In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence.

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides.

Effective in Vivo Targeting of Influenza Virus through a Cell-Penetrating/Fusion Inhibitor Tandem Peptide Anchored to the Plasma Membrane.

The impact of influenza virus infection is felt each year on a global scale when approximately 5-10% of adults and 20-30% of children globally are infected. While vaccination is the primary strategy for influenza prevention, there are a number of likely scenarios for which vaccination is inadequate, making the development of effective antiviral agents of utmost importance. Anti-influenza treatments with innovative mechanisms of action are critical in the face of emerging viral resistance to the existing drugs. These new antiviral agents are urgently needed to address future epidemic (or pandemic) influenza and are critical for the immune-compromised cohort who cannot be vaccinated. We have previously shown that lipid tagged peptides derived from the C-terminal region of influenza hemagglutinin (HA) were effective influenza fusion inhibitors. In this study, we modified the influenza fusion inhibitors by adding a cell penetrating peptide sequence to promote intracellular targeting. These fusion-inhibiting peptides self-assemble into ∼15-30 nm nanoparticles (NPs), target relevant infectious tissues in vivo, and reduce viral infectivity upon interaction with the cell membrane. Overall, our data show that the CPP and the lipid moiety are both required for efficient biodistribution, fusion inhibition, and efficacy in vivo.

Prevention of measles virus infection by intranasal delivery of fusion inhibitor peptides.

UNLABELLED: Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV H and the fusion (F) envelope glycoprotein; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can potently inhibit MV infection at the entry stage. We show here that specific features of H's interaction with its receptors modulate the susceptibility of MV F to peptide fusion inhibitors. A higher concentration of inhibitory peptides is required to inhibit F-mediated fusion when H is engaged to its nectin-4 receptor than when H is engaged to its CD150 receptor. Peptide inhibition of F may be subverted by continued engagement of receptor by H, a finding that highlights the ongoing role of H-receptor interaction after F has been activated and that helps guide the design of more potent inhibitory peptides. Intranasal administration of these peptides results in peptide accumulation in the airway epithelium with minimal systemic levels of peptide and efficiently prevents MV infection in vivo in animal models. The results suggest an antiviral strategy for prophylaxis in vulnerable and/or immunocompromised hosts. IMPORTANCE: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that parenterally delivered fusion-inhibitory peptides protect mice from lethal CNS MV disease. Here we show, using established small-animal models of MV infection, that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. Since the fusion inhibitors are stable at room temperature, this intranasal strategy is feasible even outside health care settings, could be used to protect individuals and communities in case of MV outbreaks, and could complement global efforts to control measles.

Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

Current animal models: cotton rat animal model.

The cotton rat (Sigmodon hispidus) model has proven to be a suitable small animal model for measles virus pathogenesis to fill the niche between tissue culture and studies in macaques. Similar to mice, inbred cotton rats are available in a microbiologically defined quality with an ever-increasing arsenal of reagents and methods available for the study of infectious diseases. Cotton rats replicate measles virus in the respiratory tract and (depending on virus strain) in lymphoid organs. They can be infected with vaccine, wild-type, and recombinant measles viruses and have been used to study viruses with genetic modifications. Other areas of study include efficacy testing of antivirals and vaccines. The cotton rat also has been an informative animal model to investigate measles virus-induced immune suppression and suppression of vaccination by maternal antibodies. In addition, the cotton rat promises to be a useful model for the study of polymicrobial disease (interaction between measles virus and secondary pathogens).

Mouse models of human T lymphotropic virus type-1-associated adult T-cell leukemia/lymphoma.

Human T-lymphotropic virus type-1 (HTLV-1), the first human retrovirus discovered, is the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of lymphocyte-mediated inflammatory conditions including HTLV-1-associated myelopathy/tropical spastic paraparesis. Development of animal models to study the pathogenesis of HTLV-1-associated diseases has been problematic. Mechanisms of early infection and cell-to-cell transmission can be studied in rabbits and nonhuman primates, but lesion development and reagents are limited in these species. The mouse provides a cost-effective, highly reproducible model in which to study factors related to lymphoma development and the preclinical efficacy of potential therapies against ATL. The ability to manipulate transgenic mice has provided important insight into viral genes responsible for lymphocyte transformation. Expansion of various strains of immunodeficient mice has accelerated the testing of drugs and targeted therapy against ATL. This review compares various mouse models to illustrate recent advances in the understanding of HTLV-1-associated ATL development and how improvements in these models are critical to the future development of targeted therapies against this aggressive T-cell lymphoma.

Expression of tumor invasion factors determines systemic engraftment and induction of humoral hypercalcemia in a mouse model of adult T-cell leukemia.

Infection with human T-cell leukemia virus type 1 (HTLV-1) leads sometimes to the development of adult T-cell lymphoma/leukemia (ATL), which is invariably fatal and often associated with humoral hypercalcemia of malignancy. The transformation of infected CD4 T cells and the pathogenesis of leukemia have been studied with great limitation in tissue culture and patients. To better understand the pathogenesis and perform preclinical drug studies, animal models of ATL are urgently needed. In mice, inoculation of HTLV-1 cell lines mostly leads to development of localized lymphomas. To develop an ATL animal model with leukemic spread of ATL cells, mouse strains with different well-defined immune deficiencies were inoculated intraperitoneally with different HTLV-1-infected cell lines (ACH.2, C8166, MT-2, MET-1). Inoculation of MET-1 cells into NOD/SCID mice provided the best model system for slowly developing T-cell leukemia with multiple organ involvement. In leukemic mice, an increase in serum calcium levels correlated with expression of receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand on leukemic cells and secretion of parathyroid hormone-related protein and interleukin-6. In contrast to the other cell lines that did not spread systemically, MET-1 expressed both the adhesion molecules CD11a (LFA-1alpha) and CD49d (VLA-4alpha) and produced or induced expression of matrix metalloproteinases 1, 2, 3, and 9, thus underlining the importance of these molecules in the spread of adult T-cell leukemia cells. The MET-1/NOD/SCID model will be useful for developing interventions against invasion and spread of leukemic cells and subsequent humoral hypercalcemia of malignancy.

Measles virus induced immunosuppression: targets and effector mechanisms.

A profound, transient suppression of immune functions during and after the acute infection is the major cause of more than one million cases of infant deaths associated with measles worldwide. Concommittant with the generation of an efficient measles virus (MV) specific immunity, immune responses towards other pathogens are strongly impaired and provide the basis for the establishment and severe course of opportunistic infections. The molecular basis for MV-induced immunosuppression has not been resolved as yet. Similar to other immunosuppressive viruses, MV is lymphotropic and viral nucleic acid and proteins are detectable in peripheral blood mononuclear cells (PBMC). It is considered central to MV-induced immunosuppression that PBMC isolated from patients largely fail to proliferate in response to antigen specific and polyclonal stimulation. The low abundancy of MV-infected PBMC suggests that MV-induced immunosuppression is not directly caused by infection-mediated cell loss or fusion, but rather by indirect mechanisms such as deregulation of cytokines or surface contact-mediated signaling which may lead to apoptosis or impair the proliferative response of uninfected PBMC. Evidence for a role of any of these mechanisms was obtained in vitro, however, much has still to be learned about the tropism of MV and its interactions with particular host cells such as dendritic cells in vivo.

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis.

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.

Influenza vaccination in MS: absence of T-cell response against white matter proteins.

BACKGROUND: Natural infections bear the risk of triggering MS bouts, whereas epidemiologic studies have not delineated an increased risk for disease activity after influenza virus vaccination. OBJECTIVE: To examine influenza A virus-specific and myelin protein-reactive T-cell frequencies by interferon gamma (IFNgamma)-enzyme-linked immunospot and the response of these cells by IFNgamma-reverse transcription (RT) PCR after immunization and any incidental upper respiratory tract infection (URI) in 12 patients with MS (seven with a relapsing-remitting course; five with a secondary progressive course; Kurtzke Expanded Disability Status Scale [EDSS] score from 1.0 to 6.5, without immunosuppressive treatment) and 28 healthy volunteers. RESULTS: A cellular immune response against influenza A virus was mounted in both populations at 2 weeks after vaccination. Patients with MS showed a higher relative increase (p = 0.008) than controls with respect to the number of influenza-specific T cells. Mean antibody responses against influenza A virus were increased in both populations after 2 weeks (p < 0.01). Despite these virus-specific reactions, no increase in T-cell frequencies responsive to human myelin basic protein (MBP) or recombinant human myelin oligodendrocyte protein (MOG) was observed after immunization, arguing against a general immune stimulation by influenza vaccination. In contrast, MBP-specific T-cell responses became detectable in several individuals after febrile infection. CONCLUSION: These data support the clinical observations that influenza vaccination is effective and safe in patients with MS with respect to cellular immunoreactivity against two main CNS myelin proteins.

Cotton rats (Sigmodon hispidus): an animal model to study the pathogenesis of measles virus infection.

Measles is still the most lethal infectious disease of infants worldwide. In spite of research efforts, two major problems associated with measles virus (MV) infection have not been resolved. One is the marked immune suppression leading to subsequent (often lethal) opportunistic infections and the second is waning of maternal antibodies which do not protect against wild type virus infection any longer, but impair vaccination. Monkeys are an animal model in which MV infection most closely resembles the human disease. The use of monkeys is restricted by ethical and financial reasons and their availability. A cost-effective alternative is the cotton rat (Sigmodon hispidus). Cotton rats are the only rodents which replicate measles virus in lung tissue after intranasal infection. Our research has shown that cotton rats are a valid model to study MV induced immune suppression and to test vaccine candidates. It is also useful for comparing various wild type measles virus strains as well as recombinant measles viruses.

Characterization of human foamy virus proteins expressed by recombinant vaccinia viruses.

We report the generation of recombinant vaccinia viruses (VVs) expressing the gag, pol, bel-1, and bet open reading frames of human foamy virus (HFV), and the establishment of a transient, VV-T7 RNA polymerase-directed expression system for the HFV env gene. The correct expression of the HFV proteins was demonstrated by radioimmunoprecipitation using monospecific rabbit antisera, by analysis of the subcellular distribution (for VVgag, VVpol, VVbel-1, and VVbet), and by the ability to induce syncytium formation (for the env expression system). The HFV pol gene was successfully expressed using its own ATG start codon. Foamy viruses are regarded as retroviruses with intracytoplasmatic capsid assembly. However, when VVgag and VVpol were used to study the HFV Gag-Pol protein interaction and particle formation, no HFV capsid structures were observed in singly or doubly infected cells. In addition, no cleavage of the Pr74gag precursor molecule by the pol-encoded protease was detected in doubly infected cells. Our results indicate that foamy virus particle assembly is fundamentally different from that of other retroviruses.

Novel bacterial systems for the delivery of recombinant protein or DNA.

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.

Two functionally linked amino acids in the stem 2 region of measles virus haemagglutinin determine infectivity and virulence in the rodent central nervous system.

Rodent brain-adapted measles virus (MV) strains, such as CAM/RB and recombinant MVs based on the Edmonston strain containing the haemagglutinin (H) of CAM/RB, cause acute encephalitis after intracerebral infection of newborn rodents. We have demonstrated that rodent neurovirulence is modulated by two mutations at amino acid positions 195 and 200 in the H protein, one of these positions (200) being a potential glycosylation site. In order to analyse the effects of specific amino acids at these positions, we introduced a range of individual and combined mutations into the open reading frame of the H gene to generate a number of eukaryotic expression plasmids. The functionality of the mutant H proteins was assessed in transfected cells and by generating recombinant viruses. Interestingly, viruses caused acute encephalitis only if the amino acid Ser at position 200 was coupled with Gly at position 195, whereas viruses with single or combined mutations at these positions, including glycosylation at position 200, were attenuated. Neurovirulence was associated with virus spread and induction of neuronal apoptosis, whereas attenuated viruses failed to infect brain cells. Similar results were obtained by using primary brain-cell cultures. Our findings indicate that a structural alteration in the stem 2 region of the H protein at position 195 or 200 interferes with infectivity of rodent neurons, and suggest that the interaction of the viral attachment protein with cellular receptors on neurons is affected.

Respiratory syncytial virus replication is prolonged by a concomitant allergic response.

Epidemiological studies show an association between early exposure to respiratory syncytial virus (RSV) and the development or exacerbation of asthma. This idea is supported by studies in mice that demonstrate worsened airway hyper-reactivity (AHR) when RSV-infected animals are exposed to allergen. The effect of allergen on RSV disease, however, has not been reported. Cotton rats (Sigmodon hispidus) that have been used as a model to study RSV pathogenesis were sensitized to extracts of Aspergillus fumigatus (Af), a common household mould. The allergic response to Af included eosinophilia, formation of granulomas and induction of Th2 type cytokines. RSV infection prior to allergen challenge resulted in exacerbation of the inflammatory response as well as increased airway responsiveness to methacholine. The exacerbated response was indeed dependent on virus replication. Virus replication in turn was influenced by the allergic response, with persistence in the noses for 2 days longer in animals challenged with allergen. This diminished clearance corresponded to decreased induction of mRNA for IFN-gamma, a Th1-type cytokine that is characteristic of viral infection. Treatment of RSV-infected Af-challenged animals with recombinant IFN-gamma reduced the allergic inflammatory response as well as the relative levels of Th1 and Th2 cytokine mRNA. However, this treatment did not reduce airway reactivity, showing that these pathologic and physiologic measures of exacerbated disease are independent. We speculate that the reciprocal effect of the allergic response on viral immunity may benefit the host by limiting exacerbation of physiologic responses that are IFN-gamma-dependent.

A mouse model of persistent brain infection with recombinant Measles virus.

Measles virus (MV) nucleocapsids are present abundantly in brain cells of patients with subacute sclerosing panencephalitis (SSPE). This invariably lethal brain disease develops years after acute measles as result of a persistent MV infection. Various rodent models for MV infection of the central nervous system (CNS) have been described in the past, in which the detection of viral antigens is based on histological staining procedures of paraffin embedded brains. Here, the usage of a recombinant MV (MV-EGFP-CAMH) expressing the haemagglutinin (H) of the rodent-adapted MV-strain CAM/RB and the enhanced green fluorescent protein (EGFP) is described. In newborn rodents the virus infects neurons and causes an acute lethal encephalitis. From 2 weeks on, when the immune system of the genetically unmodified animal is maturating, intracerebral (i.c.) infection is overcome subclinically, however, a focal persistent infection in groups of neurons remains. The complete brain can be analysed in 50 or 100 microm slices, and infected autofluorescent cells are readily detected. Seven and 28 days post-infection (p.i.) 86 and 81% of mice are infected, respectively, and virus persists for more than 50 days p.i. Intraperitoneal immunization with MV 1 week before infection, but not after infection, protects and prevents persistence. The high percentage of persistence demonstrates that this is a reliable and useful model of a persistent CNS infection in fully immunocompetent mice, which allows the investigation of determinants of the immune system.

Studying experimental measles virus vaccines in the presence of maternal antibodies in the cotton rat model (Sigmodon hispidus).

The inhibition of vaccine-induced seroconversion after vaccination is one of the problems associated with measles virus (MV) immunization. In cotton rats, after transfer of human MV specific antibodies, vaccine-induced seroconversion is inhibited. With this model, it was shown that plasmid immunization (although successful in seronegative animals) was inhibited by maternal antibodies. In contrast, immunization via a mucosal surface with a vesicular stomatitis virus expressing the MV hemagglutinin induced seroconversion in the presence of maternal antibodies and subsequent protection.

Recognition of measles virus-infected cells by CD8+ T cells depends on the H-2 molecule.

H-2d mice are resistant to measles virus-induced encephalitis (MVE) and develop Ld-restricted CD8+ T cells which lyse target cells infected with measles virus or with a vaccinia virus recombinant expressing the nucleocapsid protein of measles virus (vvN). In contrast, H-2k mice are susceptible to MVE and generate CD8+ T cells which lyse target cells infected with vvN, but not those infected with MV. We were able to demonstrate that this difference is not due to a defect in the antigen processing machinery, but that Kk molecules require 100-fold more peptide to sensitize target cells for lysis by CTL. vvN replicates well in target cells and therefore enhances the level of epitope peptide available for CTL recognition. In contrast, MV infection is abortive in mouse cells and low levels of epitope peptide are produced. As Ld requires 100-fold less peptide than Kk to sensitize target cells for lysis, the low level of epitope peptide is enough to induce lysis by CD8+ T cells, whereas for recognition via Kk, increased synthesis of protein is required. We propose that the differences in peptide binding between the two H-2 molecules will have consequences for the kinetics of the generation of CD8+ T cells as well as the absolute numbers of CD8+ T cells generated.

Evolution in a chronic RNA virus infection: selection on HTLV-I tax protein differs between healthy carriers and patients with tropical spastic paraparesis.

HTLV-I causes T-cell leukemia and tropical spastic paraparesis (TSP) in a minority of infected people, whereas the majority remain healthy. The virus differs little in sequence between isolates but has been shown to have a quasispecies structure. Using the Nei and Gojobori algorithm, we have shown that the proportion of nonsynonymous to synonymous changes in HTLV-I proviral tax gene sequences from healthy seropositive subjects (Dn/Ds = 0.9 to 1.3) is significantly higher than those from TSP patients (Dn/Ds = 0.3 to 0.6). Here we show that the distinction between healthy seropositives and TSP patients can only be seen with proviral tax sequences, but not with cDNA, the amino-terminal or carboxy-terminal half of tax, or the rex gene. The Dn/Ds ratio of proviral tax sequences was used to analyze two TSP patients with atypical features and to investigate the influence of cytotoxic T cells (CTL) on the viral quasispecies.

Selective in vivo suppression of T lymphocyte responses in experimental measles virus infection.

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.