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Summary Regenerative medicine has great potential. The pace of scientific advance is exciting and the medical opportunities for regeneration and repair may be transformative. However, concerns continue to grow, relating to problems caused both by unscrupulous private clinics offering unregulated therapies based on little or no evidence and by premature regulatory approval on the basis of insufficient scientific rationale and clinical evidence. An initiative by the InterAcademy Partnership convened experts worldwide to identify opportunities and challenges, with a focus on stem cells. This was designed to be inclusive and consensus outputs reflected the diversity of the global research population. Among issues addressed for supporting research and innovation while protecting patients were ethical assessment; pre-clinical and clinical research; regulatory authorization and medicines access; and engagement with patients, policy makers, and the public. The InterAcademy Partnership (IAP) identified options for action for sharing good practice and building collaboration within the scientific community and with other stakeholders worldwide., Highlights • An inclusive IAP study examined global stem cell opportunities and challenges • Great potential leads to concerns for evidence base in unregulated clinics • Also concerns for premature regulatory approval without sufficient evidence • Recommendations for responsible research and innovation and collaborations, In this article, Fears and colleagues describe an inclusive global initiative by the InterAcademy Partnership examining stem cell opportunities and challenges. Great therapeutic potential brings concerns about the inadequate evidence base used by unregulated clinics. Concerns are also expressed about premature regulatory approval without sufficient evidence. Recommendations cover ethical assessment; pre-clinical and clinical research; regulatory authorization and medicines access; and engagement with patients, policy makers, and the public.

An EASAC working group on genome editing recommends that regulators should focus on specific applications of these new techniques rather than attempting to regulate genome editing itself as a new technology.

N-methyl-D-aspartate (NMDA) receptor activation is involved in the pathogenetic cascades of neurodegenerative disorders including human immunodeficiency virus (HIV) dementia. Memantine, an uncompetitive NMDA receptor antagonist, which has been recently approved for the treatment of Alzheimer's disease, is being discussed as a potential adjunctive therapeutic substance for HIV dementia. We used simian immunodeficiency virus-infected rhesus macaques to assess the effects of memantine on brain dysfunction and brain pathology within 3-5 months after initial infection during early asymptomatic stage of disease. We had shown previously that within this time frame, marked changes were evident in the dopaminergic systems. Memantine was administered two weeks post infection, at peak viremia, in order to prevent early NMDA receptor activation due to immune mediators. We found that memantine prevented onset of dopamine deficits in the brains of SIV-infected macaques, without affecting early brain pathology or peripheral course of infection. Memantine specifically upregulated mRNA and protein expression of the neurotrophic factor brain-derived neurotrophic factor (BDNF), suggesting that the protective effect of memantine on dopamine function may be mechanistically remote from NMDA receptor antagonism. This novel pharmacological action of memantine may also be relevant for other neurodegenerative disorders and supports the involvement of neurotrophic factors in adult brain neuroprotection.

Movement disorders are a common neurological complication of immunodeficiency virus infection and are thought to result from dopaminergic dysfunction in the basal ganglia. We measured levels of dopamine, and its metabolites homovanillic acid and 3,4-dihydroxyphenylacetic acid, in the putamen of healthy and simian immunodeficiency virus (SIV)-infected rhesus monkeys from infection until the development of AIDS. Changes in expression levels of cAMP response element binding protein (CREB), a transcription factor involved in the signalling pathway of dopamine, were also examined. Furthermore, we isolated microglia from the same animals and investigated their activation status in order to explore whether neurochemical findings are associated with immune activation. Plasma and CSF viral RNA load, T-cell analysis and basal ganglia histopathology provided information about disease progression in the animals. Putamen dopamine content was significantly reduced within 3 months of SIV infection, due to decreased dopamine synthesis initially, followed by loss of tyrosine hydroxylase-positive cells in substantia nigra, and accompanied by a decrease in total CREB expression. Pharmacological manipulation of dopaminergic tone with L-DOPA and selegiline showed that the reduction in CREB expression was due to reduced levels of dopamine. These neurochemical changes were significantly correlated with microglia activation in the absence of gross histopathological lesions. Our data demonstrate that putamen dopaminergic function is impaired during SIV infection and indicate that microglia may trigger endogenous mechanisms involved in the dysfunction of dopaminergic systems.

After passive transfer of measles virus (MV)-specific antibodies, vaccine-induced seroconversion and subsequent protection is inhibited in cotton rats (Sigmodon hispidus). In this system, an attenuated, recombinant vesicular stomatitis virus expressing the MV haemagglutinin (VSV-H) was found previously to induce neutralizing antibodies and protection against MV challenge after intranasal (i.n.) immunization. Here it is demonstrated that, after i.n. immunization, VSV-H is found in both lung and brain tissue in the absence of clinical signs. Intratracheal inoculation, which does not lead to infection of the brain, proved that immunization via the lung mucosa is sufficient to protect. To reduce or eliminate infection of the brain after i.n. inoculation, stepwise-attenuated VSV-H mutants with truncated cytoplasmic tails of the G protein were tested in cotton rats. A mutant with 9 aa in the G cytoplasmic tail was found at much lower levels in the brain and was protective in the absence or presence of MV-specific antibodies. A more attenuated mutant containing only 1 aa in its tail was not found in brain tissue after inoculation, but it still induced protective antibody to measles in the absence of MV-specific antibody. However, its ability to induce MV-neutralizing antibodies in the presence of passively transferred MV-specific antibodies and its protective capacity was abolished unless higher-dose immunizations were used. This study demonstrates that a lower degree of attenuation is required to be able to immunize in the presence of MV-specific antibodies.

After infection of CEM174.T2 cells [deficient for the transporter of antigen presentation (TAP)] with measles virus (MV) the nucleocapsid protein is recognized by Ld-restricted cytotoxic T cells in a TAP-independent, chloroquine-sensitive fashion. Presentation via the TAP-independent pathway requires virus replication. During MV infection of the cell the nucleocapsid as well as the matrix protein enter the endolysosomal compartment as indicated by colocalization with the lysosomal-associated membrane protein 1 (LAMP-1). Similarly, the nucleocapsid protein of canine distemper virus (CDV) is recognized in a TAP-independent fashion. In addition, a recombinant MV expressing bacterial β-galactosidase protein is able to introduce the recombinant antigen into the TAP-independent pathway whereas a vaccinia virus expressing β-galactosidase is not. These data and a report about TAP-independent recognition of parainfluenza virus type 1 suggest that members of the Paramyxoviridae family regularly introduce viral proteins into the TAP-independent antigen-processing pathway.

CD46 (or membrane cofactor protein) protects autologous cells from complement-mediated lysis and has been expressed as a transgene in pigs to overcome complement-mediated hyperacute rejection of porcine organs upon transplantation into primates. Since CD46 has been identified as a receptor for measles virus (MV), the susceptibility of CD46-transgenic (tg) pig peripheral blood mononuclear cells (PBMC) to infection with MV strains which do and do not use CD46 as receptor was investigated. Surprisingly, it was found that MV vaccine strains (e.g. Edmonston) bound to tg as well as non-tg pig PBMC. Phytohaemagglutinin-stimulated CD46-tg and non-tg pig PBMC were equally well infected with MV vaccine strains irrespective of CD46 expression. Upon infection, tg CD46 was downregulated from the cell surface. In contrast, the binding capacity for MV wild-type strains to pig and human PBMC was low, irrespective of CD46 expression. These MV strains did not infect tg or non-tg pig cells. Expression of endogenous pig CD46 was detected with polyclonal sera against human CD46. After infection of pig PBMC with MV strain Edmonston, endogenous pig CD46 was also downregulated. This suggests an interaction between MV Edmonston and pig CD46. However, polyclonal CD46 sera did not inhibit infection with MV Edmonston indicating that CD46 may not exclusively act as a receptor for MV on these cells. Interestingly, similar results were observed using human PBMC. Data suggest that CD46 downregulation after interaction with MV may also occur in porcine organs which express endogenous and/or human CD46 as a means of protection against complement-mediated damage.

In humans, maternal antibodies inhibit successful immunization against measles, because they interfere with vaccine-induced seroconversion. We have investigated this problem using the cotton rat model ( Sigmodon hispidus ). As in humans, passively transferred antibodies inhibit the induction of measles virus (MV)-neutralizing antibodies and protection after immunization with MV. In contrast, a recombinant vesicular stomatitis virus (VSV) expressing the MV hemagglutinin (VSV-H) induces high titers of neutralizing antibodies to MV in the presence of MV-specific antibodies. The induction of neutralizing antibodies increased with increasing virus dose, and all doses gave good protection from subsequent challenge with MV. Induction of antibodies by VSV-H was observed in the presence of passively transferred human or cotton rat antibodies, which were used as the models of maternal antibodies. Because MV hemagglutinin is not a functional part of the VSV-H envelope, MV-specific antibodies only slightly inhibit VSV-H replication in vitro. This dissociation of function and antigenicity is probably key to the induction of a neutralizing antibody in the presence of a maternal antibody.

Plasmids that expressed the nucleocapsid, haemagglutinin and fusion proteins of measles virus (MV) were used to immunize cotton rats (Sigmodon hispidus) against intranasal MV infection. After immunization with all three plasmids, T cell responses and MV-specific antibodies were induced. A reduction in virus titre was observed in lung tissue from animals immunized with plasmids expressing the viral glycoproteins. Histologically, however, a moderate peribronchitis was observed after immunization with the plasmid expressing the fusion protein whereas, after immunization with plasmids expressing haemagglutinin or both glycoproteins, only mild or focal peribronchitis was seen. Immunization with the nucleocapsid did not reduce virus titres, probably because of the failure to induce neutralizing antibodies. A disadvantage of plasmid immunization was its inefficacy in the presence of MV-specific ‘maternal’ antibodies. This indicates that genetic immunization has to be improved to be a useful alternative vaccine against measles.

To longitudinally determine T cell activation and turnover in early simian immunodeficiency virus (SIV) infection of macaques, immunological and virological parameters were monitored in 10 SIV-infected animals starting before infection until 40 weeks postinfection (wpi). Lymphocyte subsets in blood and lymph nodes (LNs) were characterized by three-color flow cytometry for expression of markers of activation, proliferation, and differentiation. As early as 1 wpi, CD69 expression was upregulated both on CD4+ and CD8+ T cells, indicative of an early activation of these cells. Whereas this activation led to increased proliferation, determined by expression of Ki-67, and absolute numbers of CD8+ T cells, CD4+ T cells showed a decreased expression of Ki-67 and reduced counts in blood at 2 wpi. Later, the percentage of Ki-67-expressing CD4+ T cells in blood and LNs increased again above preinfection levels in most animals but remained low in two monkeys progressing to AIDS. These findings suggest that T cells are activated after SIV infection, leading to increased T cell proliferation already in the early asymptomatic phase. In addition, we found a correlation between the capacity to regenerate CD4+ T cells by peripheral proliferation and the disease course. Moreover, our data indicate that the increased peripheral T cell proliferation during immunodeficiency virus infection is probably not caused by the effort of the immune system to maintain T cell homeostasis but may be a reflection of the ongoing immune response against the virus.

On the basis of attenuated intracellular bacteria, we have developed two delivery systems for either heterologous proteins or DNA vaccine vectors. The first system utilizes attenuated strains of Gram-negative bacteria which are engineered to secrete heterologous antigens via the alpha-hemolysin secretion system of Escherichia coli. The second system is based on attenuated suicide strains of Listeria monocytogenes, which are used for the direct delivery of eukaryotic antigen expression vectors into professional antigen presenting cells (APC) like macrophages in vitro as well as in vivo.

A pathogenetic hallmark of retroviral neurodegeneration is the affinity of neurovirulent retroviruses for microglia cells, while degenerating neurons are excluded from retroviral infections. Microglia isolated ex vivo from rats peripherally infected with a neurovirulent retrovirus released abundant mature type C virions; however, infectivity associated with microglia was very low. In microglia, viral transcription was unaffected but envelope proteins were insufficiently cleaved into mature viral proteins and were not detected on the microglia cell surface. These microglia-specific defects in envelope protein translocation and processing not only may have prevented formation of infectious virus particles but also may have caused further cellular defects in microglia with the consequence of indirect neuronal damage. It is conceivable that similar events play a role in neuro-AIDS.

Measles virus (MV)-induced immune suppression during acute measles often leads to secondary viral, bacterial and parasitic infections which severely complicate the course of disease. Previously, we have shown that cotton rats are a good animal model to study MV-induced immune suppression, where proliferation inhibition after ex vivo stimulation of cotton rat spleen cells is induced by the viral glycoproteins (fusion and haemagglutinin proteins). We have now tested a variety of putative mechanisms of MV-induced immune suppression in this animal model. Proliferation inhibition is not due to fusion mediated by the MV glycoproteins and subsequent lysis of cells. Other putative mechanisms like classical anergy (unresponsiveness towards IL-2) or apoptosis do not seem to play a role in MV-induced immune suppression. In contrast, it was shown that spleen cells from infected animals preferentially accumulate in the G0/G1 phase and progress more slowly through the cell cycle after mitogen stimulation in comparison to cells from non-infected animals. These data indicate a retardation of the cell cycle which is correlated with proliferation inhibition and might have severe consequences in mounting an effective immune response.

Cerebrospinal fluid (CSF) samples were collected from 24 uninfected and 24 SIV251 MPBMC-infected rhesus monkeys during early infection and from 6 animals in a longitudinal design up to 7 months postinfection to investigate excitatory and inhibitory amino acid neurotransmitter levels. During the early infection period CSF amino acid concentrations of infected animals were not significantly different from those of uninfected animals. However, long-term studies demonstrated that gamma-aminobutyric acid (GABA) concentrations were decreased while glutamate concentrations were increased late in infection compared with the preinfection values of the same animals. Moreover, we showed that the source of increased glutamate in animals with AIDS is, at least partially, microglial cells. Our data support the hypothesis that excitotoxicity is involved in immunodeficiency virus-induced neurological disease and propose microglia as a contributor to excitotoxic damage.

Acute measles (for review see: Griffin and Bellini, 1996) can be accompanied by early or late central nervous system (CNS) complications. These include the acute postinfectious measles encephalitis...

Live attenuated measles virus (MV) vaccines are ineffective in young infants because of neutralization by maternal antibody. An immunization strategy that may permit priming for T cell memory for MV in infants at or shortly after birth uses recombinant bacille Calmette-Guérin expressing the full-length MV nucleocapsid (N) protein (rBCG::N). C3H/He mice immunized with rBCG::N developed T cell responses and ELISA antibodies to the N protein and low levels of neutralizing antibody after intracranial infection with MV strain CAM/R40. There was considerable reduction in the virus titer recovered from brain homogenates, a decrease in the incidence and severity of histologic encephalitis, and a decrease in mortality in rBCG::N-printed C3H/He mice compared with control mice. Given the limitations of existing live attenuated MV vaccines, these results encourage the further testing of rBCG::N vaccines in primate models.

The questions how a viral infection induces cellular autoimmune reactions (CMAI) and which components of both virus and auto-antigen play part in this process were addressed in our animal model of measles virus (MV)-induced CMAI against myelin basic protein (MBP) during subacute measles encephalitis (SAME). In an attempt to define whether cellular or humoral immune responses are involved in the occurrence of the autoimmune based disease process, Lewis rats were treated with different combinations of antibodies and T cells reactive with either MV and its structural proteins or MBP and MBP-peptides. The only treatment combination after which experimental allergic encephalomyelitis (EAE)-like disease and pathology developed was when non-encephalitogenic T cells reactive against residues 69–81 of MBP were adoptively transferred into MV-infected Lewis rats. The results of the study show that T cells which are non-encephalitogenic in the normal central nervous tissue are capable of inducing an allergic encephalomyelitis in animals with a viral infection involving the brain.

The European Academies Science Advisory Council has published a series of reports on infectious disease policy issues, analyzing priorities for building the science base as part of public health strategy. Among current challenges facing the European Union are the needs to tackle antibiotic resistance, promote vaccine innovation, prepare for the emergence of novel zoonoses, and integrate research approaches to human and animal health. The scientific community must help public policy-makers to address the organization, balance, and sustainability of research funding and infrastructure; encourage the creation of a more supportive regulatory environment for translational medicine; and evaluate new models for public-private partnership to facilitate innovation.

An infectious molecular clone (pHSRV) of the human Spumaretrovirus (HSRV) was constructed using viral DNA and cDNA clones. The infectivity of pHSRV was proven by transfection of cell cultures and subsequent infection of susceptible cultures with cell free transfection derlved virus. pHSRV derived virus produced foamy virus typical cytopathic effects in susceptible cultures. lnfected cells could be stained specifically with foamy virus antisera by means of indirect immunofluorescence. Radiolmmuno­ precipltatlon revealed the presence of characteristic HSRV structural proteins in pHSRV infected cultures. By cotransfection of pHSRV and an indicator plasmid it was found that pHSRV is able to transactivate the viral L TR. Viral transcripts were found to be approximately 200 bases Ionger in pHSRV infected cultures compared to wildtype infected cultures. This difference is most likely due to an Insertion of DNA of non-viral origin ln the U3 region of the 3'L TR of the infectious clone.

Glutamate-mediated neurodysfunction in human immunodeficiency virus (HIV) infection has been primarily suggested by in vitro studies. The regulation of glutamatergic neurotransmission in inflammation is a complex interaction between activation of immune mediators and adaptive changes in the functional elements of the glutamatergic synapse. We have used simian immunodeficiency virus (SIV)-infected macaques to answer the questions (i) whether perturbation of glutamate neurotransmission is evident during progression of immunodeficiency disease and (ii) what are the mechanisms underlying this impairment. Disease progression in SIV-infected macaques both in the periphery and in the brain was documented by clinical and general pathological examination, plasma and brain viral RNA load, T-cell analysis and brain histopathology. We report for the first time, disruption of excitatory amino acid transporters (EAATs), the cardinal glutamate clearing system, during SIV infection and a dramatic loss of EAATs associated with development of rapid acquired immunodeficiency syndrome (AIDS). EAATs impairment was correlated with activation status of microglia. Our data support the glutamate hypothesis for the development of HIV dementia and suggest that the pathogenetic mechanism for the neurodysfunction is the impairment of glutamate clearing which occurs in the stage of AIDS and which is associated with activated microglia.

Measles virus infection reduces or abolishes delayed type hypersensitivity reactions (DTH) in humans. We have previously shown that the primary 2,4-dinitrofluorobenzene (DNFB) response is temporarily suppressed by measles virus in cotton rats. Here, we demonstrate that also the secondary DNFB response (cutaneous hypersensitivity [CHS]) is suppressed in cotton rats by measles virus infection. As in mice, DNFB specific CD8 T cells are the predominant T cell response in cotton rats. After MV infection, CD8 T cells are reduced in their proliferative capacity whereas the CD4/CD8 ratio, the number and activation status of CD8 T cells is not affected. As a result of impaired proliferation of DNFB specific T cells the DTH response (measured as ear swelling) is reduced in measles virus infected cotton rats. At the same time as DNFB specific T cell responses are suppressed, spontaneous proliferation of lymphocytes as evidence for immune activation is found.

Measles virus (MV) remains among the most potent global pathogens, killing more than 1 million children annually. The virus induces a profound suppression of immune functions that favours the establishment of, and aggravates the course of, secondary infections. By contrast, MV-specific immune responses are efficiently generated, and these clear the virus from the organism and confer a long-lasting immunity. As sensitisers of pathogen encounter and instructors of the adaptive immune response, professional antigen-presenting cells (APCs) such as dendritic cells play a decisive role in the induction and quality of the MV-specific immune response. However, key features of immune suppression associated with MV are compatible with interference with APC maturation and function, and subsequent qualitative and quantitative alterations of T-cell activation.

Immunization via the oral route with an attenuated Yersinia enterocolitica strain expressing a fragment of the measles virus nucleocapsid protein (aa 79–161) via its type III protein secretion system induced a T helper type 1 response in immunized C3H mice, which conferred protection against measles virus-induced encephalitis in a time- and dose-dependent manner.

Infection of humans with wild-type measles virus leads to strong immune suppression and secondary infections, whereas immunization with an attenuated vaccine strain does not. Using the cotton rat model ( Sigmodon hispidus ), we investigated whether vaccine and wild-type viruses differ in viral spread and whether this is correlated with inhibition of of proliferation of spleen cells ex vivo after mitogen stimulation. After intranasal infection of cotton rats with wild-type and vaccine strains, it was found that wild-type virus replicates better in lung tissue, spreads to the mediastinal lymph nodes, and induces a more pronounced and longer-lasting inhibition of proliferation of spleen cells ex vivo after mitogen stimulation than does vaccine virus. To induce the same degree of proliferation inhibition, 1,000-fold less wild-type virus was required than vaccine virus. With this system, the virulence of various measles virus isolates and recombinant viruses was tested. Four (in humans and/or monkeys) highly pathogenic virus strains were immunosuppressive, whereas viruses of vaccine virus genotype A were not. Using virus pairs which, due to passage on fibroblasts versus lymphoid cells or due to a point mutation in the hemagglutinin (N481 → Y), differed in their usage of the two receptor molecules CD46 and CD150 on human cells, it was found that viruses using exclusively CD150 in vitro spread to mediastinal lymph nodes and induced strong immune suppression. These data demonstrate that important parameters of virulence seen in humans, such as viral spread and immune suppression, are reflected in the cotton rat model.

HIV infection leads to reduced numbers and increased turnover of CD4+ T cells in blood. However, blood represents only 2% of the total lymphocyte pool, and information about other organs is lacking, leading to controversy about the effects of HIV infection on T-cell homeostasis. Therefore, we have determined phenotype and turnover of lymphocyte subsets in various tissues of macaques. Infection with simian immunodeficiency virus (SIV) resulted in increased proliferation rates of T cells in all organs. Despite reduced CD4 counts in blood, absolute numbers of CD4+ T cells were increased in spleen and lymph nodes and remained stable in nonlymphoid organs such as liver, lung, bone marrow, and brain during the asymptomatic phase, indicative for an altered tissue distribution. In animals killed with first signs of AIDS, total body CD4 counts and proliferation rates had returned to control levels, whereas thymocytes were almost completely absent. Our data show that a drastically increased turnover in the early stages of HIV infection, driven by a generalized immune activation rather than a homeostatic response to CD4+ T-cell destruction, is followed by exhaustion of the regenerative capacity of the immune system.

In the course of acute measles, an efficient virus-specific immune response is generated which mediates viral clearance from the host and confers protection against reinfection. Paradoxically, a general immunosuppression is also induced favoring secondary infections, which are the major reason for the annual high morbidity and mortality rates associated with measles. The magnitude and duration of immune activation and immune suppression differ between natural or experimental infection and vaccination (20, 60). Studies addressing measles virus (MV)-induced immune suppression mainly have focused on alterations of T-cell functions and viability as a consequence of direct MV infection or contact-mediated signaling (53). In vitro observations suggest that MV infection also enhances apoptosis of monocytes and dendritic cells (DC) and affects their antigen-presenting capacity and cytokine release (31, 53). MV interaction with DC and monocytes is, however, also associated with their maturation or activation, respectively, and thus is important for induction of virus-specific immune responses (32, 39, 45, 54, 56). Strains expressing an MV wild-type-derived hemagglutinin (H) protein reveal a particular tropism for DC and are more efficient in inducing both DC maturation and immunosuppression (32, 48, 54). The mechanisms by which MV leads to these functional alterations are largely unknown. Downregulation of interleukin-12 (IL-12) production in monocytes was linked to MV- or antibody-mediated cross-linking of CD46, the receptor for certain MV strains (29). Lymphotropic MV wild-type strains and clinical isolates, with few known exceptions (43), fail to interact with CD46 but require CD150 for cell entry (15, 26, 49, 59). This molecule is absent from unstimulated monocytes and immature DC (33, 45, 48), and it is thus unknown how infection of these cells by CD150-dependent MV strains occurs. Mammalian Toll-like receptors (TLRs) were implicated in the innate immune recognition of a variety of bacterial pathogens and bacterial products (2). Ten TLR family members were discovered, and several of these appear to play important roles in the activation of cells by various bacterial products. TLR2 is responsible for recognition of gram-positive bacteria (57, 65), bacterial lipoproteins (12, 42), and lipoteichoic acid (38, 55). TLR4 appears to be the main receptor for lipopolysaccharide (LPS) lipid A from gram-negative bacteria (41), TLR6 might be a coreceptor for TLR2 in recognizing certain bacterial structures (50, 58), and TLR9 and TLR3 mediate responses to CpG bacterial DNA and double-stranded RNA (dsRNA), respectively (3, 24). Hence, these receptors are able to discriminate between different bacteria and bacterial structures and thereby direct a proper immune response to a specific pathogen. Intracellular domains of the TLRs share motifs with the protein families of the IL-1 receptors, and a common intracellular pathway leading to activation of NF-κB and mitogen-activated protein kinases involves MyD88, IRAK, and TRAF6 (2). However, other signaling pathways upstream of NF-κB have been described which also include utilization of the phosphatidylinositol-3/Akt-kinase pathway by TLR2 (4). It has recently been demonstrated that mammalian TLR signaling can also be regulated by viral gene products. Vaccinia virus encodes gene products that interfere with proximal TLR signaling in the cytoplasm (11), and the fusion protein of respiratory syncytial virus (RSV) was found to activate cells via TLR4 and CD14 (35). Using CHO cells stably overexpressing TLR2 or TLR4, we found that MV wild-type strains specifically activated cells via TLR2, and this was dependent on the expression of the H protein of the MV wild-type strain, WTF. The failure of attenuated MV strains to induce TLR2 activation correlated with a single amino acid exchange at position 481 which is, in turn, essential for the usage of CD46 as receptor by these strains. Importantly, MV expressing the wild-type H protein induced activation of TLR-responsive genes such as IL-1α/β, IL-6, and IL-12 p40 in monocytes and also the expression of CD150, the receptor for all MV strains. Activation of TLR signaling by wild-type MV H protein may thus essentially contribute to the immune activation during measles, and loss of the capability to activate TLR2 may be considered as an attenuation marker.

In order to investigate which measles virus (MV)-strains use CD46 and/or CD150 (signalling lymphocytic activation molecule, SLAM) as receptors, CHO cells expressing either recombinant CD46 or SLAM were infected with a panel of 28 MV-strains including vaccine strains, wild-type strains with various passage histories and recombinant viruses. We found that SLAM served as a common receptor conferring virus uptake and syncytium formation for all MV-strains tested. Predominantly vaccine and laboratory adapted strains, but also a minor fraction of the wild-type strains tested, could utilize both CD46 and SLAM. Using recombinant viruses, we demonstrate that the single amino acid exchange in the haemagglutinin (H) protein at position 481 Asn/Tyr (H481NY) determines whether the virus can utilize CD46. This amino acid alteration has no affect on the usage of SLAM as receptor, and as such demonstrates that the binding sites for SLAM and CD46 are distinct.

Acute measles, a well known disease usually contracted during early childhood, is still the major cause of vaccine-preventable infant deaths worldwide. There are about 40 million cases of acute measles per year, with more than one million cases of infant death as a consequence of measles. These are mainly due to opportunistic infections which develop on the basis of a generalized suppression of the cellular immunity in the course and after the acute disease. Lymphopenia, a general proliferative unresponsiveness of T cells ex vivo and cytokine imbalance, are considered as major hallmarks of measles virus (MV) induced immunosuppression. These findings are compatible with modulation of T cell responses by viral interference with professional antigen-presenting cells such as dendritic cells or direct effects on T cells by suppression of survival or proliferation signals. In vitro, MV interaction causes a variety of effects on dendritic cells, including maturation and loss of their allostimulatory functions. Whether there is an additional impact on the quality of T cell responses is unknown as yet. It is clear, however, that surface interaction of lymphocytes with the MV glycoprotein complex is necessary and sufficient to induce a state of proliferative unresponsiveness in T cells. This surface contact mediated signal essentially interferes with the propagation of the interleukin 2 receptor signal by blocking the activation of the protein kinase B, also called Akt kinase, both in vitro and after experimental infection.

Recombinant measles viruses (MV) in which the authentic glycoprotein genes encoding the fusion and the haemagglutinin (H) proteins of the Edmonston (ED) vaccine strains were swapped singly or doubly for the corresponding genes of a lymphotropic MV wild-type virus (strain WTF) were used previously to investigate MV tropism in cell lines in tissue culture. When these recombinants and their parental strains, the molecular ED-based clone (ED-tag) and WTF, were used to infect cotton rats, only viruses expressing the MV WTF H protein replicated in secondary lymphatic tissues and caused significant immunosuppression. In vitro, viruses containing the ED H protein revealed a tropism for human peripheral blood lymphocytes as documented by enhanced binding and virus production, whereas those containing the WTF H protein replicated well in monocyte-derived dendritic cells (Mo-DC). This did not correlate with more efficient binding of these viruses to DC, but with an enhancement of uptake, virus spread, accumulation of viral antigens and virus production. Thus, replacement of the ED H protein with WTF H protein was sufficient to confer the DC tropism of WTF to ED-tag in vitro. This study suggests that the MV H protein plays an important role in determining cell tropism to immune cells and this may play an important role in the induction of immunosuppression in vivo.

In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.

Measles virus (MV) infection promotes maturation of dendritic cells (DC), but also interferes with DC functions, and MV renders the DC inhibitory for T cell proliferation. We now describe that MV infection triggers the release of type I IFN from monocyte-derived DC (Mo-DC) which contributes to DC maturation. There is no evidence that soluble mediators are released interfering with the stimulatory activity of uninfected DC. Since inhibition of allogeneic T cell proliferation was unaffected by a fusion inhibitory peptide (Z-fFG), MV infection of T cells did not contribute to inhibition. Allogeneic T cell proliferation depended on the percentage of DC expressing MV F/H glycoproteins within the DC population and their surface expression levels, was induced upon addition of UV-inactivated MV to a mixed lymphocyte reaction stimulated by lipopolysaccharide-matured DC, and was not induced by DC infected with a recombinant MV encoding the ectodomain of vesicular stomatitis virus G protein (MG/FV) instead of the MV glycoproteins. Similarly, DC infected with MV, but not with MG/FV inhibited mitogen-induced proliferation of T cells. Thus, a dominant inhibitory signal is delivered to T cells by the MV glycoproteins on the surface of DC overcoming positive signals by co-stimulatory molecules promoted by maturation factors released from infected DC.

Depending on their major histocompatibility complex (MHC) haplotype, inbred mouse strains are either resistant (H2-d, BALB/c), susceptible (H2-k, C3H) or partially resistant (H2-d×k, BaCF1) to intracerebral infection with the neurotropic rodent-adapted measles virus (MV) strain CAM/RBH. Here, mortality is demonstrated to be correlated directly with virus spread and virus replication in the CNS and to be inversely correlated with the activation of MV-specific T cells. Previously, it has been shown that primary CD4+ T cells alone are protective in the resistant background. In the susceptible background, CD4+ T cells acquire protective capacity after immunization with a newly defined CD4+ T cell epitope peptide. In the partially resistant mice, CD4+ T cells provide help for CD8+ T cells and protect in cooperation with them. It seems that the lytic capacity of CD8+ T cells is crucial in providing protection, as MV-specific Ld-restricted CD8+ T cells, which are highly lytic in vitro after transfer, protect naive animals against MV-induced encephalitis (MVE). In contrast, Kk-restricted CD8+ T cells with low lytic capacity do not protect. In the MVE model, CD4+ T cells are able to protect either alone (resistant mice), through cooperation with CD8+ T cells (intermediate susceptible) or after immunization as secondary T cells (susceptible mice). CD8+ T cells are able to protect alone after immunization if they are cytolytic. Thus, susceptibility and resistance depend upon the functional composition of CD4+ and CD8+ T cells governed by the MHC haplotype.

Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.

During and after measles virus (MV) infection humans are highly susceptible to opportunistic infections because of a marked immunosuppressive effect of the virus. The mechanisms by which the virus induces this phenomenon is not well understood. In particular, detailed information is missing on the targets of suppression in relation to antigen-specific T and B cell responses. Because such studies require animal experiments, we used the cotton rat model, in which the MV causes a respiratory tract infection. Primary as well as secondary T cell responses were impaired in vivo and ex vivo by MV infection. The proliferation of T cells was greatly reduced, but their effector functions, such as cytolysis or cytokine secretion, were not. In contrast, primary and secondary B cell responses in vivo as measured by the frequency of antigen-specific plasma cells in an enzyme-linked immunospot (ELISPOT) assay were not altered by MV infection. Only the secretion of immunoglobulins was reduced slightly in animals primarily infected with MV after 2 weeks. These data demonstrate that MV-induced immunosuppression acts primarily on the T cell responses in vivo .

The pathogenesis of human immunodeficiency virus-associated motor and cognitive disorders is poorly understood. In this context both a protective and a harmful role of the immune system has been discussed. This question was addressed in the present study by correlating the occurrence of neurologic disease in simian immunodeficiency virus (SIV)-infected macaques with disease progression and the humoral and cellular intrathecal antiviral immune response. Overt neurologic signs consisting of ataxia and apathy were observed at a much higher frequency in rapid progressor animals (6 of 12) than in slow progressors (1 of 7). Whereas slow progressors mounted a strong antiviral antibody (Ab) response as evidenced by enzyme-linked immunosorbent and immunospot assays, neither virus-specific Ab titers nor Ab-secreting cells could be found in the cerebrospinal fluid (CSF) or brain parenchyma of rapid progressors. Similarly, increased infiltration of CD8 + T cells and cytotoxic T lymphocytes specific for viral antigens were detected only in the CSF of slow progressors. The finding that neurologic signs develop frequently in SIV-infected macaques in the absence of an antiviral immune response demonstrates that the immune system does not contribute to the development of motor disorders in these animals. Moreover, the lower incidence of neurologic symptoms in slow progressors with a strong intrathecal immune response suggests a protective role of the virus-specific immunity in immunodeficiency virus-induced central nervous system disease.

Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314–322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V−), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats ( Sigmodon hispidus ) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.

Subacute sclerosing panencephalitis (SSPE), a very rare but lethal disease caused by measles viruses (MV) persisting in the human central nervous system (CNS) is characterized by lack of viral budding, reduced expression of the viral envelope proteins and spread of MV genomes through the CNS despite massive immune responses. The five major MV genes from several SSPE cases were cloned and sequenced, the two transmembrane envelope glycoproteins hemagglutinin (H) and fusion protein (F) were expressed and their maturation, cellular localization and functionality analyzed. We conclude that 1) mutations in the MV genes arise not only individually, by errors of the MV polymerase, but also in clusters as hypermutations, presumably due to RNA unwinding/modifying activity altering accidentally formed double-stranded RNA regions, 2) MVs spread in SSPE brains after clonal selection, 3) the MV matrix (M) gene is most heavily mutated and dispensable, 4) the two genes encoding envelope transmembrane proteins give rise to functional but altered proteins (typically F is heavily altered in its cytoplasmic domain), 5) H protein is transported poorly to the cell surface, 6) F and H proteins maintain tightly interdepending fusion functions, presumably to allow local cell fusion and MV ribonucleoprotein (RNP) spread through the CNS.

The effect of measles virus (MV) infection on mRNA expression and protein synthesis of cytokines in human malignant glioma cell lines (D-54 and U-251) was investigated. Primary MV infections led in both cell lines to the induction of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interferon-beta (IFN-beta), and tumor necrosis factor-alpha (TNF-alpha). In contrast, persistently infected astrocytoma lines continually produced IL-6 (two out of 12 lines high levels) and IFN-beta, whereas only 1 out of 12 lines synthesized TNF-alpha and none IL-1 beta. The pathways for induction of IL-1 beta and TNF-alpha expression were not suppressed by the persistent MV infection, since IL-1 beta and TNF-alpha could be induced by external stimuli like diacylglycerol analog plus calcium ionophore. Interestingly, persistently infected astrocytoma cells synthesized considerably higher levels of IL-1 beta and TNF-alpha than uninfected cells after additional external induction. These results suggest that in the central nervous system (CNS) of SSPE patients a percentage of persistently infected astrocytes may continually synthesize IL-6 and IFN-beta, and in the presence of additional external stimuli, as possibly provided by activated lymphocytes, might overexpress the inflammatory cytokines IL-1 beta and TNF-alpha. This may be of pathogenetic significance in CNS diseases associated with persistent MV infections.

Human immunodeficiency virus (HIV) infection is associated with a progressive dementia, in addition to motor and behavioural deficits. Cognitive deterioration and motor impairments have been observed also in simian immunodeficiency virus (SIV)-infected monkeys, an animal model for HIV infection. We found recently that choline acetyltransferase activity is markedly reduced in brains of SIV-infected monkeys. We report now that selegiline, completely restores the reduced choline acetyltransferase activity which encourages for a meaningful anti-dementia therapeutic strategy.

Temperature-sensitive mutants of the murine coronavirus JHM induced a subacute demyelinating encephalomyelitis (SDE) in young rats. Neurological symptoms were associated with marked lesions of primary demyelination in the white matter of the central nervous system (CNS), and developing after an incubation time of several weeks to months. Many rats survived this infection and recovered completely from this CNS disease. Among 43 survivors of SDE, 9 rats developed a relapse 27-153 days after onset of the first attack. Neuropathological examination of these animals revealed areas of fresh demyelination together with old remyelinated lesions. Viral antigens were detectable in the neighbourhood of fresh lesions and in some cases infectious virus was re-isolated from rats revealing low antibody titers to JHM virus. These results demonstrate that mutants of JHM virus can induce a relapsing demyelinating disease process, associated with a persistent infection, which possesses some similarities to chronic experimental allergic encephalomyelitis.

The present investigation describes the biological and biochemical properties of a persistent SSPE virus infection. Persistently infected cells were derived by cocultivation of infected brain cells and uninfected Vero cells, and cultures were maintained by normal subculturing methods. No infectious virus was ever released from these cultures, and all attempts to induce infectious virus release were unsuccessful. Biological assays showed that infected cells contained nucleocapsid and salt-dependent hemagglutinin antigens, whereas the normal hemagglutinin appeared not to be present. Electron microscopic examination demonstrated the presence of both intranuclear and cytoplasmic nucleocapsids together with the release of virus particles (defective?) from the cell membrane. Biochemical analysis demonstrated that approximately 90% of the intracellular genomic RNA was defective or subgenomic although a small quantity of infectious genomes was present. It is proposed that the large quantities of defective genomes in the infected cells are the major factor in the maintenance of this persistent infection.

H-2 antigen compatibility of effector spleen cells and target cells was necessary for cell-mediated cytotoxicity (CMC) after both 1 and 2 intraperitoneal inoculations of 6/94 virus into mice. T cells were responsible for both primary and secondary CMC reactions. Neither reaction was influenced by the presence of virus-specific antibody added to the test system. Secondary CMC peaked earlier than the primary response and declined more rapidly. In vitro stimulation of primed spleen cells could also be used to detect the secondary CMC response.

Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000- and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecular-weight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000- and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000- and 23,000-molecular-weight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA.

Summary The synthesis of virus-specific polypeptides and messenger RNA in cell cultures persistently infected with an isolate of measles virus from a patient with subacute sclerosing panencephalitis (SSPE) has been compared to that found in a lytic infection with the homologous virus. The persistent infection described here was chosen as its biological characteristics reflect those of virus-infected brain cells from SSPE patients. The synthesis of H, N and possibly F protein was seen in both lytic and persistent infections, but the synthesis of M protein was only detected in the lytic infection. However, messenger RNA isolated from either the lytic or persistent infection directed the synthesis in a cell-free translation system of all structural polypeptides, including M, and also three non-structural polypeptides, with mol. wt. of 34000, 30000 and 18000. Messenger RNAs coding for the virus-specific polypeptides were also shown to be polyadenylated. In addition, those polypeptides made in vitro which were antigenically related to the haemagglutinin, demonstrated structural changes after passage through a persistent infection.

6/94 virus, isolated originally from a multiple sclerosis (MS) patient, was compared antigenically with related parainfluenza type 1 strains. These included two Sendai strains of mouse and two Sendai strains of reported human origin as well as the HA2 strain. By standard hemagglutination inhibition (HI) or hemadsorption neutralization (HAD-N) tests or by the complement-fixation (CF) cross-block titration test for detecting surface antigens, 6/94 virus and the Sendai virus strains were indistinguishable from each other but distinct from the HA2 strain. By the kinetic HI test, however, 6/94 virus could be readily differentiated from the Sendai viruses isolated from mice and more closely resembled those recovered from man.

Our previous experiments showed that a variant virus with a larger envelope glycoprotein encoded by a larger mRNA3 (cl-2) multiplied predominantly in the brain of rats after wild type (wt) JHMV infection ( F. Taguchi, S. Siddell, H. Wege, and V. ter Meulen, 1985 , J. Virol. , 54 , 429–435). We could isolate similar but not identical variant virus after infection of cultured neural cells from rat brain with wt JHMV (designated CNS virus), which also had a larger mRNA3 and produced larger envelope E2 glycoprotein in infected cells. CNS virus multiplied to a higher degree in cultured astrocytes from rat than wt JHMV and cl-2. During infection with these variant viruses in neural cells, virus populations generated did not change, in contrast to consistent selection of viruses with larger mRNA3 after wt JHMV infections. CNS virus produced abundant mRNA2a as well as 65K glycoprotein while the productions of both were trace in cl-2 infected cells. The present experiments, together with our previous observation, suggest that the larger E2 glycoprotein may be of importance for the replication in rat brain cells.

We have recently observed strain differences in interferon-gamma (IFN-gamma) induction of Ia on astrocytes in rats and mice that are susceptible or resistant to experimental allergic encephalomyelitis (EAE) (Massa et al., 1987). We now found that keratinocytes cultured from rat skin show the same strain differences as astrocytes. Keratinocytes of EAE-susceptible Lewis rats are induced to higher levels of Ia expression compared to relatively resistant Brown-Norway (BN) rats, independent of the dose of IFN-gamma applied. This difference in regulation is specific for class II histocompatibility molecules because no such differences are seen with class I antigen induction by IFN-gamma. That keratinocytes reflect similar strain differences as astrocytes may be explained by a common ectodermal origin of these epithelial-like cells. This observation allows the determination of the genetic predisposition of an autoimmune response in the central nervous system by skin biopsy. This approach may be of importance in studies of immunopathological phenomena of brain tissue in man such as multiple sclerosis.

SUMMARY Maintenance of measles (SSPE-Lec) virus persistently infected C6 rat glioma cells in medium containing polyclonal measles antiserum resulted in the loss of detectable expression of all measles virus proteins. Removal of these cells from antiserum, however, led to a re-expression of virus proteins and the production of infectious virus. Cloning of antibody-modulated non-expressing cells in the presence of antiserum showed that re-expression of virus proteins was not due to an incomplete curing process following the addition of antiserum, as a large number of non-expressing cell clones developed the capacity to express measles virus antigen at different periods after removal of antiserum. Irradiation of persistently infected cells to give a non-growing culture showed that modulation was not mediated by a selection and outgrowth of a small percentage of non-expressing cells originally present in the culture. Antibody directed against C6 membrane proteins did not lead to modulation and it was also shown that only monoclonal antibodies with neutralizing activity could affect intracellular antigen expression. Immunoglobulin Fab fragments with neutralizing activity also had modulating activity. Although all modulated cell clones were more susceptible to homologous virus infection than control C6 cells, it was not possible to rescue any defective measles virus which may have been maintained in the culture.

SummaryThe effect of 6-azauridine, an inhibitor of RNA synthesis, has been shown by 2 experimental methods to inhibit the replication of measles virus within mammalian cells in vitro. In a direct virus titration in which the drug was incorporated in the agar overlay, 6-AZ not only reduced the infectivity of measles virus but also delayed plaque development. Experiments designed to test the effectiveness of 6-azauridine when added to cultures at intervals after infection, showed that this uridine analog was capable of reducing the amount of infectious virus produced by each infected cell, and that the drug is more effective when added early in the infection cycle.