Hunein F. Maassab

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. Using this model, there was evidence of significant airway epithelial cell damage and sloughing, along with mucus production. The airway hyperreactivity response was significantly increased by 8 days postinfection, peaked during days 10–12, and began to resolve by day 14. When the local production of Th1- and Th2-associated cytokines was examined, there was a significant increase, primarily in IL-13, as the viral response progressed. Treatment of RSV-infected mice with anti-IL-13 substantially inhibited airway hyperreactivity. Anti-IL-4 treatment had no effect on the RSV-induced responses. Interestingly, when IL-13 was neutralized, an early increase in IL-12 production was observed within the lungs, as was a significantly lower level of viral Ags, suggesting that IL-13 may be regulating an important antiviral pathway. The examination of RSV-induced airway hyperreactivity in STAT6−/− mice demonstrated a significant attenuation of the response, similar to the anti-IL-13 treatment. In addition, STAT6−/− mice had a significant alteration of mucus-producing cells in the airway. Altogether, these studies suggest that a primary factor leading to chronic RSV-induced airway dysfunction may be the inappropriate production of IL-13.

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°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. The derivation is based on the concept of 6/2 where 6 signifies the six internal genes of the master strain and 2 refers to the two genes coding for the two surface glycoproteins HA and NA of the circulating influenza virus. The advantages of this vaccine were demonstrated to be (1) proper level of attentuation, (2) non‐transmissibility, (3) genetic stability, (4) presence of the ca and ts markers and (5) immunogenicity involving both local and the cell‐mediated immune responses. The clinical trials in infants, children, adults and elderly have provided the necessary data for eventual licensing of this vaccine. The ease of administration (intranasal) safety and high efficacy make this vaccine suitable to prevent influenza virus infection in all age groups. Copyright

Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2).

Nucleotide sequences have been obtained for RNA segments encoding the PB2, PB1, PA, NP, M1, M2, NS1, and NS2 proteins of the influenza A/Ann Arbor/6/60 (H2N2) wild-type (wt) virus and its cold-adapted (ca) derivative that has been used for preparing investigational live attenuated vaccines. Twenty-four nucleotide differences between the ca and wt viruses were detected, of which 11 were deduced to code for amino acid substitutions in the ca virus proteins. One amino acid substitution each was predicted for the PB2, M2, and NS1 proteins. Two amino acid substitutions were predicted for the NP and the PA proteins. Four substitutions were predicted for the PB1 protein. The biological significance of mutations in the PB2, PB1, PA, and M2 genes of the ca virus is suggested by currently available genetic data, a comparison with other available influenza gene sequences, and the nature of the predicted amino acid changes. In addition, the sequence data confirm the close evolutionary relationship between the genomes of influenza A (H2N2) and influenza A (H3N2) viruses.

Comparative studies of wild-type and cold-mutant (temperature-sensitive) influenza viruses: Nonrandom reassortment of genes during preparation of live virus vaccine candidates by recombination at 25° between recent H3N2 and H1N1 epidemic strains and cold-adapted A/Ann Arbor/6/60

Abstract Genetic compositions of 35 recombinant cold-adapted influenza A(H3N2 and H1N1) candidate live attenuated vaccine strains have been determined. The viruses, which had been obtained by recombination (reassortment) at 25° between contemporary epidemic wild-type strains and cold-adapted A/Ann Arbor/6/60(H2N2), followed by selection for growth at 25° of virus with wild-type HA and NA, have a highly restricted genetic composition. Eighteen of the thirty-five recombinants had RNAs coding for the three polymerase (P) proteins, NP, M, and NS, from the cold-adapted mutant A/Ann Arbor/6/60 had only the HA and NA of the wild-type strains. Only 4 out of 64 theoretically possible combinations of genes coding for nonglycoprotein viral products were detected. The restricted genetic composition of cold-adapted recombinants produced at 25° supports the evaluation of this method of preparing live vaccine strains to determine whether recombinants with constant gene composition have predictable levels of attenuation for man.

Prevention of influenza by the intranasal administration of cold-recombinant, live-attenuated influenza virus vaccine : importance of interferon-γ 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. Subjects were mostly seropositive before vaccination. In 29 (80.6%) of the 36 examinees showing a prevaccination HI antibody titre of less than 1:128, the titre increased more than four times after vaccination. On the other hand, an increase of more than four times was found in four (8.2%) of the 49 individuals who had shown a prevaccination titre of more than 1:128. The IgA antibody was negligibly detected in the nasal wash specimens before vaccination, and was induced by vaccination in some cases. Lymphocyte proliferation and interleukin 2 (IL-2) production in cultured lymphocytes of the same subjects stimulated by H1N1 virus in vitro were correlated with the HI antibody titre. However, the interferon gamma (IFN-gamma) production was low before vaccination, regardless of the HI antibody titre, and showed a significant increase after vaccination. It was suggested that local IgA response and IFN-gamma production play important roles in the prevention of influenza. Since there was the outbreak of influenza A (H1N1) in Kochi Prefecture after completion of blood samples 6-8 weeks after the second vaccination, we examined the above hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Rubella Antibodies in Human Serum: Detection by the Indirect Fluorescent-Antibody Technique

Antibodies for rubella virus were detected in human serum and titrated by the indirect method of immunofluorescence; a chronically infected, continuous line of monkey kidney cells was used as antigen. Positive reactions were obtained with serums from convalescent patients or persons who had been exposed to the virus while serums from patients in the acute stage of the disease and those from unexposed individuals were negative

Sequence comparison of wild-type and cold-adapted B/Ann Arbor/1/66 influenza virus genes

Consensus sequences for both wt and ca B/Ann Arbor/1/66 viral PB2, PB1, PA, NP, M, and NS genes were directly determined from vRNA using a combination of chemical and chain-termination sequencing methods. There were 105 sites of difference between the wt and ca sets of these six RNA genes. The differences resulted in 26 amino acid substitutions distributed over the six proteins. The sequence changes were compared to the sequences of other known influenza type B wt viruses to pinpoint those changes that were unique to the ca B/ann Arbor/1/66 virus. Of the 26 amino acid differences, only 11 were unique to the cold-adapted virus. These unique sites were distributed among five of the six genes. The NS protein had no amino acid substitutions. The sequence changes are discussed in terms of their probable mode of origin and selection, and in terms of their importance to the cold-adapted, temperature-sensitive, and attenuation phenotypes of ca B/AA/1/66 virus. The sequence and organization of the PB2 gene and predicted protein are also given. The PB2 gene was 2396 nucleotides long, and it encoded a predicted protein of 770 amino acids with a molecular weight of 88,035 Da for the wt virus and 88,072 Da for the ca virus. Both proteins were predominantly hydrophilic, and each had an overall charge of +24.5 at pH 7.0.

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. The two virus lines have the same parent but different passage histories. The first line is both temperature sensitive (ts) and attenuated in ferrets and the second line (after multiple passages in chick kidney cells, eggs and mice) is non-ts and virulent in ferrets. Both lines of viruses have been further differentiated by sequence analysis. We have identified point mutations common to all virulent viruses but absent from the attenuated viruses. This was accomplished by comparing the nucleotide sequences of the six internal genes in three different attenuated passages of A/AA/6/60 with those of five different virulent passages of the same virus. The corresponding nucleotides of the attenuated viruses, therefore, represent candidate attenuating lesions: 6 in the basic polymerase genes (5 in PB1, 1 in PB2), 2 in the acidic polymerase gene (PA), 1 in the matrix (M) gene, 2 in the non-structural (NS) gene, and none in the nucleoprotein (NP) gene. Two of the 5 attenuating lesions in PB1 are silent; 1/2 in PA is silent; and 1/2 in NS is silent. Further changes which might be identified by comparing nucleotide and amino acid sequences of the A/AA/6/60 viruses with those of other influenza viruses may also contribute to the attenuation of the ca virus. Our study identifies nucleotides which more precisely define virulence for this virus and suggests that growth of the virus at low temperature may have preserved a non-virulent virus population rather than attenuating a virulent one.

Rubella Virus: Inhibition in vitro by Amantadine Hydrochloride

Amantadine (or 1-adamantanamine) hydrochloride, a compound reported to be active against influenza viruses and Sendai virus, inhibited the growth of rubella virus in tissue culture. The antiviral activity appears at an early phase of the infection and is not due to direct inactivation of the virus.

A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype

Previously a spontaneous 36 nucleotide deletion in the coding region of NS1 was detected in the NS gene of a reassortant virus (CR43-3) recovered from a dual infection by the influenza A/Ann Arbor/6/60 cold-adapted (ca) mutant and wild-type (wt) influenza A/Alaska/6/77 (H3N2). The hemagglutinin, neuraminidase and NS genes were derived from the wild type virus parent while the other 5 genes were derived from the ca parent. The CR43-3 reassortant virus exhibited: (i) a host range (hr) phenotype, i.e. the reassortant replicated efficiently in avian cells in tissue culture but failed to grow in mammalian (MDCK) cell culture and (ii) an attenuation (att) phenotype, i.e., the reassortant was restricted in replication in the upper and lower respiratory tract of ferrets and hamsters. Since the CR43-3 reassortant possessed 5 genes from the ca parent which are each known to contain one or more mutations, it was not possible to assign the hr and att phenotypes solely to the NS deletion mutant gene. In order to determine the phenotype(s) specified solely by the mutant NS gene, it was transferred into a reassortant virus (143-1) which derived its seven other genes from the homologous wild type A/Alaska/6/77 virus. The deletion mutant NS gene specified only a partial hr phenotype manifested by a reduction in plaque size in MDCK tissue, but not a reduction in plaque number. Thus, the complete hr manifested by the CR43-3 parent virus is specified by the mutant NS1 gene acting in concert with one or more genes derived from the ca virus. The clone 143-1 virus exhibited the ts phenotype and was restricted in plaque formation at 37 degrees C in MDCK cells, a level of temperature sensitivity previously shown with other ts mutants to correlate with significant restriction of viral replication in the lower respiratory tract of hamsters. However, the clone 143-1 virus grew almost as well as the wt virus in the upper and lower respiratory tracts of hamsters and chimpanzees and thus did not possess the att phenotype. The finding that the ts phenotype was not manifest in vivo in animals with a 37 degrees C core temperature indicates that the mutated NS1 gene specifies a host dependent ts phenotype with replication restricted in vitro (MDCK tissue culture) at 37 degrees C but not in vivo in the lungs of hamsters and chimpanzees. ts+ virus was readily recovered from infected hamsters and chimpanzees indicating that the ts phenotype specified by the 36-base deletion was not stable following replication in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)