Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells.

We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40 degreesC selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.

Protective responses in mice to vaccination with multiply administered cold-adapted influenza vaccine reassortants and wild-type viruses.

Protective responses to influenza vaccine reassortants derived from the cold-adapted (ca) donor strains A/Leningrad/134/17/57 and B/USSR/60/69 and wild-type epidemic viruses were studied in two strains of mice. Preliminary experiments revealed that, when mixtures of three viruses were inoculated intranasally to mice with 50 microliters containing 10(6) EID50 per 200 microliters (10(5.4) EID50 per mouse), interference between strains did not occur. However, interference with the growth of the influenza reassortant B/60/32/R took place if its concentration in the mixture was reduced to 10(5) (10(4.4) per mouse) or if it was inoculated at 10(6) EID50 (10(5.4) per mouse) in the presence of the influenza reassortant R/34 and two other influenza A epidemic strains; interference was unrelated to serological responses to infection with B/60/32/R. Despite evidence of interference, mice inoculated with the same mixtures in two identical doses, three weeks apart, were able to clear a challenge from each of seven homotypic and heterotypic influenza A and B strains. Heterotypic clearance of influenza A challenge viruses was greater following mixed infection, indicating that common determinants within the surface antigen glycoproteins contributed to immune responses which were broader than could be expected to be induced by parenteral vaccination.

Genetic stability of cold-adapted A/Leningrad/134/47/57 (H2N2) influenza virus: sequence analysis of live cold-adapted reassortant vaccine strains before and after replication in children.

We previously reported that the A/Leningrad/134/47/57 (H2N2) cold-adapted virus (A/Len/47) used in preparing reassortant live attenuated vaccines for children acquired 14 (11 coding) mutations in genes coding for proteins other than haemagglutinin and neuraminidase during cold-adaptation. Preservation of these mutations in genomes of viruses isolated from children on the second, fifth, or eighth day after vaccination was examined by sequence analysis. The sequence data demonstrated that all nine coding mutations selected for examination were conserved in the genomes of all 11 strains investigated, indicating that the mutations accompanying cold-adaptation and attenuation of the A/Len/47 master vaccine are highly stable.

Nuclear accumulation of MDM2 protein in well-differentiated papillary thyroid carcinomas.

Papillary thyroid cancer is the most common endocrine malignancy. Of all solid cancers presenting in adults, papillary thyroid cancer generally carries the best long-term prognosis. However, very little is understood about the molecular pathogenesis of this neoplasm. We recently hypothesized that increased nuclear levels of MDM2 protein might occur in well-differentiated papillary thyroid carcinomas (Gerasimov et al., Exp. Mol. Pathol. 62, 52-62, 1995). MDM2 is known to complex with and inactive the p53 tumor suppressor protein. Since p53 inactivation by gene mutation has an established role in the pathogenesis of undifferentiated (anaplastic) thyroid carcinoma, we reasoned that abrogation of p53 function by nuclear MDM2 protein accumulation might participate in the pathogenesis of certain well-differentiated thyroid cancers such as papillary cancer. In the present report we present the first direct evidence of MDM2 protein accumulation in the nuclei of papillary thyroid carcinoma cells in a subset of tumors. Using the IF-2 monoclonal antibody, which reacts specifically with human MDM2 protein, we studied 24 well-differentiated papillary thyroid carcinomas and 26 benign lesions (nodular goiters, adenomas, thyroiditis). Nuclear staining was quantitated using the CAS computerized image analysis system. We found positive nuclear MDM2 immunoreactivity in 8 (33%) of the carcinomas. In contrast, MDM2 staining was negative in all benign lesions (P = 0.001, two-tailed Fisher exact test). Normal thyroid tissue was also negative. These data suggest that nuclear accumulation of MDM2 protein might be implicated in the pathogenesis of a subset of papillary carcinomas. Further studies to investigate this possibility are warranted.

Nuclear p53 immunoreactivity in papillary thyroid cancers is associated with two established indicators of poor prognosis.

The tumor suppressor protein, p53, protects somatic cells against the accumulation of genomic alterations. Cells harboring mutant or inactivated wild-type p53 protein are at risk for the development of genomic instability. Nuclear accumulation of p53 protein is associated with the stepwise dedifferentiation of papillary carcinoma. We asked whether nuclear p53 accumulation is associated with two known indicators of poor prognosis in papillary carcinoma. We studied 55 consecutive papillary cancers (28 from Russia, and 27 from upstate New York). Nuclear p53 immunoreactivity was assessed using a monoclonal antibody, DO-1, on Formalin-fixed paraffin-embedded specimens. The DNA index was determined by computerized image analysis of Feulgen-stained sections. Nearly all cases were well differentiated and none were associated with distant metastases or extrathyroidal invasion. All primary lesions were less than 4 cm in diameter, and almost all patients were female. Nuclear p53 immunoreactivity was associated with a high-risk group characterized by two known indicators of poor prognosis: age > 50, aneuploid DNA content, or both. In the high-risk group (N = 24) 33% of cases displayed nuclear p53 positivity, compared with only 6% in a low-risk group (N = 31) which lacked both features (P = 0.015, two-tailed Fisher exact test). Nuclear accumulation of immunoreactive p53 protein is associated with two established indicators of poor prognosis in papillary carcinoma of the thyroid. This result is consistent with the idea that aberrations in p53 function are associated with the stepwise loss of differentiation in this cancer.

Nucleotide sequences of the neuraminidase genes of influenza A/Leningrad/134/57 (H2N2) virus and two of its live, attenuated, cold-adapted variants.

The nucleotide sequences of the neuraminidase (NA) genes of the A/Leningrad/134/57 (H2N2) wild-type (Len/wt) virus as well as two of its live attenuated, cold-adapted (ca) variants, A/Leningrad/134/17/57 (Len/17) and A/Leningrad/134/47/57 (Len/47), were determined. In comparison with Len/wt, one nucleotide change (C-225 to A) was found in the NA gene of Len/17. This change codes for a Thr-to-Asn substitution at position 69 of NA. The NA gene of the more attenuated Len/47 ca virus has one silent (T-814 to C) and two coding nucleotide substitutions, C-78 to T (Ala-20 to Val) and C-225 to A (Thr-69 to Asn). These sequence data were used to design a PCR-restriction technique to determine the origin of the NA gene in candidate live, attenuated vaccine reassortants made by reassorting these ca strains with current field viruses.

Preferential expression of the cell adhesion molecule CD44 in papillary thyroid carcinoma.

CD44 is a polymorphic family of immunologically related integral membrane glycoproteins associated with cell matrix adhesion, lymphocyte activation and targeting, and tumor growth and metastasis. We studied CD44 expression in 114 formalin-fixed paraffin-embedded thyroid tumors using the A3D8 anti-human CD44 monoclonal antibody. Sixty-five of 67 papillary carcinomas (97%) strongly expressed CD44 with an intense plasma membrane pattern. Thirty-seven of these cases originated from Albany, New York, and 30 cases from Russia. Immunoreactivity was also observed in 9 of 16 follicular adenomas (56%); 4 of 8 Hurthle cell neoplasms (50%); 5 of 15 medullary carcinomas (33%); and 3 of 8 follicular carcinomas (38%). These results show that among thyroid neoplasms, papillary carcinomas preferentially display the CD44 antigen (P < or = 0.001). Nonneoplastic follicular epithelium exhibited a low to moderate level of staining. To further characterize the CD44 isoform, we tested a subset of cases with the 2F10 anti-human CD44 variant 6 monoclonal antibody, which recognizes a CD44 variant exon (CD44v6) implicated in tumor metastasis. Eleven of 11 papillary carcinomas tested were 2F10 positive, and 1 of the follicular carcinomas was positive. These results suggest the hypothesis that deregulated CD44v6 expression on the plasma membrane of papillary carcinoma cells contributes to the ability of those cells to metastasize to regional lymph nodes and then to remain dormant for years. Our results suggest that human papillary thyroid cancer will be an interesting model in which to further study the role of CD44 isoforms, particularly those containing CD44v6, in tumor metastasis and lymphatic invasion.

Interference between cold-adapted (ca) influenza A and B vaccine reassortants or between ca reassortants and wild-type strains in eggs and mice.

Interference between pairs of cold-adapted (ca) influenza A and B vaccine reassortant viruses and between ca influenza B reassortant viruses and wild-type influenza strain A/PR/8/34 was studied in embryonated eggs and/or in the respiratory tract of three strains of mice. Interference in eggs between ca A and B reassortants occurred after 2 days but was masked by later cycles of growth. When doses of 10(6) EID50 of the reassortant viruses B/60/32/R and A/47/6/R were administered to outbred mice, interference with the growth of A/47/6/R occurred in the turbinates; interference with the growth of B/60/32/R did not occur. However, interference with the growth of B/60/32/R was noted in the lungs of Balb/c mice after co-infection with 10(6) EID50 of both A/PR/8/34 and B/60/32/R. Interference did not occur when the dose of B/60/32/R was increased to 10(7) EID50 per mouse. When CBA mice were co-infected with 10(6) EID50 of both B/60/32/R and the influenza A ca reassortant R/34, or 10(6) EID50 of B/60/32/R,R/34 and A/Leningrad/134/57, interference did not occur. Interference could not be detected in mice following dual infection from serum responses to individual viruses.

Sequence changes in the live attenuated, cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus.

Nucleotide sequences were determined for the RNA segments coding for proteins other than the hemagglutinin and neuraminidase of the A/Leningrad/134/57 (H2N2) wild-type (A/Len/wt) virus and its two cold-adapted (ca) and attenuated variants, A/Leningrad/134/17/57 (A/Len/17/ca) and A/Leningrad/134/47/57 (A/Len/47/ca) that are used in the U.S.S.R. in the preparation of reassortant live attenuated vaccines. Ten nucleotide differences were detected between the sequences of the A/Len/wt and A/Len/17/ca viruses; of these, eight were deduced to encode amino acid (aa) substitutions. One aa substitution each was predicted for the PB2, M1, M2, and NS2 proteins, whereas two aa substitutions each were predicted for the PB1, and PA proteins of the A/Len/17/ca virus. Four additional nucleotide changes were found in the genome of the A/Len/47/ca virus; three of these were detected to code for one additional aa substitution each for the PB2, PB1, and NP proteins.

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. This virus exhibited a moderate level of attenuation in the animals, in contrast to the B/Ann Arbor/1/66 cold-adapted virus, which was fully attenuated. Reassortant viruses deriving the surface antigens of the contemporary wild type virus B/Ann Arbor/1/86 and most or all of their other genes, from one or other cold-adapted parent, were virtually indistinguishable from their respective cold-adapted parents. The B/Leningrad/14/55 reassortant was slightly more attenuated than its cold-adapted parent in ferrets. These studies extend knowledge of the properties of viruses used to prepare experimental live influenza B human vaccines.

Class-specific antibody responses in school children vaccinated with an A/Brazil/11/78 (H1N1)-like recombinant influenza virus prepared from the A/Leningrad/134/57 paediatric cold-adapted donor strain.

Forty-three school children from 8 to 11 years old were vaccinated intranasally with two doses of a paediatric attenuated influenza vaccine developed by reassortment between cold-adapted A/Leningrad/134/57(H2N2) and an A/Brazil/11/78(H1N1)-like strain. Two vaccine doses were administered 1 month apart in a randomized, blind, placebo-controlled study. Although the first vaccine dose had a low infectivity titre, overall 65% of children who received two doses of vaccine showed serological evidence of infection by HI tests. Serum IgA antibody responses against the vaccine strain were detected in nearly 50% of the vaccines and serum IgG antibody responses were detected in approximately equal to 40% by an enzyme immunoassay.

Study of live recombinant cold-adapted influenza bivalent vaccine of type A for use in children: an epidemiological control trial.

Live cold-adapted recombinant bivalent vaccine of influenza type A was studied in a controlled field trial in 1982-1983 among nearly 30,000 children 3-15 years old. The bivalent vaccine consisted of recombinants 47/25/1 (H1N1) and 47/7/2 (H3N2) of wild-type viruses A/Brazil/11/78 (H1N1) and A/Bangkok/1/79 (H3N2) with cold-adapted donor A/Leningrad/134/47/57 (H2N2). The recombinants which received mutant nonglycoprotein genes from cold-adapted donor did not suppress each other after simultaneous inoculation of children and stimulated antibody response to both strains. The bivalent vaccine was completely attenuated for children. It caused less than 1% transient febrile reactions during five days after the first vaccination, including double seronegative individuals with low antibody titres to both vaccinal strains. The cold-adapted bivalent vaccine tested proved to be safe for children according to the analysis of morbidity studies among vaccines and a control group performed during the five days and the following six months after the first immunization. There is a similar distribution of non-influenza illnesses and a statistically significant decrease in influenza-like diseases among vaccines compared to the control group. In the four months after the immunization programme was completed, epidemics of influenza A H1N1 and H3N2 occurred. The incidence of influenza-like diseases was approximately 50% less in the vaccinated than in the control groups. This is the first evidence of safety and protective efficacy of recombinant live influenza vaccine for children 3-15 years of age.

Comparative studies of A/Leningrad/134/57 wild-type and 47-times passaged cold-adapted mutant influenza viruses: oligonucleotide mapping and RNA-RNA hybridization studies.

Direct biochemical evidence for the existence of mutations in five of the RNA segments of the A/Leningrad/134/57 cold-adapted 47th passage mutant as compared with its wild-type progenitor has been obtained using two techniques. T1 oligonucleotide mapping of total viral RNA as well as of individual RNA segments revealed changes in RNAs 4, 5 and 6. Analysis of S1 nuclease-treated RNA-RNA hybrids on polyacrylamide gels revealed changes in at least one of the polymerase genes as well as in RNAs 4, 5, 6 and 7. These findings provide a direct demonstration for the existence of multiple mutations in the cold-adapted mutant vaccine strain.

Recombinant cold-adapted attenuated influenza A vaccines for use in children: molecular genetic analysis of the cold-adapted donor and recombinants.

A previously described cold-adapted attenuated virus, A/Leningrad/134/17/57 (H2N2), was further modified by 30 additional passages in chicken embryos at 25 degrees C. This virus had a distinct temperature-sensitive (ts) phenotype, grew well in chicken embryos at 25 degrees C, and failed to recombine with reference ts mutants of fowl plague virus containing ts lesions in five genes coding for non-glycosylated proteins (genes 1, 2, 5, 7, and 8). Recombination of A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79 (H1N1) and A/Bangkok/1/79(H3N2) yielded ts recombinants 47/25/1(H1N1) and 47/7/2 (H3N2). These recombinants inherited their ts phenotype and ability to reproduce in chicken embryos at 25 degrees C from the cold-adapted parent. Analysis of the genome composition of the recombinants obtained by recombination of the cold-adapted donor with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) showed that recombinants 47/25/1(H1N1) and 47/7/2 (H3N2) inherited five and six genes, respectively, from the cold-adapted parent, and hemagglutinin and neuraminidase genes from the wild-type strains.

Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees.

Reactogenicity and antigenic activity of recombinants obtained by crossing cold-adapted donor of attenuation A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) were studied. The recombinants were areactogenic when administered as an intranasal spray to children aged 3 to 15, including those who lacked or had only low titers of pre-existing anti-hemagglutinin and anti-neuraminidase antibody in their blood. After two administrations of vaccines at a 3-week interval, both strains induced antibody in 75 to 95% of the children. On coinfection of chicken embryos with both recombinants, only weak interference was observed. Administration to children of the bivalent vaccine containing H1N1 and H3N2 recombinants induced efficient production of antibody to H1 and H3 hemagglutinins and N1 and N2 neuraminidases without adverse reactions. The recombinants studied were genetically stable as judged by retention of the temperature-sensitive phenotypes and a lack of reversion of the genes carrying temperature-sensitive mutations in all of the reisolates from vaccinated children.

Location of ts defects in the genome of cold-adapted recombinant influenza A virus vaccine strains.

The ts phenotype and location of ts mutations were studied in the genome of parent viruses and those obtained by recombination of cold-adapted strains A/Leningrad/134/17/57 or A/Leningrad/134/47/57 with epidemic H1N1 and H3N2 influenza A virus strains. The epidemic H1N1 and H3N2 strains under study possessed a ts phenotype and contained ts mutations in one or two genes. The ts phenotype was lost following three clonings at 40 degrees C, suggesting that influenza virus strains isolated from humans may be heterogeneous and contain virions either carrying or not carrying the ts mutations in their genomes. Two cold-adapted strains possessing a distinct ts phenotype contained ts mutations in three (A/Leningrad/134/17/57 virus after 17 passages at 25 degrees C) or in five (A/Leningrad/134/47/57 variant after 30 additional passages at 25 degrees C) genes coding for non-glycosylated proteins. When compared with cold-adapted donor strains, the recombinants had either the same set or additional ts mutations. However, no ts mutation was detected in a gene which had been inherited from the donor strain. It is suggested that, in addition to the analysis of the genome composition, in cold-adapted recombinant influenza virus strains recommended as vaccine candidates it is necessary to control the number of genes containing ts mutations.

Peculiarities of obtaining and characterization of a cold-adapted A/PR/8/34 influenza virus variant.

The dynamics of alterations in biological properties of A/PR/8/34 virus strain was studied in the process of prolonged adaptation to the growth in chick embryos (CE) at a lowered (25 degrees C) cultivation temperature. The variant selected after 59 passages and subsequent cloning at the above temperature retained the high reproductive capacity in CE at optimal temperature (34 degrees C) - a characteristic of the original strain. Unlike to the latter, it showed a distinctly reduced ability to reproduce at 40 degrees C and a lower level of pathogenicity for white mice and CE. Analysis of genes of the cloned cold-adapted A/PR/8/34 strain detected 5 ts mutations in genes 1, 3, 5, 6 and 7 coding for P3, P2, NP, NA and M proteins, respectively.

Attenuated influenza B virus recombinants obtained by crossing of B/England/2608/76 virus with a cold-adapted B/Leningrad/14/17/55 strain.

Crossing of an attenuated influenza B virus strain (B/Leningrad/14/17/55) passaged at a low temperature with a virulent influenza B virus strain (B/England/2608/76) yielded recombinants similar in the antigenic specificity of their haemagglutinin (HA) and neuraminidase (NA) to B/England/2608/76 strain, but possessing an RCT37.5 marker alike to the attenuated donor. Analysis of the genome composition of 2 recombinants has shown that they inherited genes coding for P (1, 2, 3) and M (7) proteins from the attenuated parent, but genes coding for HA (4), NA (6), NP (5) and NS (8) proteins from the virulent parent. All recombinants proved to be areactogenic for adult volunteers with no pre-existing antibody to the corresponding HA (less than or equal to 8); however, they had a reduced immunogenicity as compared to parent viruses.

Analysis of genome composition and reactogenicity of recombinants of cold-adapted and virulent virus strains.

Temperature-sensitive (ts) mutations occurring in three genes, 1, 2, and 7, and 1, 5 and 7 were found in two cold-adapted (ts) attenuated influenza virus strains A/Leningrad/9/37/46 (H0N1) and A/Leningrad/134/17/57 (H2N2) respectively. The recombinants, obtained by crossing these cold-adapted strains with virulent influenza virus strains, had different genome structures and inherited from one to six genes from the cold-adapted parents. Tests of reactogenicity of recombinants in volunteers showed all the recombinants to be non-reactogenic irrespective of the number of genes inherited from the cold-adapted parent.

Electrophoretic migration rate differences of polypeptides of human influenza A viruses: partial analysis of the genome of influenza vaccine recombinant viruses.

Electrophoretic migration rate differences were detected in high resolution SDS polyacrylamide gels for nucleoprotein (NP), matrix protein (M), non structural protein (NS1), haemagglutinin (HA) annd, less regularly, for the polymerase polypeptides P1, P2 and P3 induced by different influenza A viruses. The technique allowed parental assignation of the corresponding genes in certain recombinant viruses including A/PR/8/34 (H0N1)--A/HK/117/77 (H1N1), A/Okuda/57 (H2N2)--A/HK/119/77 (H1N1) and A/Leningrad/76 (H3N2)--A/Leningrad/46 (H0N1) recombinants, thus considerably extending the technique which had been applied previously to A/PR/8/34--A/HK/68 (H3N2) only. Agreement in gene assignment between three recombinants of the former group and 11 of 17 recombinants in the A/Okuda/57--A/HK/119/77 group was noted when the data obtained using the polypeptide method was correlated with a direct genetic analysis by others using RNA:RNA hybridisation techniques. The polypeptide method appears to have wide application for the initial rapid analysis of influenza A virus recombinants obtained using parents of different influenza subtypes although complete analysis of the total genome requires the use of RNA hybridisation techniques. Two additional virus induced proteins are described, a phosphorylated form of NS 1 and a non structual polypeptide with a molecular weight of 16K daltons.