalization of Core and NS5A with lipids. Treatment with interferon-alpha2b but not ribavirin or amantadine showed a significant antiviral effect. Infection with all genotypes could be blocked by specific antibodies against the putative coreceptors CD81 and scavenger receptor class B type I in a dose-dependent manner. Finally, neutralizing antibodies in selected chronic phase HCV sera had differential effects against genotype 1-7 viruses. CONCLUSION: We completed and characterized a panel of JFH1-based cell culture systems of all seven major HCV genotypes and important subtypes and used these viruses in comparative studies of antivirals, HCV receptor interaction, and neutralizing antibodies.

Estimation of Y haplotype frequencies with lower order dependencies.

Estimating Y haplotype population frequencies is a demanding task in forensic genetics. Despite the suggestion of various methods, none these have yet reached a level of accuracy and precision that is acceptable to the forensic genetics community. At the basis of this problem is the complex dependency structure between the involved STR loci. Here, we approximate this structure by the use of specific graphical models, namely t-cherry junction trees. We apply trees of order three by which dependencies between three STR loci can be taken into account, thereby extending the Chow-Liu method which is restricted to pairwise dependencies. We show that the t-cherry tree method outperforms the Chow-Liu method as well as the well-established discrete Laplace method in estimation accuracy.

Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens.

Cervical cancer develops as a result of infection with high-risk human papillomavirus (HPV) through persistent expression of early proteins E6 and E7. Our group pioneered a recombinant viral vector system based on Semliki Forest virus (SFV) for vaccination against cervical cancer. The most striking benefit of this alphavirus vector-based vaccine platform is its high potency. DNA vaccines on the other hand, have a major advantage with respect to ease of production. In this study, the benefits associated with both SFV-based vaccines and DNA vaccines were combined with the development of a DNA-launched RNA replicon (DREP) vaccine targeting cervical cancer. Using intradermal delivery followed by electroporation, we demonstrated that DREP encoding for E6,7 (DREP-E6,7) induced effective, therapeutic antitumor immunity. While immunizations with a conventional DNA vaccine did not prevent tumor outgrowth, immunization with a 200-fold lower equimolar dose of DREP (0.05 µg of DREP) resulted in approximately 85% of tumor-free mice. To overcome the safety concern of potential malignant transformation at the vaccination site, we evaluated the anti-tumor effect of a DREP vaccine encoding a shuffled version of E7 (DREP-E7sh). DREP-E7sh delayed tumor growth yet not to the same extent as DREP-E6,7. In addition, inclusion of a helper cassette and an ER targeting signal (sigHelp) did not significantly further enhance the suppression of tumor outgrowth in the long term, albeit exhibiting better tumor control early after immunization. Collectively, this study points towards the clinical evaluation of DREP encoding HPV antigens as a potent immunotherapy for patients with HPV16 (pre)-malignancies.

Increased 4R tau expression and behavioural changes in a novel MAPT-N296H genomic mouse model of tauopathy.

The microtubule-associated protein tau is implicated in various neurodegenerative diseases including Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, which are characterized by intracellular accumulation of hyperphosphorylated tau. Mutations in the tau gene MAPT cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In the human central nervous system, six tau isoforms are expressed, and imbalances in tau isoform ratios are associated with pathology. To date, few animal models of tauopathy allow for the potential influence of these protein isoforms, relying instead on cDNA-based transgene expression. Using the P1-derived artificial chromosome (PAC) technology, we created mouse lines expressing all six tau isoforms from the human MAPT locus, harbouring either the wild-type sequence or the disease-associated N296H mutation on an endogenous Mapt-/- background. Animals expressing N296H mutant tau recapitulated early key features of tauopathic disease, including a tau isoform imbalance and tau hyperphosphorylation in the absence of somatodendritic tau inclusions. Furthermore, N296H animals displayed behavioural anomalies such as hyperactivity, increased time in the open arms of the elevated plus maze and increased immobility during the tail suspension test. The mouse models described provide an excellent model to study the function of wild-type or mutant tau in a highly physiological setting.

Transforming growth factor-beta inhibits coxsackievirus-mediated autoimmune myocarditis.

Clinical myocarditis is a precursor to dilated cardiomyopathy and a principal cause of heart failure. Nearly 30% of all recently diagnosed cases of myocarditis are attributable to infection with coxsackie B virus (CBV), the most frequently associated pathogen. CBV initially replicates in the pancreas and quickly spreads to the heart, inducing chronic autoimmunity. To determine whether immunosuppressive cytokines could act to limit the extent of autoimmunity to the heart, we infected transgenic mice that express immunosuppressive cytokines in the pancreas. Herein, we demonstrate that transgenic expression of transforming growth factor-beta (1) (TGF-beta) within the pancreatic beta cells prevented mice from developing autoimmune myocarditis after CBV infection. In contrast, transgenic expression of interleukin-4 did not inhibit virus-mediated heart disease. Furthermore, we show that TGF-beta expression reduced viral replication while promoting the recruitment of macrophages into the pancreas. These results illustrate the benefit of TGF-beta in controlling not only viral replication, but also CBV-mediated autoimmunity.

Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA) or with HIV gp140 protein antigen.

Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 µg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 µg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 µg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

Intradermal electroporation of RNA.

This chapter describes the in vivo delivery of conventional mRNA or alphaviral replicon RNA via intradermal electroporation. The use of RNA in clinical applications has several potential advantages compared to DNA. For instance, RNA cannot integrate into the host genome, and it does not contain bacterial sequence motifs such as CpG often present in plasmid DNA backbones that can potentially trigger autoimmune responses. Intradermal electroporation is well tolerated and causes only minor trauma compared to intramuscular electroporation. As the skin houses high concentrations of antigen-presenting cells, vaccines could especially benefit from intradermal administration of RNA.