Mouse Model Of Encephalitis Research Articles

The Cellular Immune Response Plays an Important Role in Protecting Against Dengue Virus in the Mouse Encephalitis Model

For several years, researchers have known that the generation of neutralizing antibodies is a prerequisite for attaining adequate protection against dengue virus. Nevertheless, the cellular immune response is the principal arm of the adaptive immune system against non-cytopathic viruses such as dengue, as once the virus enters into the cell it is necessary to destroy it to eliminate the virus. To define the role of the cellular immune response in the protection against dengue, we selected the mouse encephalitis model. Mice were immunized with a single dose of infective dengue 2 virus and different markers of both branches of the induced adaptive immunity were measured. Animals elicited a broad antibody response against the four dengue virus serotypes, but neutralizing activity was only detected against the homologous serotype. On the other hand, the splenocytes of the infected animals strongly proliferated after in vitro stimulation with the homologous virus, and specifically the CD8 T-cell subset was responsible for the secretion of the cytokine IFN-gamma. Finally, to define the role of T cells in in vivo protection, groups of animals were inoculated with the depleting monoclonal antibodies anti-CD4 or anti-CD8. Only depletion with anti-CD8 decreased to 50% the level of protection reached in the non-depleted mice. The present work constitutes the first report defining the role of the cellular immune response in protection against dengue virus in the mouse model.

Humanized Monoclonal Antibodies Derived from Chimpanzee Fabs Protect against Japanese Encephalitis Virus In Vitro and In Vivo

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.

Cognitive dysfunction in HIV encephalitic SCID mice correlates with levels of Interferon-α in the brain

Interferon alpha (IFNalpha) is an antiviral cytokine produced in response to viral infection. IFNalpha also acts as a neuromodulatory molecule in the central nervous system (CNS). Elevated IFNalpha in the CNS causes cognitive deficits. To determine if elevated levels of IFNalpha in an HIV encephalitis mouse model correlate with cognitive deficits. C57BL/6J SCID mice were inoculated intracerebrally (i.c.) with HIV infected or uninfected (control) macrophages and cognitively tested in a water escape radial arm maze. After behavioral testing was completed, immunohistochemistry and ELISA were used to examine brain pathology and IFNalpha expression. Mice injected i.c. with HIV infected macrophages exhibited significantly more working memory errors, particularly in trials with the highest memory load. Immunohistochemistry indicated increased mouse IFNalpha staining prevalent on neurons and glial cells in the brains of mice with HIV infected macrophages compared to mice with uninfected control macrophages. In addition, IFNalpha levels in the brain correlated directly with working memory errors for mice with HIV infected macrophages. These data suggest that the cognitive deficit noted for the C57BL/6J SCID mice with HIV infected macrophages is mediated by the infection induced increase in IFNalpha.

Measles virus-specific CD4 T-cell activity does not correlate with protection against lung infection or viral clearance.

Acute measles in children can be prevented by immunization with the live attenuated measles vaccine virus. Although immunization is able to induce CD4 and CD8 T cells as well as neutralizing antibodies, only the latter have been correlated with protective immunity. CD8 T cells, however, have been documented to be important in viral clearance in the respiratory tract, whereas CD4 T cells have been shown to be protective in a mouse encephalitis model. In order to investigate the CD4 T-cell response in infection of the respiratory tract, we have defined a T-cell epitope in the hemagglutinin (H) protein for immunization and developed a monoclonal antibody for depletion of CD4 T cells in the cotton rat model. Although the kinetics of CD4 T-cell development correlated with clearance of virus, the depletion of CD4 T cells during the primary infection did not influence viral titers in lung tissue. Immunization with the H epitope induced a CD4 T-cell response but did not protect against infection. Immunization in the presence of maternal antibodies resulted in the development of a CD4 T-cell response which (in the absence of neutralizing antibodies) did not protect against infection. In summary, CD4 T cells do not seem to protect against infection after immunization and do not participate in clearance of virus infection from lung tissue during measles virus infection. We speculate that the major role of CD4 T cells is to control and clear virus infection from other affected organs like the brain.

In vitro and in vivo infection of neural cells by a recombinant measles virus expressing enhanced green fluorescent protein.

This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell spread was studied by virtue of the resulting EGFP autofluorescence, using real-time confocal microscopy. Cells were differentiated to a neuronal phenotype, and extended processes, which interconnected the cells, were observed. It was also possible to infect the differentiated neuroblastoma cells (dTMN) with MVeGFP. Single autofluorescent EGFP-positive cells were selected at the earliest possible point in the infection, and the spread of EGFP autofluorescence was monitored. In this instance the virus used the interconnecting processes to spread from cell to cell. Human neuroblastoma cells (SH-SY-5Y) were also infected with MVeGFP. The virus infected these cells, and existing processes were used to initiate new foci of infection at distinct regions of the monolayer. Transgenic animals expressing CD46, a measles virus receptor, and lacking interferon type 1 receptor gene were infected intracerebrally with MVeGFP. A productive infection ensued, and the mice exhibited clinical signs of infection, such as ataxia and an awkward gait, identical to those previously observed for the parental virus (Edtag). Mice were sacrificed, and brain sections were examined for EGFP autofluorescence by confocal scanning laser microscopy over a period of 6 h. EGFP was detected in discrete focal regions of the brain and in processes, which extended deep into the parenchyma. Collectively, these results indicate (i) that MVeGFP can be used to monitor virus replication sensitively, in real time, in animal tissues, (ii) that infection of ependymal cells and neuroblasts provides a route by which measles virus can enter the central nervous system in mouse models of encephalitis, and (iii) that upon infection, the virus spreads transneuronally.

The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection.

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1beta, IL-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed 20- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

Effect of recombinant human interferon α B/D (rHu-IFN-α B/D) in combination with acyclovir in experimental HSV-1 encephalitis

The efficacy of recombinant human interferon α B/D in experimental HSV-1 encephalitis was investigated in the murine system. Recombinant Hu-IFN-α B/D significantly reduced the mortality in a mouse encephalitis model (about 30%, P=0.021), whereas natural mouse interferon was inactive. Combination of acyclovir with Hu-IFN-α B/D had an additive effect.

Fine specificity of the antibody response to a synthetic peptide from the fusion protein and protection against measles virus-induced encephalitis in a mouse model.

A synthetic peptide representing residues 397-420 from the measles virus (MV) fusion (F) protein was tested for its structure, immunogenicity and protective capacity against intracerebral challenge with a neuroadapted strain of MV. Analysis of the peptide by mass spectrometry showed that it was linear, despite the presence of two cysteine residues in the sequence. Circular dichroism spectroscopy highlighted a weak preference for the peptide to adopt an alpha-helical conformation. The peptide was shown to be immunogenic in BALB/c and C57BL/6 mice after intraperitoneal immunization in Freund's adjuvant, and anti-peptide antibodies from both strains of mice reacted with the MV as a solid phase antigen on an ELISA plate. When the fine specificity of the anti-peptide antibody response was examined using overlapping 8-mer peptides, serum antibodies from BALB/c mice recognized the region between residues 407-417 whereas antibodies from C57BL/6 mice recognized the region 408-420 of the 397-420 peptide sequence. Although anti-397-420 antibodies had no demonstrable neutralizing activity, protection against challenge with a neuroadapted strain of MV was demonstrated following active immunization with peptide in C57BL/6 mice or after passive transfer of anti-peptide antibodies in BALB/c mice. These findings highlight the importance of the 397-420 region in the induction of protective antibodies in the MV encephalitis mouse model, and suggest that this epitope might be a good candidate for inclusion in a future MV synthetic peptide vaccine.

Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis

The protective immunity conferred by a set of recombinant vaccinia viruses containing the entire coding sequence of dengue virus type 4 nonstructural glycoprotein NS1 plus various flanking sequences was evaluated by using a mouse encephalitis model. Mice immunized with recombinant vNS1-NS2a, which expresses authentic NS1, were solidly protected against intracerebral dengue virus challenge. However, mice immunized with recombinants vNS1-15%NS2a and vRSVG/NS1-15%NS2a, which express aberrant forms of NS1, were only partially protected (63 to 67% survival rate). Serologic analysis showed that mice immunized with vNS1-NS2a developed high titers of antibodies to NS1 as measured by radioimmunoprecipitation, enzyme-linked immunosorbent assay, and complement-mediated cytolytic assays. In addition, a pool of sera from these animals was protective in a passive transfer experiment. Lower titers of NS1-specific antibodies were detected in sera of animals immunized with vNS1-15%NS2a or vRSVG/NS1-15%NS2a by all three assays. These data support the view that protection against dengue virus infection in mice may be mediated at least in part by NS1-specific antibodies through a mechanism of complement-mediated lysis of infected cells. Additionally, immunization with two recombinant viruses expressing authentic NS1 of dengue virus type 2 conferred partial protection (30-50%) against dengue virus type 2 challenge.

Synthesis and anti-herpes-virus activity of acyclic 2'-deoxyguanosine analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.

Several "sugar" modified acyclic nucleoside analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 2) were synthesized and evaluated for antiviral activity. The preparation generally involved the condensation of the acetoxymethyl ether of alcohols 6c-g and 10-12a with diacetylguanine to give adducts 7c-g and 14-16, which were then deprotected to afford analogues 9c-g and 17-19. Alternatively, alcohols 12a and 13a were converted to iodides via their tosylates 12b and 13b and then reacted with the sodium salt of guanine to afford, after deprotection, analogues 22 and 23. A crossed aldol-Cannizzaro reaction on aldehyde 27 readily afforded 28, which was deprotected to give analogue 29. An in vitro assay against HSV-1 showed that all compounds tested were less active than DHPG, though several were good substrates for the viral thymidine kinase. The more promising acyclic nucleosides 9c, 19, and 29 were evaluated in a mouse encephalitis model and proved ineffective at preventing death at a dose of 20 mg/kg.

Passive immunotherapy for encephalitis caused by herpes simplex virus.

Passive administration of antiviral antibody has been assessed in animal models as a potential form of treatment for infections due to herpes simplex virus (HSV). Previous investigative work has demonstrated the beneficial effect of immune serum administered either before or after HSV infection of mice. In some studies the protective effect of antibody was abrogated in the absence of T lymphocytes, an observation suggesting that cellular effector mechanisms may be necessary for antibody efficacy. In the present study of a mouse model of encephalitis caused by HSV type 2, a 0.5-ml dose of human immunoglobulin (1,250 mg of immunoglobulin G/kg of body weight) prepared at pH 4.25 for intravenous administration reduced mortality and prolonged survival when administered by intraperitoneal injection 24 hr before or up to 8 hr after intranasal viral challenge. The serum titer of HSV-neutralizing antibody and the response to therapy were dose dependent. Clinical trials of passive immunotherapy for severe HSV infections may be warranted.

ChemInform Abstract: SYNTHESIS AND ANTIHERPES SIMPLEX VIRUS ACTIVITY OF 9‐((1,3‐DIHYDROXY‐2‐PROPYLTHIO)METHYL)GUANINE

AbstractAusgehend von dem Bis‐ether (I) gelangt man zu der im Titel genannten Verbindung (IV), die Wirkung gegen Viren vom Typ HSV‐1, HSV‐2 und HCMV zeigt.

Acyclic analogues of 2'-deoxynucleosides related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine as potential antiviral agents.

A series of acyclic analogues of 2'-deoxynucleosides related in structure to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) have been synthesized and evaluated for antiviral activity against herpes simplex virus type 1 (F strain). Additionally, the ability of these analogues to function as substrates for the virus-specified thymidine kinase was examined. Phosphorylation by this kinase is essential for antiviral activity. Although the acyclic 4-oxopyrimidine nucleosides were substrates for the kinase, they were devoid of antiviral activity. In the purine series, most analogues similar in structure to DHPG did exhibit significantly lower antiviral activity, indicating that even small modifications in the purine substituents substantially reduce the antiviral potency. The most active agent, 2,6-diaminopurine 27, was only poorly phosphorylated by the viral kinase; therefore, its activity was most likely due to a prior enzymatic deamination to give DHPG. Evaluation of 27 in a mouse encephalitis model has shown it to be nearly as potent as DHPG (1).

Experimental Encephalitis Caused by Herpes Simplex Virus: Comparison of Treatment with Tilorone Hydrochloride and Phosphonoacetic Acid

A mouse model of encephalitis caused by herpes simplex virus was used to compare the antiviral activity of tilorone hydrochloride with that of phosphonoacetic acid. These compounds were also administered simultaneously to determine whether the combination had a synergistic effect. Rates of survival and concentrations of virus in brain were used as criteria for judging the effectiveness of treatment. The fatal course of encephalitis was not altered by any treatment protocols in which tilorone hydrochloride was used alone. Four days of treatment with phosphonoacetic acid resulted in a long-term survival rate of about 15% of the infected, treated animals, and extension of this therapy for an additional three days resulted in an overall survival rate of about 35%. No increase in survival rate was obtained by use of phosphonoacetic acid and tilorone hydrochloride in combination. The concentration of virus in the brains of tilorone hydrochloride treated animals did not differ significantly from that in the untreated, infected control animals. Treatment with phosphonoacetic acid resulted in a reduction in titer of virus in brain and in an increased rate of survival.