Evaluation of cotton rats as a model for severe acute respiratory syndrome.

Experimental studies were conducted to evaluate two species of cotton rats, Sigmodon hispidus and Sigmodon fulviventer, as a model for severe acute respiratory syndrome (SARS). Blood and turbinate wash samples, and lung tissue were collected from each animal at different time points after SARS coronavirus (CoV) infection for determining the growth curve of virus, if any, by the standard infectivity assay in Vero E6 cells. In addition, sections of the lung, liver, spleen, and kidney were taken and used for histology analysis. All animals were observed daily for signs of illness, and in some experiments, animals were weighed on the day when they were sacrificed. The results indicated that the cotton rat species, S. hispidus and S. fulviventer, were not a useful model for either SARS-CoV infection or disease. This observation was supported by the absence of any signs of illness, the failure to consistently demonstrate virus in the blood and tissues, and the absent of any notable histopathology. However, infected animals were capable of producing neutralizing antibodies against SARS-CoV, suggesting the seroconversion did occur. Further studies are warranted to consider other animal species in efforts to find better animal models for the evaluation of SARS-CoV vaccines and antiviral drugs.

Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors.

Between December 1996 and September 1998, 13 patients with advanced recurrent malignant brain tumors (9 with glioblastoma multiforme, 1 with gliosarcoma, and 3 with anaplastic astrocytoma) were treated with a single intratumoral injection of 2 x 10(9), 2 x 10(10), 2 x 10(11), or 2 x 10(12) vector particles (VP) of a replication-defective adenoviral vector bearing the herpes simplex virus thymidine kinase gene driven by the Rous sarcoma virus promoter (Adv.RSVtk), followed by ganciclovir (GCV) treatment. The VP to infectious unit ratio was 20:1. Our primary objective was to determine the safety of this treatment. Injection of Adv.RSVtk in doses <==2 x 10(11) VP, followed by GCV, was safely tolerated. Patients treated with the highest dose, 2 x 10(12) VP, exhibited central nervous system toxicity with confusion, hyponatremia, and seizures. One patient is living and stable 29.2 months after treatment. Two patients survived >25 months before succumbing to tumor progression. Ten patients died within 10 months of treatment, 9 from tumor progression and 1 with sepsis and endocarditis. Neuropathologic examination of postmortem tissue demonstrated cavitation at the injection site, intratumoral foci of coagulative necrosis, and variable infiltration of the residual tumor with macrophages and lymphocytes.

Use of cotton rats for preclinical evaluation of measles vaccines.

The continued prevalence and medical impact of measles worldwide has created interest in the development of new generations of measles vaccines. Monkeys can be used for preclinical testing of these vaccines. However, a more practical and less expensive animal model is highly desirable, particularly for initial vaccine development and evaluation. Cotton rats have been shown to support the replication of different strains of measles virus (MV), and thus may be useful for these purposes. To test this concept, the immunogenicity and protective efficacy of two standard (Moraten and trivalent measles, mumps, rubella) and four experimental (two recombinant ALVAC, one ISCOM subunit and live attenuated Edmonston-Zagreb) MV vaccines were evaluated in naïve cotton rats, and cotton rats with passively acquired MV-specific neutralizing serum antibodies. All of the test vaccines were immunogenic and protected naíve animals from pulmonary infection and viral dissemination. However, under the conditions utilized, only the Edmonston-Zagreb vaccine provided such protection to animals with significant levels of passively acquired MV-specific neutralizing antibodies. The results of these tests and the potential of using cotton rats as an animal model for preliminary testing of MV vaccines are discussed.

Use of cotton rats to evaluate the efficacy of antivirals in treatment of measles virus infections.

No practical animal models for the testing of chemotherapeutic or biologic agents identified in cell culture assays as being active against measles virus (MV) are currently available. Cotton rats may serve this purpose. To evaluate this possibility, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and poly(acrylamidomethyl propanesulfonate) (PAMPS), two compounds that have been reported to inhibit MV in vitro, and ribavirin, an established antiviral drug with MV-inhibitory activity, were evaluated for their antiviral activities against MV and respiratory syncytial virus (RSV) in tissue culture and in hispid cotton rats. A single administration of PAMPS markedly inhibited pulmonary RSV or MV replication (>3 log(10) reduction in pulmonary titer compared to that for controls), but only if this compound was administered intranasally at about the time of virus inoculation. Both EICAR and ribavirin exhibited therapeutic activity against RSV and MV in cotton rats when they were administered parenterally. However, both of these compounds were less effective against MV. On the basis of the pulmonary virus titers on day 4 after virus inoculation, the minimal efficacious dose of EICAR against MV (120 mg/kg of body weight/day when delivered intraperitoneally twice daily) appeared to be three times lower against this virus than that of ribavirin delivered at a similar dose (i.e., 360 mg/kg/day). These findings correlated with those obtained in vitro. The data obtained suggest that cotton rats may indeed be useful for the initial evaluation of the activities of antiviral agents against MV.

Replication of clinical measles virus strains in hispid cotton rats.

An alternative model to nonhuman primates to study measles virus (MV) pathogenesis, to evaluate potential MV vaccines, or to screen for potential antivirals effective against this virus is highly desirable. The laboratory-adapted Edmonston strain of MV has been reported to replicate in the lungs of hispid cotton rats following intranasal inoculation, immunosuppress infected animals, and disseminate widely from the lungs, making these animals a candidate model. However, clinical MV strains have generally not been found to grow in these animals, limiting the utility and acceptance of this model. In the present studies we demonstrate reproducible replication of several clinical MV strains in hispid cotton rats. As with the Edmonston strain, leukocytes appear to be the primary target cells of these viruses following intranasal inoculation, and extrapulmonary dissemination is common. It is also demonstrated that prior MV infection or immunization of test animals with MV vaccine prevents pulmonary tract infection. These findings should make the MV-cotton rat model more acceptable.

Respiratory syncytial virus (RSV) disease and prospects for its control.

Respiratory syncytial virus (RSV) is a major virus pathogen of infants and young children, an important cause of disease in adults and is responsible for a significant amount of excess morbidity and mortality in the elderly. It also can be devastating in immunosuppressed populations. Vaccines are being developed, but none are currently licensed. Moreover, even if one or more are approved, they may not be suitable for some populations vulnerable to RSV (e.g. very young infants and the immunosuppressed). Ribavirin and immunoglobulin preparations with high titers of RSV-specific neutralizing antibodies are currently approved for use to treat and prevent RSV infection. However, neither of these is cost-effective or simple to administer. New agents are needed to reduce the impact of RSV. This review is concerned with the means currently available for controlling RSV, the search for new agents effective against this virus, and future prospects for preventing and treating RSV infections.

CL387626 exhibits marked and unusual antiviral activity against respiratory syncytial virus in tissue culture and in cotton rats.

CL387626 (4,4'-Bis[4,6-di[3-aminophenyl-N,N-bis(2-carbamoylethyl)-sulfon ilimino]-1,3,5-triazine-2-ylamino-bi-phenyl-2,2'-disulfonic acid, disodium salt), a compound synthesized by Wyeth-Ayerst Research Laboratories, was tested for its cytotoxicity and antiviral activity against respiratory syncytial virus (RSV) in tissue culture and in cotton rats. The median cell inhibitory (IC50) and median efficacious (EC50) concentrations of CL387626 against RSV in proliferating HEp2 or Vero tissue culture cells were determined to be 375 and 0.25 microg/ml, respectively, giving the compound an apparent selective index (S.I.) of 1500. This compound also exhibited uncommon antiviral activity against RSV in cotton rats. In multiple experiments, a single 30 mg/kg dose of CL387626 administered intranasally 4 or 5 days prior to virus challenge, significantly inhibited pulmonary replication of RSV compared to that seen in control animals inoculated similarly with placebo (i.e. water). In contrast to these results, most lots of CL387626 failed to significantly inhibit pulmonary RSV replication when administered utilizing therapeutic administration schedules. Although some cytotoxicity was noted in tissue culture assays, no overt toxic effects were noted in any test animal, including those inoculated with > 300 mg CL387626/kg, a dose approximately 150 times the apparent minimal efficacious dose (i.e. 1.9 mg/kg).

Common emergence of amantadine- and rimantadine-resistant influenza A viruses in symptomatic immunocompromised adults.

The importance and significance of amantadine- or rimantadine-resistant influenza viruses in immunocompromised patients was studied in a population of adult bone marrow transplant (BMT) recipients and patients with leukemia prospectively cultured for respiratory viruses. Influenza A viruses were isolated from 29 patients with acute respiratory illness (14 BMT recipients and 15 patients with leukemia). Fifteen patients (52%) received amantadine (n = 4) or rimantadine (n = 11) therapy. All influenza isolates recovered from six patients shedding virus for > or = 3 days were screened for antiviral susceptibility; resistant isolates were further genetically characterized. Initial influenza isolates were susceptible to amantadine or rimantadine, but subsequent isolates from five of six patients were resistant. Influenza-associated mortality was similar among patients with and without documented antiviral resistance (2 of 5 vs. 5 of 24). We conclude that development of antiviral resistance in immunocompromised individuals should be considered when they have been treated with antivirals and have shed influenza virus for a prolonged period. Isolation procedures should be instituted for all immunocompromised patients with influenza, both during and after therapy with amantadine or rimantadine.

Genetic inactivation of an extracellular cysteine protease (SpeB) expressed by Streptococcus pyogenes decreases resistance to phagocytosis and dissemination to organs.

Streptococcal pyrogenic exotoxin B (SpeB), a conserved cysteine protease expressed by virtually all Streptococcus pyogenes strains, has recently been shown to be an important virulence factor (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574-2580, 1997). Genetic inactivation of SpeB significantly decreased the lethality of a serotype M49 strain for mice and abolished the lethality of a serotype M3 strain after intraperitoneal (i.p.) injection. In the present study, a wild-type M3 isolate and an M3 speB mutant derivative were used to investigate the mechanism responsible for altered virulence. Following i.p. injection, the mutant and wild-type strains induced virtually identical cellular inflammatory responses, characterized largely by an influx of polymorphonuclear leukocytes (PMNs). In addition, the mutant and wild-type strains rapidly entered the blood and were recovered from all organs examined. However, significantly fewer (P < 0.05) CFUs of the isogenic mutant derivative than of the wild-type parent strain were recovered from blood and organs. PMNs effectively cleared the M3 speB mutant from the peritoneum by 22 h, thereby sparing the host. In contrast, the wild-type M3 strain continued to replicate intraperitoneally and had the ability to kill phagocytes. This process allowed the wild-type strain to continuously disseminate, resulting in host death. Our results indicate that genetic inactivation of the cysteine protease decreased the resistance of the mutant to phagocytosis and impaired its subsequent dissemination to organs. These results provide insight into the detrimental effect of SpeB inactivation on virulence.

Distribution, persistency, toxicity, and lack of replication of an E1A-deficient adenoviral vector after intracardiac delivery in the cotton rat.

Adenoviral vectors were inoculated via intracardiac injection into 5- to 1O-week-old cotton rats (Sigmodon hispidus) to evaluate the effects of systemic delivery. Cotton rats were chosen as a model because they are semipermissive to the replication of human adenoviruses. The vector used was AdV.RSV-tk, a replication-deficient adenovirus with a herpes simplex virus thymidine kinase gene inserted in the E1 region. Vector doses were 3 x 10(8), 3 x 10(9), and 3 x 10(10) viral particles per animal with and without ganciclovir at 10 mg/kg twice a day. Animals were sacrificed and necropsied at 24 hours, 7 days, and 14 days postinoculation. Gross and microscopic pathologic observations in dosed groups were compared with an unmanipulated control group. From each animal, 10 different organ systems were analyzed for histopathology and vector distribution. The only significant microscopic lesions observed were epicardial inflammation and splenic hemosiderosis. Vector sequences persisted throughout the 14-day assay with preponderance in the heart, lung, and lymphoid organs. Infectious virions were detected for 24 hours, and these virions were only detected at the site of injection of two animals in the highest dose group. No viral replication was detected. Therefore, systemic delivery of up to 3 x 10(11) viral particles/kg was well tolerated in this semipermissive host model and did not result in any significant pathology.

Neurotoxicity of intracerebral injection of a replication-defective adenoviral vector in a semipermissive species (cotton rat).

The neurotoxicity of an adenoviral vector (Adv.RSVtk) carrying the gene for herpes simplex virus thymidine kinase (HSVtk) was tested in the cotton rat, a semipermissive host. Adv.RSVtk was injected intracerebrally in cotton rats at a dose of 5.0 x 10(6) or 7.5 x 10(7) p.f.u. No signs of illness were observed. Histological inspection at 12 and 28 days after injection showed inflammation of the ependyma and choroid plexus and at the injection site. No demyelination, viral inclusions, cerebral edema, necrosis, cavities or vascular necrosis were seen in the brains. There was no significant difference between animals injected with 5.0 x 10(6) or 7.5 x 10(7) p.f.u., nor was there a difference between animals analyzed at 12 or 28 days after vector injection. This inflammation was similar in animals that had been preimmunized with wild-type virus and in animals that had been treated with ganciclovir. No histopathology, was observed in the lungs of the animals and no replication-competent virus was detected. These experiments indicate that Adv.RSVtk has limited neurotoxicity which would not prohibit its use in a limited phase I clinical trial in humans that have malignant tumors of the central nervous system.

Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.

Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.

Recombinant superoxide dismutase (SOD) administered by aerosol inhibits respiratory syncytial virus infection in cotton rats.

Recombinant (r) human (hu) manganese (Mn) and copper-zinc (CuZn) superoxide dismutase (SOD) were evaluated for their cytotoxicity and antiviral activity against respiratory syncytial virus (RSV) in tissue culture and in cotton rats. No apparent cytotoxicity or inhibition of RSV was observed in the tissue culture studies (both compounds had IC50 and EC50 values > or = 1000 micrograms/ml and a selective index = 1). However, significant reductions in mean pulmonary RSV titers (ranging between 0.5 and 1.9 log10/g of lung compared with the mean pulmonary viral titers detected in similarly inoculated, placebo-treated control animals) were seen in most of the experiments, in which experimentally infected cotton rats were exposed to continuous small-particle aerosols (reservoir concentrations > or = 20 mg/ml) containing either rhuMnSOD or rhuCuZnSOD. This protective effect was dose dependent and not observed when either rSOD compound was administered parenterally (intraperitoneally) or intranasally. No toxic effects were noted in any of the cotton rats exposed to aerosols of either rhuMn or CuZnSOD; nor was any evidence of drug-induced histopathology observed in sections of lung prepared from these animals.

Evaluation of the protective efficacy of reshaped human monoclonal antibody RSHZ19 against respiratory syncytial virus in cotton rats.

Reshaped human MAb RSHZ19, which is specific for the surface fusion protein of respiratory syncytial virus (RSV) is in clinical development for the prevention and treatment of RSV-induced disease in human infants. The current studies profile lung virus clearance and evaluate lung histopathology in MAb-treated, RSV-infected cotton rats, a well characterized model of RSV infection. The highest dose of this MAb (10 mg/kg) administered parenterally 24 h before infection decreased subgroup A or B RSV lung titers to below detectable levels (> or = 2.3 log10 reduction), and significantly reduced lung virus titers (> or = 2.0 log10 reduction) when administered 96 h postinfection. Prophylactic administration of 10 mg/kg RSHZ19 was significantly more protective than 1000 mg/kg conventional human immune serum globulin (HSIg), and protective serum-neutralizing titers in MAb-treated animals (1:32, which correlated with approximately 40 micrograms/ml determined by anti-idiotype ELISA) were significantly lower than those reported previously for HSIg or for convalescent human serum (1:200-1:400). MAb concentration in lung lavages was determined by ELISA to be approximately 1% of the serum MAb concentration, but was not detectable by neutralization assay. The degree of lung histopathology in MAb-treated cotton rats was proportional to lung virus titer, and inversely proportional to the RSHZ19 dose administered. There was no evidence of exacerbated disease in the lungs of MAb-treated animals. These studies thus support the potential clinical utility of RSHZ19 MAb in the prevention and treatment of RSV-induced disease in humans.

Protection of mice against lethal viral infection by synthetic peptides corresponding to B- and T-cell recognition sites of influenza A hemagglutinin.

Previously, we reported 12 synthetic T- and B-cell recognition regions representing surface areas of the hemagglutinin (HA) of X31 influenza virus. In the present study, four of these peptides were examined in Balb/c mice for their ability to produce protective immunity against lethal infection with a dose equivalent to 10 LD50 of influenza virus. These peptides corresponded to the following sequences: 23-36 (HA1-1); 138-152 (HA1-3); 183-199 (HA1-6) and 1-11 (HA2-10). Each of the selected peptides, in their free form, evoked anti-peptide antibodies that cross-react with intact X31 virus. Two of the peptides, HA1-1 and HA1-3, also elicited virus-specific delayed type hypersensitivity (DTH) responses. These two peptides, when injected into mice, not only failed to protect the immunized mice against challenge with influenza virus, but in fact caused greater susceptibility to viral infection as compared to control animals that had been injected with saline. In contrast, peptides HA1-6 and HA2-10, which were unable to induce adequate virus-specific DTH responses, conferred 42-46% and 54-73% protection, respectively, compared to the control group that received only saline (P < 0.03 to P < 0.01).

Evidence that measles virus hemagglutinin initiates modulation of leukocyte function-associated antigen 1 expression.

Measles virus (MV), human immunodeficiency virus, Epstein-Barr virus, and other leukotropic viruses can modulate the expression of leukocyte function antigen 1 (LFA-1) on the surface of infected and nearby leukocytes. This ability to induce changes in LFA-1 expression may play an important role in the pathogenesis of these viruses. However, the mechanism(s) involved in virus-mediated regulation of LFA-1 is unknown. Evidence is presented in this report that it is the MV hemagglutinin (H) protein that initiates up-regulation of LFA-1 expression in leukocyte cultures infected with this virus. Indeed, comparison of the abilities of different MV strains to modulate LFA-1 expression, examination of published nucleotide sequences for the H proteins of different vaccine strains, and competitive inhibition assays using oligopeptides homologous or heterologous to a region of the H protein gene encompassing amino acid 116 (from the amino terminus) all suggest that it is this portion of the H protein that is responsible for MV-induced alteration of LFA-1. These comparisons also support the hypothesis that there is a relationship between the abilities of different MV strains to alter LFA-1 expression and their pathogenic potentials.

Evaluation of the antiviral activity of N-(phosphonoacetyl)-L-aspartate against paramyxoviruses in tissue culture and against respiratory syncytial virus in cotton rats.

N-(phosphonoacetyl)-L-aspartate (PALA), a potent inhibitor of L-aspartic acid transcarbamoylase, was evaluated for cytotoxicity and antiviral activity against three different paramyxoviruses in tissue culture, and for antiviral efficacy and toxicity in vivo using a cotton rat-respiratory syncytial virus (RSV) model. Significant in vitro cytotoxicity was observed in proliferating cultures of HEp-2 (IC50 = 250 micrograms/ml) and Vero cells (IC50 = 32 micrograms/ml), but was less evident in cultures containing confluent monolayers (i.e., stationary cells) of these cells, or in cultures of Madin Darby canine kidney (MDCK) cells (these IC50 values were all > or = 750 micrograms/ml, with 1000 micrograms/ml being the maximum concentration tested). Mean selective indices (ratio of the median cytotoxic dose: median efficacious dose) of 1, 72 and 146 were obtained against parainfluenza virus type 3, RSV and measles virus, respectively, when PALA was tested against these viruses using confluent HEp-2 and Vero cell monolayers. In cotton rats, significant reductions in pulmonary titers (0.8-1.4 log10/g lung) compared to pulmonary viral titers in placebo-treated control animals, were consistently seen in cotton rats given > or = 10 mg of PALA/kg/day (b.i.d.) intraperitoneally on days 1-3 postinfection with either subtype A or B RSV. No toxic effects were noted even in animals given 100 mg of PALA/kg/day for 7 consecutive days.

A prototype recombinant vaccine against respiratory syncytial virus and parainfluenza virus type 3.

We have produced a genetically-engineered chimeric protein composed of the external domains of the respiratory syncytial virus (RSV) fusion (F) protein and the parainfluenza virus type 3 (PIV-3) hemagglutinin-neuraminidase (HN) protein in insect cells using the baculovirus expression system. The yield of the soluble chimeric FRSV-HNPIV-3 protein could be increased approximately 2-fold by using Trichoplasia ni (High Five) insect cells in place of Spodoptera frugiperda (Sf9) for expression. The chimeric protein, purified from the supernatant of baculovirus-infected High Five cells by immunoaffinity chromatography was correctly processed at the F2-F1 proteolytic cleavage site. Immunochemical analysis of the chimera with a panel of anti-F and anti-HN monoclonal antibodies suggested that the antigenicity of the major F and HN neutralization epitopes of the chimeric protein was preserved. Immunization of cotton rats with two 1 or 10 micrograms doses of the chimeric protein adsorbed to aluminum phosphate elicited strong PIV-3 specific HAI responses as well as PIV-3 and RSV specific neutralizing antibodies, and at either dose completely protected against challenge with live RSV and PIV-3.