Influenza virus directly infects, inflames, and resides in the arteries of atherosclerotic and normal mice.

OBJECTIVE: Influenza can trigger heart attacks, and vaccination against influenza reduces the risk of cardiovascular events. Currently, it is believed that influenza virus in general does not disseminate to extra-pulmonary tissues. We assessed the vascular effects of influenza infection and whether the virus can directly infect atherosclerotic arteries in mice. METHODS/RESULTS: We intranasally infected 4 different types of mice--atherosclerotic apo E-deficient (our primary model), LDL receptor knockout, C57BL/6, and outbred Swiss--with influenza A/HK (H3/N2) virus. On day 7 after infection, we cultured viable virus from lung, aorta, and heart tissue, but not from the blood of apo E-deficient mice. Immunofluorescence studies showed influenza A virus NP1 protein and real time polymerase chain reaction (PCR) assay showed RNA in the aorta of infected apo E-deficient mice. Infected mice had significantly higher blood levels of chemokines and cytokines than control mice. At the local level, gene expression for several chemokines and cytokines was increased and eNOS expression was decreased. Infected mice had a higher density of macrophages in plaque than did control mice. CONCLUSIONS: We have shown for the first time that influenza virus can directly infect and reside in atherosclerotic arteries and that infection was associated with systemic and arterial-level pro-inflammatory changes.

Contrasting effects of type I interferon as a mucosal adjuvant for influenza vaccine in mice and humans.

To identify an adjuvant that enhances antibody responses in respiratory secretions to inactivated influenza virus vaccine (IVV), a comparison was made of responses to intranasal vaccinations of mice with IVV containing monophosphoryl lipid A (MPL), type I interferon (IFN) or cholera toxin B (CTB). Antibody in nasal secretions and lung wash fluids from mice was increased after vaccination and lung virus was significantly reduced after challenge to a similar level in each adjuvant group. Interferon was selected for a trial in humans. Trivalent inactivated influenza vaccine was given intranasally to healthy adult volunteers alone or with 1 million units (Mu) or 10 Mu of alpha interferon. Vaccinations were well tolerated but neither serum hemagglutination-inhibiting nor neutralizing antibody responses among the vaccine groups were significantly different. Similarly, neither neutralizing nor IgA antibody responses in nasal secretions were significantly different. Thus, despite exhibiting a significant adjuvant effect in mice, interferon did not exhibit an adjuvant effect for induction of antibody in respiratory secretions of humans to inactivated influenza virus vaccine given intranasally.

Chimeric coronavirus-like particles carrying severe acute respiratory syndrome coronavirus (SCoV) S protein protect mice against challenge with SCoV.

We tested the efficacy of coronavirus-like particles (VLPs) for protecting mice against severe acute respiratory syndrome coronavirus (SCoV) infection. Coexpression of SCoV S protein and E, M and N proteins of mouse hepatitis virus in 293T or CHO cells resulted in the efficient production of chimeric VLPs carrying SCoV S protein. Balb/c mice inoculated with a mixture of chimeric VLPs and alum twice at an interval of four weeks were protected from SCoV challenge, as indicated by the absence of infectious virus in the lungs. The same groups of mice had high levels of SCoV-specific neutralizing antibodies, while mice in the negative control groups, which were not immunized with chimeric VLPs, failed to manifest neutralizing antibodies, suggesting that SCoV-specific neutralizing antibodies are important for the suppression of viral replication within the lungs. Despite some differences in the cellular composition of inflammatory infiltrates, we did not observe any overt lung pathology in the chimeric-VLP-treated mice, when compared to the negative control mice. Our results show that chimeric VLP can be an effective vaccine strategy against SCoV infection.

Protection against influenza infection by cytokine-enhanced aerosol genetic immunization.

BACKGROUND: Conventional vaccine development for newly emerging pandemic influenza virus strains would likely take too long to prevent devastating global morbidity and mortality. If DNA vaccines can be distributed and delivered efficiently, genetic immunization could be an attractive solution to this problem, since plasmid DNA is stable, easily engineered to encode new protein antigens, and able to be quickly produced in large quantities. METHODS: We compared two novel genetic immunization methods in a mouse model of influenza to evaluate protective effects: aerosol delivery of polyethylenimine (PEI)-complexed hemagglutinin (HA)-expressing plasmid and intravenous (IV) delivery of the plasmid complexed with macroaggregated albumin/PEI. Serial serum samples were obtained for assay of neutralizing antibodies against HA. Mice were then challenged in the airway with influenza virus, and production of infectious virus in the lungs was titered. RESULTS: Most mice immunized with HA plasmid alone by aerosol and all mice immunized IV developed protective immune responses, whereas none administered control plasmid were protected. Aerosol co-administration of HA plasmid with plasmids encoding the cytokines interleukin 12 (IL12) and granulocyte-macrophage colony stimulating factor (GM-CSF) markedly increased neutralizing antibody responses, so that all aerosol immunized mice were protected from high level virus proliferation. CONCLUSIONS: Cytokine-enhanced aerosol delivery of plasmid vaccines can elicit robust protective immune responses against influenza. Thus, aerosol delivery has the potential to address the need for rapid widespread immunization against new influenza virus strains, and may have applications for other infectious and toxic disease processes.

Respiratory syncytial virus fusion inhibitors. Part 3: Water-soluble benzimidazol-2-one derivatives with antiviral activity in vivo.

The introduction of acidic and basic functionality into the side chains of respiratory syncytial virus (RSV) fusion inhibitors was examined in an effort to identify compounds suitable for evaluation in vivo in the cotton rat model of RSV infection following administration as a small particle aerosol. The acidic compounds 2r, 2u, 2v, 2w, 2z, and 2aj demonstrated potent antiviral activity in cell culture and exhibited efficacy in the cotton rat comparable to ribavirin. In a BALB/c mouse model, the oxadiazolone 2aj reduced virus titers following subcutaneous dosing, whilst the ester 2az and amide 2aab exhibited efficacy following oral administration. These results established the potential of this class of RSV fusion inhibitors to interfere with infection in vivo following topical or systemic administration.

A novel inhibitor of respiratory syncytial virus isolated from ethnobotanicals.

A novel low molecular weight compound, CJ 4-16-4, isolated from ethnobotanicals using bioassay-guided fractionation, was found to be a potent inhibitor of respiratory syncytial virus (RSV) in vitro and in vivo. In vitro, a very low micromolar efficacious dose was obtained against at least four of subtype A (RSV-Long, RSV A2, and RSV A6 57754) and one of subtype B (Washington) RSV strains without seeing any significant cytotoxicity to Hep-2, MDCK or Vero cell lines. The drug inhibits growth of RSV in Hep-2 cells maintained in tissue culture at a very low concentration (approximately 0.07 microM) with cell toxicity >400 microM (TI>5880). In a cotton rat model of RSV infection, the drug was able to reduce viral titers by approximately 1 log at dose 12.5 and 25 mg/kg/day, and by >2 log at 100 mg/kg/day. This antiviral activity was specific as influenza A and B and herpes simplex 1 and 2 viruses were not inhibited. The results obtained indicate that CJ 4-16-4 warrants clinical development.

Antiviral efficacy of VP14637 against respiratory syncytial virus in vitro and in cotton rats following delivery by small droplet aerosol.

VP14637, the lead compound in a series of substituted bis-tetrazole-benzhydrylphenols developed by ViroPharma Incorporated, was evaluated for antiviral efficacy against respiratory syncytial virus (RSV) in vitro in cell culture and in vivo in cotton rats. A selective index of >3000 (> or =2000 times greater than that observed for ribavirin) was determined in the in vitro studies for this compound against both RSV A and B subtypes. In cotton rats, animals given as little as 126 microg drug/kg by small droplet aerosol in divided doses starting 1 day after experimental virus infection with either a RSV A or B subtype consistently had significantly lower mean pulmonary RSV titers and reduced histopathological findings than mock-treated animals or cotton rats given placebo (vehicle-treated animals). No cotton rat treated with aerosols of VP14637 during these studies manifested any evident untoward responses. Thus, VP14637 exhibited good selective antiviral efficacy both in vitro and in vivo.

Development of a cotton rat-human metapneumovirus (hMPV) model for identifying and evaluating potential hMPV antivirals and vaccines.

Hispid cotton rats were inoculated with two different human metapneumovirus (hMPV) subtype A strains and one subtype B hMPV. Although no overt disease was seen in any virus-inoculated animal, following an eclipse phase, significant pulmonary virus titers were observed in every hMPV-inoculated animal through day 7 post virus inoculation (p.i.) and in most through day 10. Peak virus titers occurred four days p.i., while virus-induced histopathology was most evident in lung sections obtained from animals 7 to 10 days p.i. The latter consisted primarily of desquamating and hypertrophic columnar epithelial cells lining the bronchi and bronchioles and the presence of large numbers of leukocytes in and around the bronchi and bronchioles. In fluorescent antibody studies, virus antigen-specific fluorescence was most evident in the desquamating tall columnar epithelial cells lining bronchi and bronchioles, in pneumocytes lining alveoli and in single or small groups of free cells, most probably leukocytes, present in the lumen of alveoli, bronchi and bronchioles. Virus was generally not detected in inoculated animals >10 days p.i. Although the pattern of virus replication in cotton rats was similar for all the three virus stains, the B subtype consistently grew to lower levels than the two A strains. Regardless, these findings indicate that hMPV replicates in cotton rats and that these animals may be used as a small animal model of hMPV infection and to facilitate the identification and development of vaccines and antivirals for preventing and/or ameliorating infections caused by this virus.

Comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by NMSO3 in tissue culture assays.

Human metapneumovirus (hMPV) is a recently elucidated respiratory virus pathogen for which there are no agents currently licensed to prevent or treat infections caused by it. However, NMSO3 has been reported to inhibit replication of human respiratory syncytial virus (hRSV), a virus that is closely related to hMPV, both in vitro in tissue culture cells and in vivo in cotton rats. For this reason, experiments were performed to compare the antiviral activity of NMSO3 against both hRSV and hMPV in tissue culture-based assays. Heparin and ribavirin, two other compounds known to inhibit hRSV, and two other paramyxoviruses, human parainfluenza virus type 3 (PIV3) and measles virus (MV), were included in these tests for comparison. All three compounds significantly inhibited the replication of subtype A and B strains of hRSV and serotypes 1 and 2 hMPV. However, unlike ribavirin, NMSO3 and heparin inhibited only hMPV and hRSV and not PIV3 or MV. Also unlike ribavirin, the activity of the two sulfated molecules was most effective if these materials were present during virus attachment and penetration of host cells. Interestingly, NMSO3, but not heparin, was able to limit secondary infection and spread of both viruses.

Substituted benzimidazoles with nanomolar activity against respiratory syncytial virus.

A cell-based assay was used to discover compounds inhibiting respiratory syncytial virus (RSV)-induced fusion in HeLa/M cells. A lead compound was identified and subsequent synthesis of >300 analogues led to the identification of JNJ 2408068 (R170591), a low molecular weight (MW 395) benzimidazole derivative with an EC(50) (0.16 nM) against some lab strains almost 100,000 times better than that of ribavirin (15 microM). Antiviral activity was confirmed for subgroup A and B clinical isolates of human RSV and for a bovine RSV isolate. The compound did not inhibit the growth of representative viruses from other Paramyxovirus genera, i.e. HPIV2 and Mumps Virus (genus Rubulavirus), HPIV3 (genus Respirovirus), Measles virus (genus Morbillivirus) and hMPV. Efficacy in cytopathic effect inhibition assays correlated well with efficacy in virus yield reduction assays. A concentration of 10nM reduced RSV production 1000-fold in multi-cycle experiments, irrespective of the multiplicity of infection. Time of addition studies pointed to a dual mode of action: inhibition of virus-cell fusion early in the infection cycle and inhibition of cell-cell fusion at the end of the replication cycle. Two resistant mutants were raised and shown to have single point mutations in the F-gene (S398L and D486N). JNJ 2408068 was also shown to inhibit the release of proinflammatory cytokines IL-6, IL-8 and Rantes from RSV-infected A549 cells.

Short duration aerosols of JNJ 2408068 (R170591) administered prophylactically or therapeutically protect cotton rats from experimental respiratory syncytial virus infection.

Cotton rats exposed to continuous small droplet aerosols of 2[[2-[[1-(2-aminoethyl)-4-piperidinyl]amino]-4-methyl-1H-benzimidazol-1-yl]methyl]-6-methyl-3-pyridinol (JNJ 2408068) or its hydrochloric salt for only 15 min, one day prior to virus inoculation or one day after, were significantly protected from pulmonary respiratory syncytial virus (RSV) infection compared to control animals similarly infected but exposed to aerosols of placebo at these times. No evidence of toxicity was seen in any of these animals or in cotton rats administered 10 times the minimum cotton rat efficacious dose (i.e. 10x0.39 mg of active compound per kilogram of body weight) for four continuous days. The marked selective antiviral activity observed in the cotton rats mirrored that seen for these compounds in cytotoxicity and antiviral assays performed against RSV in vitro. Plasma kinetics and tissue distribution of JNJ 2408068 in cotton rats following inhalation were determined in separate experiments performed using conditions similar to those utilized in the in vivo efficacy studies. The data from these experiments indicated that significant levels of the test compound were delivered to the lungs of exposed animals, but that extrapulmonary distribution was limited.

Comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro.

Human metapneumovirus (hMPV) is a newly recognized pathogen that like its better-known relative, human respiratory syncytial virus (hRSV), appears to be ubiquitous and an important cause of respiratory disease in diverse subpopulations. No antivirals or vaccines are currently approved for the treatment or prevention of hMPV infections. However, ribavirin is licensed to treat serious hRSV-induced infections in children and immune globulin designed for intravenous administration (i.v.IG) and palivizumab (Synagis), a humanized monoclonal antibody preparation, have been utilized as alternatives to vaccines for preventing or reducing the severity of infections caused by this virus. Because both ribavirin and i.v.IG have broad viral specificities, studies were performed to compare the ability of these two agents to inhibit the replication of hRSV and hMPV in tissue culture-based assays. Two experimental chemotherapeutic agents (i.e. VP14637 and JNJ2408068) and different antibody preparations were included in this testing for comparison. Ribavirin and the i.v.IG utilized were found to have equivalent antiviral activity against hMPV and hRSV. In contrast, except for antisera specifically raised against hMPV, all of the other materials tested had marked activity only against hRSV.

Modulation of gamma delta T cells in mouse buccal epithelium following antigen priming.

T cells using the gamma delta T cell receptor (TCR) are abundant in mucosal and epidermal tissues in mice. Most studies of mucosal gamma delta T cells, however, have examined cells from the intestinal mucosa, whereas little is known about the presence or function of gamma delta T cells in the oral cavity. To better understand the involvement of oral gamma delta T cells in immunity, we have characterized TCR variable gamma-gene usage in the buccal epithelium from normal mice, and from mice challenged locally with a non-replicating antigen (bovine serum albumin [BSA]) or by influenza-virus infection as a replicating antigen. Our findings demonstrate a restricted use of V gamma genes by buccal gamma delta T cells, consisting primarily of V gamma 1.2, V gamma 3, and V gamma 5, with minimal use of V gamma 2 and V gamma 4 genes. Of particular interest, 3-4 days post-antigen challenge with BSA, there was a precipitous drop in the level of expression of V gamma 1.2, V gamma 3, and V gamma 5 genes, and to a lesser extent for the V gamma 2 gene, whereas V gamma 4 gene expression increased between days 1 and 2 post-priming. In influenza-infected mice, a similar pattern was observed for the V gamma 2 and V gamma 5 genes, but not other V gamma genes. The immune-modulating effects of oral antigen exposure on buccal gamma delta T cells suggest that these cells are functionally involved in the local immune response to both replicating and non-replicating antigens in oral mucosal surfaces.

In vitro Inhibition of HHV-6 replication by sophocarpines.

The virostatic activity of sophocarpines and gancyclovir (GCV) was tested using HHV-6 Z29 strain and Molt-3 cells. The cytotoxic (IC(50)) and the antiviral (ED(50)) values were first experimentally determined and selective indices (SI) were then calculated. The SI values for sophocarpines 1 and 2 and GCV were in the order 184, 183, and 23, respectively. Though preliminary, these findings indicate that sophocarpines have the potential to inhibit HHV-6 replication.

An aged mouse model for RSV infection and diminished CD8(+) CTL responses.

Recent studies indicate that respiratory syncytial virus (RSV), like influenza, causes significant morbidity and mortality among elderly persons. There are currently no animal models to study the effects of aging on RSV disease and immunity. This manuscript provides an initial description of such a model. Aged and young BALB/c mice (22-24 and 2-4 months, respectively) were infected with 10(4) TCID(50) of RSV A2. RSV was detected by culture in lung and nose wash specimens obtained 4-6 days following infection at a slightly higher titer in old mice in comparison with young mice. RT-PCR assay detected RSV in the lungs and nose washes of all mice on 4, 8, and 21 days postinoculation, with only a slightly less frequency in young mice. Splenic lymphocytes from old mice exhibited significantly lower RSV-specific MHC class I-restricted CD8(+) CTL responses (P < 0.01-0001), and reduced IFN-production (P < 0.03) than young mice. Conversely, IL-4 production was somewhat elevated in old mice. These results demonstrate diminished RSV virus-specific CD8(+)CTL responses and IFN-gamma production in old mice in comparison with young. It is speculated that the deficient RSV-specific CTL responses may account for the increased morbidity and mortality from RSV infections in elderly persons. Although detailed histopathological, virological, and immunological analyses are incomplete at present, the old BALB/c RSV infection model described provides an opportunity to evaluate the role of CD8(+)CTL and cytokines in RSV disease in aging.

Chemotherapy of respiratory viruses: prospects and challenges.

Billions of people are infected with respiratory viruses annually. Infants and young children, the elderly, immunocompromised individuals and those debilitated by other diseases or nutritional deficiencies are most at risk for serious disease. There are few vaccines available for use against these viruses, and even where there are (influenza, measles and adenovirus), infections remain common. The continued prevalence of respiratory virus infections has lead to renewed efforts to find safe agents effective against the most medically important respiratory viruses: influenza, respiratory syncytial, parainfluenza, measles, rhino- and adenovirus. Copyright 1999 Harcourt Publishers Ltd.