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Interfering activity (IA) of the epidemic and attenuated influenza virus strains of different ts-phenotype was studied. IA was assessed by inhibition of vaccinia virus cytopathic effect in chick embryo chorioallantoic membrane (CAM) cells. Direct relationship was established between the temperature sensitivity (ts) index and IA of these viruses. The relationship between IA and multiplicity of infection was strain-specific.

The conditions of obtaining thermosensitive recombinants of the virulent A/Leningrad/82/76 (H3N2) virus with two donors of attenuation, A/Leningrad/134/17/57 (H2N2) and A/Leningrad/9/37/46 (H0N1), were evaluated. The recombinants were obtained by various methodical approaches (hybridization of native viruses, cross-reaction and selection of recombinants at temperatures of 25, 32 and 40 degrees C) to study their effects on the degree of attenuation and the regularity of transmission of the ts marker. The recombinants examined varied in thermosensitivity which depended on the conditions of recombination of parent viruses. All recombinants studied, irrespective of their RCT40 marker, were innocuous for men.

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.

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.

Recombination and cross-reactivation between virulent influenza viruses and a cold-adapted thermosensitive vaccine strain regularly produced genetically stable attenuated recombinants, the selection of which was based on the thermosensitivity marker. This marker, correlating with the safety of the recombinants for man was inherited independently on the properties of the haemagglutinin and neuraminidase surface antigens. There was no relationship between the thermosensitivity of the resulting recombinants and the decrease in the optimal temperature of the neuraminidase activity (OTNA marker). This indicates a separate localization in the viral genome of the mutation damages responsible for the expression of ts and OTNA markers.

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.

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.

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.

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.

Conditions of obtaining attenuated influenza virus recombinants by crossing of a cold-adapted donor with A (H1N1) influenza virus that reappeared in 1977 were studied. Temperature-sensitive recombinants suitable for intranasal immunization of adults with low titres of anti-haemagglutinin and anti-neuraminidase antibodies, and possessing sufficiently high immunogenicity were obtained by crossing of native parent strains and cross-reactivation techniques. It was confirmed that the cold-adapted A/Leningrad/134/17/57 (H2N2) influenza virus variant is a prospective donor of attenuation for obtaining recombinants--candidates for live influenza vaccine strains.

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.