Characterization of an adenovirus gene transfer vector containing an E4 deletion.

We describe the construction and characterization of an adenovirus type 2 vector, Ad2E4ORF6, which has been modified in the E4 region to contain only open reading frame 6. When assayed in cultured cells, Ad2E4ORF6 virus replication is slightly delayed but viral DNA synthesis, host-cell protein synthesis shut-off, and virus yield are indistinguishable from wild type. Late protein synthesis is normal with the exception of fiber synthesis, which is reduced approximately 10-fold. Despite the reduced fiber synthesis, Ad2E4ORF6 viral particles appear to contain a full complement of fiber protein. Virus replication in cotton rats indicates that Ad2E4ORF6 is replication defective in vivo. This may have safety implications for the development adenovirus vectors in that virus arising by recombination in the E1 region of an Ad2E4ORF6-based vector would be defective for growth in vivo. The deletion of E4 open reading frames that are not required for virus growth in vitro increases the cloning capacity of adenovirus vectors by 1.9 kb and may be generally useful for the construction of adenovirus vectors containing large cDNA inserts and/or regulatory elements. We describe the inclusion of the A2E4ORF6 modification in a recombinant adenovirus vector, Ad2/CFTR-2, for gene transfer of the human cystic fibrosis transmembrane regulator (CFTR).

Detection of respiratory syncytial virus (RSV) infected cells by in situ hybridization in the lungs of cotton rats immunized with formalin-inactivated virus or purified RSV F and G glycoprotein subunit vaccine and challenged with RSV.

The replication of RSV in unimmunized cotton rats was evaluated by quantitating the amount of infectious virus in the lung and the number of RSV infected cells in a histopathological section of lung by in situ hybridization. RSV infected cells were detected only in alveoli and bronchioles and constituted only a small minority of the cell population. The temporal patterns of rise to the peak number of infected cells (day 4) and the peak titer of infectious virus (day 3) were similar. The clearance of both infected cells and infectious virus was nearly complete by day 7. In animals previously immunized with purified RSV glycoproteins or formalin-inactivated RSV there also was a good correlation between the number of infected cells detected by in situ hybridization and the amount of infectious virus recovered. It was previously demonstrated that cotton rats immunized with formalin-inactivated vaccine developed enhanced pulmonary histopathology following challenge with RSV. In such animals, there was approximately a 90% reduction in the number of infected cells compared to control unimmunized, RSV-challenged animals. Formalin-inactivated RSV vaccine-enhanced lung histopathology developed despite the effective elimination of virus and virus-infected cells suggesting that the enhanced pathology is the result of an exaggeration of normal immune mechanisms involved in clearance of virus infection, an aberrant immune response during infection, or both.

Immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat.

Human convalescent antiserum to respiratory syncytial virus (RSV) administered intraperitoneally to cotton rats prior to RSV challenge provided near-complete protection from pulmonary infection. Antiserum given subsequent to viral challenge reduced pulmonary viral titers 100-fold or greater within 24 h. Sandoglobulin, a preparation of purified human IgG with high titer of anti-RSV neutralizing activity, produced the same effects as convalescent antiserum. Sandoglobulin was absorbed rapidly and produced a significant therapeutic reduction in virus titer within 3 h. The level of virus reduction in pulmonary and nasal tissues was directly proportional to the neutralizing antibody titer in the cotton rat serum, and was always greater in the lungs than the nose. Animals treated therapeutically with Sandoglobulin had a depressed primary antibody response to infection, but were completely resistant to reinfection with RSV. Histologic examination of pulmonary tissues from Sandoglobulin-treated animals showed no pathologic changes.

Current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses.

Vaccines against parainfluenza (PIV) and respiratory syncytial viruses (RSV) that are currently being developed include both live and subunit vaccines. Candidate live PIV vaccines that have been found to be attenuated and efficacious in rodents or primate models are (1) cold-adapted, temperature-sensitive mutants of PIV-type 3 that have been serially passaged at low temperature (20 degrees C) in simian kidney tissue culture; (2) protease-activation mutants (PIV-1-Sendai), which have mutations that decrease the cleavability of their F glycoprotein by host cell protease; (3) an animal virus, bovine PIV-3 virus, which is antigenically related to the human PIV-3 virus, and (4) vaccinia recombinant viruses bearing RSV or PIV-3 glycoproteins. Subunit RSV and PIV-3 viruses are being produced and evaluated as immunogens. A major concern with these vaccines is the possibility of disease potentiation following virus infection as occurred previously with formalin-inactivated measles and RSV vaccines. Studies indicate that PIV-3 and RSV glycoprotein vaccines are immunogenic and efficacious in animals but insufficient data exist to estimate their capacity to potentiate disease. However, since a cotton rat model is available to detect potentiated disease resulting from infection of cotton rats previously immunized with formalin-inactivated RSV vaccine, it is now possible to systematically evaluate new vaccines in experimental animals for disease potentiation before studies are initiated in humans. It is likely within the next several years that one or more of these PIV or RSV vaccines will be tested in humans for safety and immunogenicity.

Enhanced pulmonary pathology in cotton rats upon challenge after immunization with inactivated parainfluenza virus 3 vaccines.

Vaccine-induced potentiation was studied in cotton rats immunized with formalin-inactivated human parainfluenza type 3, ultraviolet light-inactivated virus, or infection with live virus. Immunized animals and unimmunized controls were later challenged by intranasal inoculation of live virus and evaluated for pulmonary pathology 4 days later. Animals immunized with either of the inactivated vaccines developed marked peribronchiolitis, perivasculitis, and an alveolar cellular infiltration much more severe than seen in animals infected previously, or in unmanipulated but challenged animals. Disease enhancement after immunization with killed virus is thus a characteristic of a member of each of three genera of the family, Paramyxoviridae, and is not restricted to immunization with formalin-inactivated virus.

An update on respiratory syncytial virus antiviral agents.

Respiratory syncytial virus (RSV), the most common cause of lower respiratory tract disease in infants and young children, is a ubiquitous respiratory pathogen, infecting or reinfecting much of the population every year and causing severe, sometimes fatal disease in high-risk populations of infants and adults, particularly in developing countries. Spurred by the medical and economic burdens of RSV disease and enticed by the economic potential of therapeutic drugs, particularly in the absence to date of a safe and effective RSV vaccine, scientists in many industrial, academic and government laboratories have developed a wide variety of candidate RSV antiviral agents. Most of these have been screened thus far only in cell culture, a few in animal models. Aside from ribavirin, however, none has proven effective in therapeutic clinical trials and even ribavirin usage has declined precipitously in recent years due to concerns over efficacy, safety, ease of use and cost. All of the antiviral compounds discussed in this review were evaluated primarily for their ability to reduce viral load, with little or no attention paid to the role of host inflammation in the pathogenesis of RSV disease. Recent research has highlighted the prominent role of inflammatory mediators and an increasing number of reports suggest that a therapeutic strategy that combines antiviral and anti-inflammatory components will be the most effective way of treating RSV disease.

Respiratory syncytial virus infections and intravenous gamma-globulins.

The respiratory syncytial virus (RSV) is a common cause of bronchiolitis and pneumonia in infants and young children. Throughout the world annual RSV epidemics result in numerous hospitalizations, substantial morbidity and some mortality. Until the recent introduction of ribavirin only supportive therapy has been available for treating these infections. The development of animal models of RSV infection and the observation that some lots of immunoglobulin prepared for intravenous administration contained substantial RSV-neutralization antibody titers, prompted a series of studies examining the safety and efficacy of immunoglobulin prepared for intravenous administration in the prophylaxis and treatment of RSV infections. This discussion will review our published, or soon to be published, studies on the use of Sandoglobulin for both immunoprophylaxis and immunotherapy of RSV infections in cotton rats. It will summarize studies utilizing both parenteral and topical (tracheal) Sandoglobulin therapy for RSV infections in owl monkeys. Finally the results of a small double blind trial of parenteral albumin or Sandoglobulin in the therapy of RSV bronchiolitis and/or pneumonia in hospitalized children will be reviewed. The data show that immunoprophylaxis and immunotherapy of RSV infections in laboratory animals was well-tolerated, was safe and induced highly significant reductions in RSV shedding from the lower respiratory tract. Further, immunotherapy of RSV infections in children was also well-tolerated, induced no short or long term evidence of toxicity or injury and caused significant improvements in oxygenation and reductions in RSV shed from the respiratory tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Palivizumab is highly effective in suppressing respiratory syncytial virus in an immunosuppressed animal model.

Respiratory syncytial virus (RSV) is widely recognized as a leading cause of pneumonia, with substantial mortality, in bone marrow transplant recipients. We tested the efficacy of a systemic monoclonal antibody (MAB) preparation possessing a high titer of anti-RSV neutralizing antibody, palivizumab (Synagis) for prophylaxis and therapy of RSV infection in cytoxan (CY) immunosuppressed cotton rats, a model in which the efficacy of a polyclonal anti-RSV product (Respigam) has been demonstrated. Both prophylaxis and therapy with this MAB were highly effective in reducing pulmonary viral replication. However, multiple sequential therapeutic doses of MAB were necessary to control rebound viral replication in continually suppressed animals.

Effectiveness of RSVIG prophylaxis and therapy of respiratory syncytial virus in an immunosuppressed animal model.

Respiratory syncytial virus (RSV) has emerged as a leading cause of pneumonia, with high mortality, in bone marrow transplant (BMT) recipients, as well as in other profoundly immunocompromised patients, such as myelosuppressed adults with leukemia. We tested the efficacy of immunoglobulin with high anti-RSV neutralizing antibody levels (RSVIG) for prophylaxis and therapy of RSV infection in cotton rats undergoing prolonged immunosuppression with cyclophosphamide. These animals experience persistent infection, a model which is similar to the disease seen in post-BMT humans. Both prophylaxis and therapy reduced pulmonary viral replication over 500-fold to nearly undetectable levels. In animals receiving continual immunosuppression, the use of multiple therapeutic doses of RSVIG was able to prevent rebound viral replication, though virus was not completely eliminated.

Inactivation of respiratory syncytial virus by generic hand dishwashing detergents and antibacterial hand soaps.

In an in-vitro test, generic liquid hand dishwashing detergents were as much as 100-fold more effective than proprietary antibacterial soaps in inactivating respiratory syncytial virus (RSV). The use of such detergents for hand washing during annual RSV epidemics, or the incorporation of their antiviral components into antibacterial soaps might be considered to limit nosocomial spread.

A cotton rat model of effectors of immunity to respiratory syncytial virus other than serum antibody.

A model for studying effectors of immunity to respiratory syncytial virus (RSV) was developed. Paris of inbred cotton rats (Sigmodon hispidus) were joined surgically using the technique of parabiosis. One week later, one animal of each pair was primed intranasally with a small volume of RSV suspension. Fourteen days after priming, both animals of each pair were bled for determination of serum neutralizing antibody titers, and challenged intranasally with a standard dose of RSV suspension. Single, unprimed cotton rats were challenged concomitantly and served as controls. Four days after challenge, all animals were sacrificed for virus titration of nasal tissues and lungs. Parabiosed cotton rats were surgically separated at varying intervals between priming and challenge (days 7, 9, 12, or 14 after priming) or were kept joined until sacrificed (day 18). Significant transfer of nasal and pulmonary immunity from primed to unprimed parabionts began 9 days after priming, gradually increasing through 18 days. Resistance to RSV challenge in spite of low levels of serum neutralizing antibody suggests that non-antibody immunologic mediators were responsible for the transferred immunity. Evidence is presented for three broad categories of RSV immunologic effectors: systemic, local with a transient systemic phase, and local without a systemic phase. These categories are now amenable to further study using the described model.

Legionella pneumophila: identification in tissue sections by a new immunoenzymatic procedure.

A modified glucose oxidase immunoenzyme technique was shown to be highly sensitive and specific for detection of serogroup 1 Legionella pneumophila in 4% formaldehyde solution-fixed, paraffin-embedded tissue sections. There was complete concordance between infection with L pneumophila and detection of the organisms in tissue sections by glucose oxidase immunoenzyme staining. The L pneumophila organisms stained blue-black and were found within phagocytic cells as well as in the extracellular space. A cloud of blue-black pigment, probably representing diffusable antigen, was present in the extracellular spaces in the area of L pneumophila localization. No false-positive or false-negative reactions were found. This technique requires no specialized equipment, may be applicable to retrospective diagnostic problems, and can be adapted to routine diagnostic practice.

Control of RSV-induced lung injury by alternatively activated macrophages is IL-4R alpha-, TLR4-, and IFN-beta-dependent.

Severe respiratory syncytial virus (RSV)-induced bronchiolitis has been associated with a mixed "Th1" and "Th2" cytokine storm. We hypothesized that differentiation of "alternatively activated" macrophages (AA-M phi) would mediate the resolution of RSV-induced lung injury. RSV induced interleukin (IL)-4 and IL-13 by murine lung and peritoneal macrophages, IL-4R alpha/STAT6-dependent AA-M phi differentiation, and significantly enhanced inflammation in the lungs of IL-4R alpha(-/-) mice. Adoptive transfer of wildtype macrophages to IL-4R alpha(-/-) mice restored RSV-inducible AA-M phi phenotype and diminished lung pathology. RSV-infected Toll-like receptor (TLR)4(-/-) and interferon (IFN)-beta(-/-) macrophages and mice also failed to express AA-M phi markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling is required for peroxisome proliferator-activated receptor gamma expression, a DNA-binding protein that induces AA-M phi genes, whereas IFN-beta regulates IL-4, IL-13, IL-4R alpha, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a cyclooxygenase-2 inhibitor increased expression of lung AA-M phi. These data suggest new treatment strategies for RSV that promote AA-M phi differentiation.

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.

Immunoprophylaxis of infections with respiratory syncytial virus: observations and hypothesis.

Infections with respiratory syncytial virus (RSV) may cause substantial morbidity and increased mortality in neonates and infants, especially those born before term and/or with pulmonary or cardiac disease. Humoral immunity was long thought to play only a small role in prevention, reduction of severity, or recovery from RSV infections of the lower respiratory tract. We recently performed a series of experiments in RSV-infected cotton rats, owl monkeys, and human infants to assess the role of humoral immunity in the prevention or recovery from RSV infections. This report summarizes studies utilizing intravenous IgG (IVIG) administered parenterally or topically (into the lower airway) for prophylaxis or treatment of RSV infections of the lower respiratory tract. The prophylactic administration (parenteral or topical) of IVIG to cotton rats or its therapeutic administration to RSV-infected cotton rats, owl monkeys, or humans significantly reduced RSV replication. The reductions in titers of virus correlated positively with titers of RSV-neutralizing antibody. Clinical trials are indicated to determine whether parenteral IVIG prophylaxis might prevent serious RSV infections in high-risk infants.

The molecular basis of adenovirus pathogenesis.

The pathology of type 5 (Ad5) pneumonia in Sigmodon hispidus cotton rats is closely similar to that in humans. Virus replicates in bronchiolar epithelial cells, but in situ hybridization shows early gene expression in macrophage/monocytes in alveoli and hilar lymph nodes. Only early gene expression is required to produce the pathology of which there is an "early" and a "late" phase. The early region 3 (E3), which does not function in viral replication, plays an important role in the natural history of at least the subgroup C adenoviruses (types 1, 2, 5, 6), which produce latent infections in host-infected lymphocytes: The 19-kDa glycoprotein markedly reduces the transport of the class I MHC to the surface of infected cells and, therefore, the attack of cytotoxic T cells, which could eliminate infected cells. When this gene is mutated, the late-phase inflammatory response to infection is markedly increased. The E3 14.7-kDa protein reduces the presence of polymorphonuclear leukocytes in the early-phase pathological inflammatory exudate. The E1B 55-kDa is essential to effect the late phase, and when its gene is mutated, the inflammation is greatly reduced although viral replication is not affected. Because only early genes are required to induce the complete pathogenesis of adenovirus infection in cotton rats, it is possible to produce the same pneumonia in lungs of mice in which only adenovirus early genes are expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Definition of cotton rat immunoglobulins: sigmodon species differ in expression of IgG isotypes and production of respiratory syncytial virus antibody.

The cotton rat (Sigmodon species) is the preferred animal model for experiments with a number of human pathogens, especially the respiratory viruses. The cotton rat is classified in the family Cricetidae (with hamsters and gerbils) and is a distant cousin of the common laboratory rat (Rattus) classified in the family Muridae (with the common laboratory mouse, Mus). Antibody reagents that are specific for cotton rat immunoglobulins have not been described. To enhance the usefulness of this model, four immunoglobulins in Sigmodon serum were characterized (IgG1, IgG2, IgA, IgM) and antisera specific for each immunoglobulin were made. Sera from three different species of Sigmodon were examined, S. hispidus (SH), S. arizoni (SA) and S. fulviventer (SF). Although IgA and IgM appeared similar in all three species, the IgG were expressed differently because normal serum levels of IgG2 were deficient in SH when compared with SA and SF and to other rodents. Similarly, IgG2 antibody response to purified protein antigen was deficient in SH although the IgG1 antibody response was superior to that in SF and SA. The three cotton rat species were infected with respiratory syncytial virus, and the kinetics of the antibody response was measured. Neutralizing antibody developed faster and to a higher titre in SH than in SA and SF. The enhanced immunoresponsiveness in SH may compensate for the IgG2 deficiency in SH and these changes appear to be relatively recent events in the evolution of this most populous species of Sigmodon.

Animal models of respiratory syncytial virus infection.

Over the past two decades, animal models of respiratory syncytial virus (RSV) infection have been developed using primates, cotton rats, mice, calves, guinea pigs, ferrets, and hamsters. Use of these models has shed light on the mechanisms of vaccine-enhanced disease seen in clinical trials of a formalin-inactivated RSV vaccine and has provided a means for testing efficacy and safety of candidate prophylactic and therapeutic strategies. The development of multiple animal models has coincided with the realization that RSV disease in humans is a multifaceted disease whose clinical manifestations and sequelae depend upon age, genetic makeup, immunologic status, and concurrent disease within subpopulations. There is no single human subpopulation in whom all forms of RSV disease manifest, nor is there a single animal model that duplicates all forms of RSV disease. The choice of an experimental model will be governed by the specific manifestation of disease to be studied.