Intracerebral Challenge Research Articles

MicroRNA-sensitive Oncolytic Measles Viruses for Cancer-specific Vector Tropism

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased tumor-cell specificity, and are currently under investigation in clinical trials including a phase I study for glioblastoma multiforme (GBM). Recent preclinical studies have shown that the cellular tropism of several viruses can be controlled by inserting microRNA-target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. Since neuron-specific microRNA-7 is downregulated in gliomas but highly expressed in normal brain tissue, we engineered a microRNA-sensitive virus containing target sites for microRNA-7 in the 3'-untranslated region of the viral fusion gene. In presence of microRNA-7 this modification inhibits translation of envelope proteins, restricts viral spread, and progeny production. Even though highly attenuated in presence of microRNA-7, this virus retained full efficacy against glioblastoma xenografts. Furthermore, microRNA-mediated inhibition protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Importantly, endogenous microRNA-7 expression in primary human brain resections tightly restricted replication and spread of microRNA-sensitive virus. This is proof-of-concept that tropism restriction by tissue-specific microRNAs can be adapted to oncolytic MV to regulate viral replication and gene expression to maximize tumor specificity without compromising oncolytic efficacy.

Possible Origin of CSF Antibodies Induced by Intrathecal Immunization and Prophylactic Effects against Intracerebral Rabies Virus Infection

Intrathecal (IT) immunization involves injecting antigens directly into the intraventricular or subarachnoid spaces, or brain, to induce antigen-specific antibodies (Ab) in the cerebrospinal fluid (CSF). In the present study, rabbits were immunized IT with inactivated rabies virus to investigate the origins of CSF Ab. The time course of Ab induction and tumor necrosis factor-alpha expression suggested the possibility that the CSF Ab originated in the serum. In addition, Ab-producing cells infiltrated around the blood vessels of the brain, suggesting local production of Ab within the central nervous system (CNS). Furthermore, subcutaneous (SC) immunization prior to IT immunization induced a rapid and magnified Ab response in the CSF compared with IT immunization alone. These results were confirmed by the fact that mice immunized SC prior to IT were more resistant to intracerebral challenge with rabies virus than mice immunized via the IT route alone. Taken together, these results suggest that combined SC and IT immunization is a more effective vaccination protocol for prophylaxis and treatment of rabies.

Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination

Our previous studies indicated that recruitment and/or activation of dendritic cells (DCs) is important in enhancing the protective immune responses against rabies virus (RABV) (L. Zhao, H. Toriumi, H. Wang, Y. Kuang, X. Guo, K. Morimoto, and Z. F. Fu, J. Virol. 84:9642-9648). To address the importance of DC activation for RABV vaccine efficacy, the genes for several DC recruitment and/or activation molecules, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein 1α (MIP-1α), were individually cloned into RABV. The ability of these recombinant viruses to activate DCs was determined in vitro and in vivo. Infection of mouse bone marrow-derived DCs with each of the recombinant viruses resulted in DC activation, as shown by increased surface expression of CD11c and CD86 as well as an increased level of alpha interferon (IFN-α) production compared to levels observed after infection with the parent virus. Intramuscular infection of mice with each of the viruses recruited and/or activated more DCs and B cells in the periphery than infection with the parent virus, leading to the production of higher levels of virus-neutralizing antibodies. Furthermore, a single immunization with recombinant RABV expressing GM-CSF or MDC protected significantly more mice against intracerebral challenge with virulent RABV than did immunization with the parental virus. Yet, these viruses did not show more virulence than the parent virus, since direct intracerebral inoculation with each virus at up to 1 × 10(7) fluorescent focus units each did not induce any overt clinic symptom, such as abnormal behavior, or any neurological signs. Together, these data indicate that recombinant RABVs expressing these molecules activate/recruit DCs and enhance protective immune responses.

Evaluation of immunogenicity elicited from two DNA vaccine candidates that expresses the prM and E genes of the dengue-3 virus

In this work, we report the evaluation of two DNA vaccines against dengue-3 virus (DENV-3). The first construction, called pVAC3DEN3, was engineered inserting the pre-membrane (prM) and envelope (E) gene of DENV-3 truncated with a restriction site between them, as previously described. The second construction was developed cloning the full gene sequence of prM and E from DENV-3 virus in pCI plasmid for mammalian expression and was denominated pVAC1WDEN3. The results showed that both constructions were capable of expressing the prM and E proteins, as demonstrated by ELISA and immunoblotting detection in cell culture transfected with the plasmids. After positive “in vitro” results, the vaccine candidates were used to immunize BALB/c mice and the elicited response was investigated. After immunization by intramuscular inoculation with three doses of each vaccinal clone the animals were sacrificed, the cytokine levels and T cell response were analyzed in the spleens, after three days of culture with stimulus, our analysis showed that the two constructions elicited T cell responses mea- sured by BrdU incorporation assay and high levels of IFN-γ, detected in the supernatant of the cultures. Moreover, both constructions induced detectable titers of neutralizing antibodies in mice. And finally the survival rate of the immunized animals after intracerebral challenge was analyzed, showing a better result in the pVAC3DEN3 group with an 80% survival compared with a 50% survival of the pVAC1 WDEN3. Thus, these data showed that our two constructions were able to induce specific immune response and protects mice against a lethal challenge with DENV-3, and these vaccine candidates can be employed to develop a viable dengue vaccine.

Evaluation of the immunogenicity of a recombinant glycoprotein-based Chandipura vaccine in combination with commercially available DPT vaccine

Chandipura virus (CHPV) belongs to family Rhabdovoridae and has emerged as an encephalitis causing pathogen with high mortality among pediatric population from three Indian states. The recombinant glycoprotein (rGp) was shown to be an excellent vaccine candidate as evaluated in a murine model. As the disease is predominantly rural, to ensure maximum coverage for Chandipura vaccine, an attempt was made to evaluate combination of rGp and a commercially available DPT vaccine (CHP–DPT). When CHP–DPT was used for immunization of mice, 90% seroconversion against rGp with high antibody titers (1:1200 by ELISA and 1:320 by neutralization test) was observed and did not differ from mice immunized with rGp alone (P>0.05). Similarly seroconversions and antibody titers against DPT were comparable in mice immunized with DPT alone or in combination with rGp. Seroconversions and antibody titers ranged from 90 to 100% and 1:1200 to 1:2400 respectively. Intracerebral challenge with homologous CHPV strain resulted in 90% survival in rGp alone and CHP–DPT groups. Lymphocyte proliferative responses were also comparable. Thus, neither components of the candidate combination vaccine inhibited immune response to the other component. Substantial decrease of CHPV RNA and absence of histopathological changes in the brains of surviving immunized mice after challenge than the unimmunized controls further confirm efficacy of the vaccine even after intracerebral challenge. In conclusion, a combination vaccine seems feasible for use in a restricted area where the disease is endemic and should be subjected to additional studies required for future use in humans.

Lymphocytic Choriomeningitis Virus-Induced Mortality in Mice Is Triggered by Edema and Brain Herniation

Although much is known about lymphocytic choriomeningitis virus (LCMV) infection and the subsequent immune response in its natural murine host, some crucial aspects of LCMV-mediated pathogenesis remain undefined, including the underlying basis of the characteristic central nervous system disease that occurs following intracerebral (i.c.) challenge. We show that the classic seizures and paresis that occur following i.c. infection of adult, immunocompetent mice with LCMV are accompanied by anatomical and histological changes that are consistent with brain herniation, likely of the uncal subtype, as a causative basis for disease and precipitous death. Both by water weight determinations and by magnetic resonance imaging of infected brain tissues, edema was detected only at the terminal stages of disease, likely caused by the leakage of cerebrospinal fluid from the ventricles into the parenchyma. Furthermore, death was accompanied by unilateral pupillary dilation, which is indicative of uncal herniation. While immunohistochemical analysis revealed periventricular inflammation and a loss of integrity of the blood-brain barrier (BBB), these events preceded seizures by 2 to 3 days. Moreover, surviving perforin knockout mice showed barrier permeability equivalent to that of moribund, immunocompetent mice; thus, BBB damage does not appear to be the basis of LCMV-induced neuropathogenesis. Importantly, brain herniation can occur in humans as a consequence of injuries that would be predicted to increase intracranial pressure, including inflammation, head trauma, and brain tumors. Thus, a mechanistic dissection of the basis of LCMV neuropathogenesis may be informative for the development of interventive therapies to prevent this typically fatal human condition.

Chronic wasting disease (CWD) susceptibility of several North American rodents that are sympatric with cervid CWD epidemics.

Chronic wasting disease (CWD) is a highly contagious always fatal neurodegenerative disease that is currently known to naturally infect only species of the deer family, Cervidae. CWD epidemics are occurring in free-ranging cervids at several locations in North America, and other wildlife species are certainly being exposed to infectious material. To assess the potential for transmission, we intracerebrally inoculated four species of epidemic-sympatric rodents with CWD. Transmission was efficient in all species; the onset of disease was faster in the two vole species than the two Peromyscus spp. The results for inocula prepared from CWD-positive deer with or without CWD-resistant genotypes were similar. Survival times were substantially shortened upon second passage, demonstrating adaptation. Unlike all other known prion protein sequences for cricetid rodents that possess asparagine at position 170, our red-backed voles expressed serine and refute previous suggestions that a serine in this position substantially reduces susceptibility to CWD. Given the scavenging habits of these rodent species, the apparent persistence of CWD prions in the environment, and the inevitable exposure of these rodents to CWD prions, our intracerebral challenge results indicate that further investigation of the possibility of natural transmission is warranted.

Repetitive Immunization Enhances the Susceptibility of Mice to Peripherally Administered Prions

The susceptibility of humans and animals to prion infections is determined by the virulence of the infectious agent, by genetic modifiers, and by hitherto unknown host and environmental risk factors. While little is known about the latter two, the activation state of the immune system was surmised to influence prion susceptibility. Here we administered prions to mice that were repeatedly immunized by two initial injections of CpG oligodeoxynucleotides followed by repeated injections of bovine serum albumin/alum. Immunization greatly reduced the required dosage of peripherally administered prion inoculum necessary to induce scrapie in 50% of mice. No difference in susceptibility was observed following intracerebral prion challenge. Due to its profound impact onto scrapie susceptibility, the host immune status may determine disease penetrance after low-dose prion exposure, including those that may give rise to iatrogenic and variant Creutzfeldt-Jakob disease.

Modified intra-cerebral challenge assay for acellular pertussis vaccines: Comparisons among whole cell and acellular vaccines

The modified intra-cerebral challenge assay for acellular pertussis vaccines is used in Japan, Korea, China and possibly other Asian countries as the potency assay for routine release of acellular pertussis (aP) and combination vaccines. National reference standards, typically of whole cell pertussis (Pw) vaccine, are in use in these countries, but there is no agreed international reference standard for acellular pertussis vaccines.We report here the results of a collaborative study initiated in September 2006 in which fourteen laboratories performing the modified intra-cerebral challenge assay took part. These laboratories compared their various national references of Pw vaccine, the third International Standard for whole cell pertussis vaccine, a previously studied two-component freeze-dried aP vaccine preparation coded JNIH-3, and four different aP vaccines in combination with diphtheria and tetanus toxoids.The results of this study show that the modified intra-cerebral challenge assay works reliably although there are inter-laboratory variations in potency estimates. Pw and aP vaccines show apparent differences in dose–response lines in some assay systems. This indicates dissimilarity in performance in at least some of these assay systems. Estimates of relative potency for aP vaccines in terms of the Pw vaccine national or in-house reference preparations differ significantly from one another. Different mouse strains were used in each country and the different strains may also differ in their responsiveness to Pw and aP vaccines. Estimates for different types of aP vaccine formulations show less inter-laboratory variation in terms of JNIH-3 than in terms of the third IS for Pw vaccine and the remaining variation is not apparently related to the different mouse strains. This study thus suggests that an aP vaccine standard would improve inter-laboratory agreement. These data do not show significant dissimilarity in dose–response lines between JNIH-3 and the various vaccine products included, irrespective of the differences in aP components. Available data indicate that JNIH-3 is sufficiently stable to serve as an International Standard. On the basis of these results and with the agreement of the participants, it was proposed that JNIH-3 should be established as an International Standard for acellular pertussis vaccine for use in the modified intra-cerebral challenge assay and other protective bioassays, with an assigned activity of 34 International Units (IU) per ampoule.A WHO Working Group on Standardization of Acellular Pertussis Vaccines: potency assay met in Beijing, China, 7–9 November 2007. This group considered the report of this study, the comments of the participants and implications of the use of JNIH-3 as a reference standard and recommended establishment of JNIH-3 as an International Standard. The results of this study and the report of the Working Group were submitted to the Expert Committee on Biological Standardization (ECBS) of WHO which established JNIH-3 as the first International Standard for acellular pertussis vaccine in the modified intra-cerebral challenge assay and other protective bioassays with an assignment of 34IU per ampoule in October 2008.

Recombinant glycoprotein based vaccine for Chandipura virus infection

Chandipura virus (CHPV) has emerged as an important pediatric encephalitis-causing pathogen with very high mortality in India. No specific vaccine or treatment is available till date. We attempted to prepare a candidate vaccine employing recombinant CHPV Glycoprotein (rGp). The Glycoprotein gene (G-gene) of CHPV was expressed using Baculovirus expression system. The rGp was purified by HPLC and used for mice immunization, 3 doses, and 4 weeks apart. One microgram rGp was found to be optimum. Sero-conversion was observed as early as 2nd week by detecting anti-CHPV IgG antibodies. Antibody titres were immunogen-concentration dependent. Intracerebral challenge of the immunized mice with 100 LD 50 of the homologous strain demonstrated 90% protection. In in vitro neutralization, antibodies from the immunized mice were able to neutralize heterologous viruses. There was 60% T cell proliferation observed against rGp in immunized mice. The study shows that rGp induces both arms of immune response and represents an ideal vaccine candidate for further evaluations.

Olfactory System Involvement in Natural Scrapie Disease

The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrP(Sc)). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrP(Sc) might display there. We report here on detection by different techniques of PrP(Sc) in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrP(Sc) was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrP(Sc) immunostaining. PrP(Sc) deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrP(Sc) deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrP(Sc) immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa.

Immunohistochemical and biochemical characteristics of BSE and CWD in experimentally infected European red deer (Cervus elaphus elaphus)

BackgroundThe cause of the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom (UK) was the inclusion of contaminated meat and bone meal in the protein rations fed to cattle. Those rations were not restricted to cattle but were also fed to other livestock including farmed and free living deer. Although there are no reported cases to date of natural BSE in European deer, BSE has been shown to be naturally or experimentally transmissible to a wide range of different ungulate species. Moreover, several species of North America's cervids are highly susceptible to chronic wasting disease (CWD), a transmissible spongiform encephalopathy (TSE) that has become endemic. Should BSE infection have been introduced into the UK deer population, the CWD precedent could suggest that there is a danger for spread and maintenance of the disease in both free living and captive UK deer populations. This study compares the immunohistochemical and biochemical characteristics of BSE and CWD in experimentally-infected European red deer (Cervus elpahus elaphus).ResultsAfter intracerebral or alimentary challenge, BSE in red deer more closely resembled natural infection in cattle rather than experimental BSE in small ruminants, due to the lack of accumulation of abnormal PrP in lymphoid tissues. In this respect it was different from CWD, and although the neuropathological features of both diseases were similar, BSE could be clearly differentiated from CWD by immunohistochemical and Western blotting methods currently in routine use.ConclusionRed deer are susceptible to both BSE and CWD infection, but the resulting disease phenotypes are distinct and clearly distinguishable.

P.044 Laboratory response to an imported case of Marburg hemorrhagic fever

After intracerebral challenge with 100 PFU of Lassa virus (strain Josiah), all infected mice (CBA/calac) died (control group). Production of pro-inflammatory cytokines (IL-1β, TNF-α) significantly increased in the blood of these mice during the infection. For neutralization of increasing concentrations of these cytokines recombinant IL-1RA was used intraperitonealy at a dose 100 μg kg−1, everyday, within 5 days from the third day after the challenge. Injections of IL-1RA decreased the concentration of IL-1β and TNF-α and resulted in survival of all infected mice (treatment group). Marburg fever (strain Popp) caused in guinea pigs by 5 LD50 of virus lead to the significant increase of TNF-α in the animal’s blood and caused a lethal outcome (control group). Treatment of infected guinea pigs by IL-1RA or anti-TNF serum decreased the concentration of TNF-α and resulted in survival of half of the animals (treatment group). For the treatment recombinant IL-1RA was used at a dose 100 μg kg−1, intramuscularly, everyday, within 6 days from the third day after the challenge or anti-TNF serum, intramuscularly 0.5 ml (2000 U ml−1; 1 U of the antiserum neutralises 0.03 ng of TNF-α), everyday, within 6 days from the third day after the challenge.

Effect of adjuvants on immunization with dengue virus-induced cytotoxic factor.

Specific active immunization with dengue type 2 virus (DV)-induced cytokine, cytotoxic factor (CF), prevents CF-mediated pathology in mice. The present study was undertaken to determine the optimum dose of CF and the effect of different adjuvants on the immune response as assessed by the study of anti-CF antibody titre by ELISA and protection against increase in capillary permeability to challenging dose of 3 micrograms CF. The maximum protection of 94 +/- 4% against increase in capillary permeability was observed at week 4 after immunization with 5 micrograms dose of CF mixed with Freund's incomplete adjuvant (FIA), which gradually decreased to 21 +/- 10% on week 24. With a dose of 10 micrograms the protection obtained was 79 +/- 5%, but persisted for a longer time at a higher level. The response was poor with 1 microgram dose of CF. The mean anti-CF antibody titres gradually decreased after reaching the peak at week 4 after immunization. Mice immunized with different adjuvants emulsified with 5 micrograms CF were challenged at different intervals with 3 micrograms CF. Maximum protection observed with CF + tetanus toxoid (TT) and 84/246 was about 93 +/- 2% and 97 +/- 2%, while that with alhydrogel was 33 +/- 12% and with bacille Calmette-Guérin (BCG) was 67 +/- 4%. At week 24 after immunization, however, the best response was obtained with 10 micrograms of adjuvant 84/246. Intracerebral challenge with 10 or 100 LD50 dose of dengue type 2 virus showed significantly prolonged mean survival time and delayed onset of signs of sickness in immunized mice compared with normal mice. The maximum survival time was with adjuvant 84/246 even at week 24. The findings thus show that the optimum dose of CF is 5 micrograms and the adjuvant of choice is 84/246.

A species barrier limits transmission of chronic wasting disease to mink (Mustela vison)

Transmissible mink encephalopathy (TME) occurs as sporadic outbreaks associated with ingestion of feed presumably contaminated with some type of prion disease. Mink lack a species barrier to primary oral challenge with bovine spongiform encephalopathy, whereas they have a barrier to such challenge with scrapie. We investigated whether mink have a species barrier to chronic wasting disease (CWD) by performing primary intracerebral (IC) and primary oral challenge with CWD-positive elk brain. Primary IC challenge resulted in clinical disease in two of eight mink at 31–33 months incubation. Affected mink had spongiform vacuolation and astrocytosis within the central nervous system and immunoreactivity to disease-associated prion protein (PrPd) in brain, retina and lymph node. CWD IC recipients had significantly lower brain vacuolation and PrPd deposition scores, significantly lower cerebrocortical astrocyte counts and significantly higher hippocampal astrocyte counts than TME IC recipients. Primary oral challenge with CWD-positive elk brain (n=22) or with CWD-negative elk brain given IC (n=7) or orally (n=23) did not result in clinical or microscopic abnormalities during 42 months observation. Novel prion gene polymorphisms were identified at codon 27 (arginine/tryptophan) and codon 232 (arginine/lysine). This study shows that, whilst CWD can cause disease when given IC to mink, the lesions are not characteristic of TME, transmission is inefficient compared with TME and oral challenge does not result in disease. The demonstration of a species barrier in cervid-to-mustelid prion transmission indicates that mink are unlikely to be involved in natural CWD transmission.

Experimental transmission of bovine spongiform encephalopathy to European red deer (Cervus elaphus elaphus)

BackgroundBovine spongiform encephalopathy (BSE), a member of the transmissible spongiform encephalopathies (TSE), primarily affects cattle. Transmission is via concentrate feed rations contaminated with infected meat and bone meal (MBM). In addition to cattle, other food animal species are susceptible to BSE and also pose a potential threat to human health as consumption of infected meat products is the cause of variant Creutzfeldt-Jakob disease in humans, which is invariably fatal. In the UK, farmed and free ranging deer were almost certainly exposed to BSE infected MBM in proprietary feeds prior to legislation banning its inclusion. Therefore, although BSE has never been diagnosed in any deer species, a possible risk to human health remains via ingestion of cervine products. Chronic wasting disease (CWD), also a TSE, naturally infects several cervid species in North America and is spreading rapidly in both captive and free-ranging populations.ResultsHere we show that European red deer (Cervus elaphus elaphus) are susceptible to intra-cerebral (i/c) challenge with BSE positive cattle brain pool material resulting in clinical neurological disease and weight loss by 794–1290 days and the clinical signs are indistinguishable to those reported in deer with CWD. Spongiform changes typical of TSE infections were present in brain and accumulation of the disease-associated abnormal prion protein (PrPd) was present in the central and peripheral nervous systems, but not in lymphoid or other tissues. Western immunoblot analysis of brain material showed a similar glycosylation pattern to that of BSE derived from infected cattle and experimentally infected sheep with respect to protease-resistant PrP isoforms. However, the di-, mono- and unglycosylated bands migrated significantly (p < 0.001) further in the samples from the clinically affected deer when compared to BSE infected brains of cattle and sheep.ConclusionThis study shows that deer are susceptible to BSE by intra-cerebral inoculation and display clinical signs and vacuolar pathology that are similar to those of CWD. These findings highlight the importance of preventing the spread to Europe of CWD from North America as this may necessitate even more extensive testing of animal tissues destined for human consumption within the EU. Although the absence of PrPd in lymphoid and other non-neurological tissues potentially limits the risk of transmission to humans, the replication of TSE agents in peripheral tissues following intra-cerebral challenge is often limited. Thus the assessment of risk posed by cervine BSE as a human pathogen or for environmental contamination should await the outcome of ongoing oral challenge experiments.

Lymphoma Chemovirotherapy: CD20-Targeted and Convertase-Armed Measles Virus Can Synergize with Fludarabine

Combination chemotherapy regimen incorporating CD20 antibodies are commonly used in the treatment of CD20-positive non-Hodgkin's lymphoma (NHL). Fludarabine phosphate (F-araAMP), cyclophosphamide, and CD20 antibodies (Rituximab) constitute the FCR regimen for treating selected NHL, including aggressive mantle cell lymphoma (MCL). As an alternative to the CD20 antibody, we generated a CD20-targeted measles virus (MV)-based vector. This vector was also armed with the prodrug convertase purine nucleoside phosphorylase (PNP) that locally converts the active metabolite of F-araAMP to a highly diffusible substance capable of efficiently killing bystander cells. We showed in infected cells that early prodrug administration controls vector spread, whereas late administration enhances cell killing. Control of spread by early prodrug administration was also shown in an animal model: F-araAMP protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Enhanced oncolytic potency after extensive infection was shown in a Burkitt's lymphoma xenograft model (Raji cells): After systemic vector inoculation, prodrug administration enhanced the therapeutic effect synergistically. In a MCL xenograft model (Granta 519 cells), intratumoral (i.t.) vector administration alone had high oncolytic efficacy: All mice experienced complete but temporary tumor regression, and survival was two to four times longer than that of untreated mice. Cells from MCL patients were shown to be sensitive to infection. Thus, synergy of F-araAMP with a PNP-armed and CD20-targeted MV was shown in one lymphoma therapy model after systemic vector inoculation.

Neonatal immunization with a single dose of recombinant BCG expressing subunit S1 from pertussis toxin induces complete protection against Bordetella pertussis intracerebral challenge

The currently used pertussis vaccines are highly efficacious; however, neonates are susceptible to whooping cough up to the sixth month. In agreement, DTP-immunized neonate mice were not protected against intracerebral challenge with Bordetella pertussis. Neonate mice immunized with either DTP or a recombinant-BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin do not show a humoral immune response against PT. On the other hand, rBCG-Pertussis induces higher PT-specific IFN-γ production and an increase in both IFN-γ + and TNF-α +-CD4 +-T cells than the whole cell pertussis vaccine and confers protection against a lethal intracerebral challenge with B. pertussis.

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP (‘ΔPrP'). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC-like protein, ‘π'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC-deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC-like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

Experimental scrapie in ‘plt’ mice: an assessment of the role of dendritic-cell migration in the pathogenesis of prion diseases

Peripherally acquired transmissible spongiform encephalopathies display strikingly long incubation periods, during which increasing amounts of prions can be detected in lymphoid tissues. While precise sites of peripheral accumulation have been described, the mechanisms of prion transport from mucosa and skin to lymphoid and nervous tissues remain unknown. Because of unique functional abilities, dendritic cells (DCs) have been suspected to participate in prion pathogenesis. In mice inoculated subcutaneously with scrapie-infected DCs, the incubation was shorter when cells were alive as compared with killed cells, suggesting that DC functions may facilitate prion neuroinvasion. However, early propagation in lymphoid tissues seemed not importantly affected by DC vitality. Mutant (plt) mice that have deficient CCL19/CCL21 expression and DC migration displayed similar infection of secondary lymphoid organs as normal mice, regardless of the route of inoculation and scrapie strain. Under certain conditions of transcutaneous inoculation, the incubation and duration of disease were moderately prolonged in plt mice. This was not related to a milder neuropathogenesis, since plt and normal mice were equally susceptible to intracerebral prion challenge. We conclude that peripheral spreading of prions appears poorly dependent on cell migration through the chemokine/receptor system CCL19/CCL21/CCR7, although DCs might be able to help prions reach sites of neuroinvasion.