Reverse genetics studies of attenuation of the ca A/AA/6/60 influenza virus: the role of the matrix gene.

The matrix (M) gene of influenza virus has been implicated in the attenuation phenotype of the cold adapted (ca) A/AA/6/60 vaccine. Previous studies have evaluated the ca M from A/AA/6/60 in different wild type (wt) virus backgrounds with varying results. In experiments described here, the ca M gene was transfected into the background of its own wt A/AA/6/60 to eliminate the possibility of confounding gene constellation effects.

RANTES (CCL5) production during primary respiratory syncytial virus infection exacerbates airway disease.

Respiratory syncytial virus (RSV) is a respiratory pathogen that causes significant morbidity in infants and young children. The importance of chemokines during RSV infection for respiratory symptoms has not been fully elucidated. The current study examined the effect of RANTES (CCL5) on airway pathophysiology after RSV infection.

Role of interleukin-12 and stat-4 in the regulation of airway inflammation and hyperreactivity in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a respiratory pathogen that can cause significant morbidity in infants and young children. Interestingly, the majority of children who acquire a RSV infection do not exhibit severe symptoms. Development of a Th1 response has been associated with resolution of symptoms in viral infections and may explain mild RSV illness.

Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms.

The development of severe childhood asthma may be influenced by several factors including environmental and infectious stimuli. The causal relationship between infectious viral responses, such as respiratory syncytial virus (RSV), and severe asthma during early childhood is unclear. In these studies, the ability for an initial RSV infection to exacerbate and promote a more severe asthmatic-type response was investigated by combining established murine models of disease.

IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent.

Airway damage and hyperreactivity induced during respiratory syncytial virus (RSV) infection can have a prolonged effect in infants and young children. These infections can alter the long-term function of the lung and may lead to severe asthma-like responses. In these studies, the role of IL-13 in inducing and maintaining a prolonged airway hyperreactivity response was examined using a mouse model of primary RSV infection.

The development of live attenuated cold-adapted influenza virus vaccine for humans.

A procedure to attenuate live influenza virus of type A and type B was developed using adaptation of the virus to grow at 25 degrees C (cold adaptation; ca). Through a series of stepwise passages, two stable mutants were obtained and designated as 'Master' strains, one for type A influenza virus (A/Ann Arbor/6/60-H2N2) and one for type B influenza virus (B/Ann Arbor/1/66). These mutants were used in genetic reassortment using either the classical method or more recently described reverse genetics to update the relevant surface antigens of the circulating strains of influenza virus.

Interleukin-12 cDNA skin transfection potentiates human papillomavirus E6 DNA vaccine-induced antitumor immune response.

Human papillomaviruses are associated with >90% of all cases of uterine cervical tumors. The E6 and E7 oncoproteins of human papillomavirus are potentially ideal targets of immune therapy for cervical cancer, because their expression is necessary for cellular transformation. Although both E6 and E7 proteins contain numerous predicted cytotoxic T lymphocyte (CTL) epitopes that are capable of binding to human leukocyte antigens, the majority of earlier in vivo tumor rejection studies have focused on E7.

Immunological properties of plaque purified strains of live attenuated respiratory syncytial virus (RSV) for human vaccine.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in infants, young children, and the elderly. Efforts to develop satisfactory live or inactivated vaccines have not yet been proven successful. Our research focuses on the development of four purified live attenuated RSV sub-type A human vaccine clones.

Creation of amantadine resistant clones of influenza type A virus using a new transfection procedure.

M2, the spliced segment of the matrix (M) gene of influenza A virus, is an integral membrane protein which functions as an ion channel both when the virus is in the host endosome and during protein processing in the trans-Golgi network. Amantadine inhibits replication of influenza A virus by blocking the activity of this ion channel. Reverse genetics were used to generate amantadine resistant virus mutants by introducing mutations into the M gene of cold adapted (ca) A/AA/6/60, an amantadine sensitive virus.

The cell receptor level is reduced during persistent infection with influenza C virus.

Persistent influenza C virus infection of MDCK cells perpetuates the viral genome in a cell-associated form. Typically, virus production remains at a low level over extended periods, in the absence of lytic effects of replication. In this study, we demonstrate that persistently infected cells are very restricted in permissiveness for superinfection.

Sequence comparisons of A/AA/6/60 influenza viruses: mutations which may contribute to attenuation.

Influenza virus infection is a worldwide public health threat. Cold-adaptation was used to develop a vaccine line (ca A/AA/6/60 H2N2) which promised to reduce the morbidity and mortality associated with influenza and to serve as a model for other live virus vaccines. This study establishes that two distinct lines of wt A/AA/6/60 viruses exist with different phenotypic and genotypic characteristics.

Prevention of influenza by the intranasal administration of cold-recombinant, live-attenuated influenza virus vaccine: importance of interferon-gamma production and local IgA response.

To clarify which immunological factors were more effective in preventing influenza virus infection, we measured immunological parameters induced by vaccination and infection in vivo and in vitro. Healthy adult subjects (n = 128) were divided into vaccinated (n = 85) and untreated (n = 43) groups. Eighty-five were vaccinated intranasally with a trivalent cold-adapted recombinant influenza virus vaccine containing type A (H1N1 and H3N2) and B viruses.

Cytokines and the acute phase response to influenza virus in mice.

This study characterized selected aspects of the acute phase response after intranasal inoculation of mice with two doses of mouse-adapted influenza virus differing in lethality. Mice given 140 plaque-forming units (PFU) of virus (58% survival) gradually decreased food and water intake to nearly zero over 6 days; survivors then slowly increased intakes. Declines in these behaviors were parallel to decreases in body temperature and general locomotor activity and were associated with elevated activities of interleukin-6 (IL-6), tumor necrosis factor-alpha, and interferons in lung lavage fluid.

Molecular and biological changes in the cold-adapted "master strain" A/AA/6/60 (H2N2) influenza virus.

The live cold-adapted (ca) A/AA/6/60 influenza vaccine is being commercially developed for worldwide use in children and is being used as a model for other live vaccines. Although it has been proven safe and immunogenic, the molecular basis of cold adaptation has never been determined. To identify sequence changes responsible for cold adaptation, we have compared the sequence of the master ca vaccine strain to its progenitor wild-type virus, wt A/AA/6/60 E2 (wt2).

A persistent infection in MDCK cells by an influenza type B virus.

A persistent infection in Madin Darby Canine Kidney (MDCK) cells by an influenza B virus (B/Tecumseh/63/80) has been established and characterized. Virus recovered from the persistent state titrated lower in relation to the parental wild-type (wt) that initiated the infection as measured by hemagglutination and egg and tissue culture infectious dose, suggesting that the virus is a less cytopathic variant of the original wt virus. The persistent virus (pv) has decreased cytopathology for both MDCK and primary chick kidney (PCK) cell lines, and exhibits different RNA and protein electrophoretic migrations.

Trivalent attenuated cold-adapted influenza virus vaccine: reduced viral shedding and serum antibody responses in susceptible adults.

Trivalent cold-adapted recombinant (CR) influenza virus vaccines containing types A (H1N1 and H3N2) and B viruses were evaluated in two double-blind, placebo-controlled trials. Susceptible adults were randomly assigned to receive the following vaccines by intranasal drops 1 month apart: two doses of trivalent vaccine, bivalent CR influenza A (Bi A) vaccine followed by monovalent B (Mono B) vaccine or vice versa, or two doses of placebo. All vaccines were well tolerated.

Evaluation of a cold-adapted influenza B/Texas/84 reassortant virus (CRB-87) vaccine in young children.

A cold-adapted (ca) influenza B reassortant virus vaccine that contained the six internal RNA segments from influenza B/Ann Arbor/1/66 ca virus and the neuraminidase and hemagglutinin genes from wild-type influenza B/Texas/1/84 virus was evaluated in children ranging in age from 8 months to 14 years. The children were vaccinated intranasally with doses ranging from 10(3.2) to 10(6.

Immunization of infants and young children with live attenuated trivalent cold-recombinant influenza A H1N1, H3N2, and B vaccine.

Seventeen triply seronegative infants and young children, and 15 infants and young children seropositive to all three influenza virus strains were vaccinated intranasally with 10(5) TCID50 of each of three (H1N1, H3N2, and B) live attenuated, cold-adapted influenza vaccine strains. Seventeen controls were given placebo. Vaccination with trivalent influenza vaccine was not associated with adverse reactions in triply seronegative or seropositive children.

New aspects of influenza viruses.

Influenza virus infections continue to cause substantial morbidity and mortality with a worldwide social and economic impact. The past five years have seen dramatic advances in our understanding of viral replication, evolution, and antigenic variation. Genetic analyses have clarified relationships between human and animal influenza virus strains, demonstrating the potential for the appearance of new pandemic reassortants as hemagglutinin and neuraminidase genes are exchanged in an intermediate host.

Dominant-negative mutants as antiviral agents: simultaneous infection with the cold-adapted live-virus vaccine for influenza A protects ferrets from disease produced by wild-type influenza A.

The attenuated cold-adapted strain of influenza A virus that is a candidate live-virus vaccine suppressed clinical disease in ferrets when given simultaneously with a virulent epidemic strain of influenza A virus. The cold-adapted virus effectively prevented disease, even when the epidemic strain was of a different subtype than the attenuated virus. In this case, ferrets given a mixed inoculum produced antibody to both subtypes in the absence of clinical disease, indicating that both viruses are replicating in the respiratory tract.

Detection of toxic viral-associated double-stranded RNA (dsRNA) in influenza-infected lung.

While many of the molecular events in viral replication are well studied, the molecular mechanisms by which viral infections trigger such constitutional symptoms as fever and 'malaise' are unknown. The hypothesis that these viral constitutional symptoms can be triggered by the toxic action of dsRNA associated with viral replication was investigated. Total lung RNA from mice acutely infected with PR8 influenza virus, but not from sham-infected mice, was shown to induce fever and altered sleep (excess slow-wave sleep, enhanced amplitudes of electroencephalographic slow waves, and reduced rapid eye movement sleep) when injected into the rabbit brain.

Evaluation of the infectivity, immunogenicity, and efficacy of live cold-adapted influenza B/Ann Arbor/1/86 reassortant virus vaccine in adult volunteers.

A cold-adapted (ca) influenza B reassortant that derived two genes encoding the hemagglutinin and neuraminidase from influenza B/Ann Arbor/1/86 wild-type virus and six internal RNA segments from ca influenza B/Ann Arbor/1/66 virus was evaluated in 66 adult volunteers having a serum hemagglutination inhibition antibody titer less than or equal to 1:8. The ca reassortant was attenuated and elicited the production of systemic and local antibodies; the 50% human infectious dose was 10(6.4) TCID50.

Differential interaction of virulent and attenuated influenza virus strains with ferret alveolar macrophages: possible role in pathogenicity.

The ferret provides a unique model for the study of human influenza. The interaction between alveolar macrophages and virus strains with different levels of virulence was examined in vitro. The greater virulence of wild-type A strains over type B and C viruses was reflected in the higher production of infectious virus progeny and subsequent cytopathology, even though the expression of viral antigens was equivalent for all strains tested.

Laboratory properties of cold-adapted influenza B live vaccine strains developed in the US and USSR, and their B/Ann Arbor/1/86 cold-adapted reassortant vaccine candidates.

The adaptation of two influenza B strains (B/Leningrad/14/55 and B/Ann Arbor/1/66) to replication at 25 degrees C is described. Comparison of the two viruses indicates that both also exhibit temperature sensitive phenotypes, although that of the virus B/Leningrad/14/55 is less pronounced. When inoculated into ferrets both viruses replicate well in the trachea, but only the B/Leningrad/14/55 cold-adapted virus replicates in the lungs.